Cardiotrofin application in liver diseases

FIELD: biology, medicine.

SUBSTANCE: invention relates to cartiotrofin (CT-1) expression enhancing in process of liver regeneration which consists with maximal hepatocyte proliferation. Also invention relates to application of CT-1 and composition based on the same as stimulator of liver regeneration, as well as hepatoprotective effect of CT-1 in various models of acute liver lesions.

EFFECT: application of CT-1 to improve liver functions at various pathologies thereof.

10 cl, 14 ex, 13 dwg

 

The technical field

This invention relates to the use of cardiotrophin (ST-1) to stimulate the regeneration of liver and protect hepatocytes from apoptosis and necrosis. In particular, this invention relates to the use of cardiotrophin for the treatment of acute, subacute, rapidly evolving and chronic hepatitis and cirrhosis of the liver, stimulate the regeneration of liver after resection and liver transplantation, but also to stimulate proliferation and trophic hepatocytes or precursors of hepatocytes in culture.

Background of the invention

The liver of humans and animals has a unique ability to regulate their growth and weight. When the destruction of harmful substance part of the liver parenchyma surviving hepatocytes are able to replicate and replace the damaged parenchyma. If liver resection or destruction of hepatocytes viral, toxic, immunological or metabolic nature addresses a very large part of the parenchyma, exceeding the regenerative capacity of the remaining liver tissue, liver failure occurs, which can lead to death. Currently, there are no medicines that affect liver protective and regenerative-stimulating effect that you can use in acute or chronic liver nedostate the property. Therefore, it is necessary to expand the range of drugs used in Hepatology, medical drugs, intended for these purposes. Hepatoprotective agent is a product or an active ingredient that can protect hepatocytes from different factors, which have a toxic effect and/or causing damage to the hepatocytes and, ultimately, necrosis or apoptosis. Thus, any liver disease introduction hepatoprotective funds in appropriate doses, should increase the survival rate of hepatocytes and consequently to facilitate the regeneration of the liver, contribute to the normalization of liver function and in extreme cases to save the life of the subject. Liver damage can be caused by toxic substances (including alcohol), viruses, autoimmune disorders, ischemia, ischemia/reperfusion (as in the case of lesions that occur in the implanted liver during transplantation) and any inflammatory processes. Good hepatoprotective agent should prevent or reduce the development of liver damage and death of liver cells in these situations.

Under the regeneration of the liver, the authors present invention understand the reaction of the liver, compensating for the loss of functional mass (reduction of tissue or loss of cells) by proliferatively hepatocytes to restore liver mass. There are several clinical cases of liver regeneration plays an important role, including liver resection during surgery (liver resection or liver transplantation from living donors) or above liver damage (toxic substances, viruses, ischemia, ischemia/reperfusion, and so on). Means of stimulating liver regeneration is a tool that is able to induce proliferation of hepatocytes that can help reduce the mortality associated with a decrease in functional liver mass.

The present invention relates to the use of cardiotrophin with liver disease.

Cardiotrophin (also referred to as CHF or factor hypertrophy of the heart) previously used for treatment of cardiac disorders, neurodegenerative and neurological diseases (WO 95/29237) as a local modulator of inflammatory processes caused LIFRβ-receptor (WO 97/30146), for the diagnosis and treatment of tumors (WO 00/43790) and for the treatment of amyotrophic lateral sclerosis and Parkinson's disease (WO 97/39629).

The present invention is not related to any of the above-mentioned applications, as its object is the use of CT-1 in therapeutic compositions intended for effects on hepatocytes, in particular, as a means of protecting these cells from APOP is oz and necrosis, and as a means of stimulating the overall regeneration of the liver.

ST-1 is one of the so-called neuropathies cytokines belonging to the family of IL-6 [1]. Receptors of cytokines of the specified collection are composed of individual subunits, but they all have the subunit gp130 [2]. Some members of this family (IL-6 and IL-11) is called homodimerization gp130 [3], while other States, such as factor inhibiting leukemia (LIF), oncostatin and ciliary neurotrophic factor (CNTF), cause heterodimerization subunit gp130 with LIF receptor size 190 kDa [4]. Receptor ST-1 contains a chain gp130, β-subunit of the LIF receptor (LIFRβand the third element, known as α-subunit receptor ST-1 [5, 6]. The last element involved in the formation of three-component complex, which reported high sensitivity and specificity of CT-1. Activation of the receptor ST-1 induces a number of intracellular signals that include early activation tyrosinekinase collection JAK (JAK-1, JAK-2 and Tyk2). The main effectors JAK is a group of cytosolic transcription factors STAT (STAT-1 and STAT-3; transducible signal activators of transcription). Activation of JAK also causes the signal transmission path, mediated by the Ras-MAP-kinase and activates the transmission of signals mediated by the PI3-K (phosphatidylinositol-3-kinase) [2].

CT-1 was originally Ident is adopted as hypertrophic factor cardiomyocytes [7, 8], as it was found that this factor stimulates the development of cardiomyocytes in the embryo and protects cardiomyocytes from apoptosis induced by hypoxia, ischemia, damage due to ischemia-reperfusion and other lesions[8, 9, 10, 11, 12]. In the scientific literature described the protective effect of CT-1 in the myocardium in the case of heart failure [10]. In addition, ST-1 stimulates the survival motor and dopaminergic neurons [13, 14].

Bibliographic references

Description of the invention

In this description of the invention used the following terminology appropriate to the purposes of the present invention:

i) active fraction ST-1 means any incomplete polypeptide sequence ST-1, which preserves the physiological effect of n is effective protein which is the object of the present invention;

ii) a polypeptide derivative having the activity of CT-1, refers to any polypeptide sequence, characterized by homology with native CT-1 more than 80% and preserving the physiological activity of a native protein, which is the object of the present invention;

iii) a polynucleotide sequence encoding specified active incomplete sequence ST-1 or polypeptide derivative ST-1, as described in paragraphs i) and ii)are also included in the scope of the present invention;

iv) cardiotrophin-1 or ST-1 means the native form of the protein, any form of recombinant protein (used alone or in sustained-release preparations), any polynucleotide form, encoding or expressing the native protein CT-1, or lengthening a segment of any of the forms described in paragraphs i), ii) and iii).

The basis of the present invention is the discovery that the gene ST-1 sverkhekspressiya in the process of liver regeneration after surgical resection of part of the liver parenchyma, reaching maximum expression at 48 hours after resection, which coincides with the moment of maximum proliferation of hepatocytes. On the basis of this discovery was the influence of CT-1 on the process of regeneration of the liver, it was found that transduction parenchyma pécs and gene sequences encoding CT-1, significantly stimulates liver regeneration after partial hepatectomy and prevents the death of the animal, after the almost total hepatectomy. Similarly, it was demonstrated that the transduction of the liver sequences encoding CT-1, provides effective protection of hepatocytes from different hepatotoxic substances, substantially reducing apoptosis/necrosis of hepatocytes. Finally, the results show that CT-1 is a potent protective agent for hepatocytes from exposure to substances that cause cell death and, in addition, has the property of stimulating the regeneration of liver.

Thus, the present invention relates to the use of CT-1, active faction ST-1, polypeptide derivative having the activity of CT-1, polynucleotide sequences encoding and expressing ST-1, active fraction ST-1 or polypeptide derivative having the activity of CT-1, to obtain compositions that can be used to stimulate the regeneration of liver after surgical resection of the liver or after liver injury caused by chemical substances, biological substances, inflammatory or immunological factors, as well as hepatoprotective means with different forms of acute, subacute, and quickly avivausa and chronic hepatitis, with toxic, viral, immunological or metabolic etiology, and for the stimulation of liver regeneration, protection of hepatocytes and improvement of liver function in case of liver cirrhosis with alcoholic, viral, metabolic or immunological etiology, and liver transplantation.

Examples

1. An adenoviral vector containing a gene sequence encoding a CT-1 (AdCT-1)

Defective adenovirus (with a deletion in positions E1 and E3), containing the gene for cardiotrophin-1 (AdCT-1), was created by the method, described in detail below. cDNA ST-1 mouse was selected from a library of muscle cDNA mouse with a probe PCR corresponding to nucleotides 20-639 cDNA sequence of the mouse (no access U18366 in the Bank of genes GenBank). The obtained cDNA was cloned in the vector pGEM-T/CT-1 and the correct clone was verified by sequencing. Then cDNA ST-1 cloned in the vector pKS with the formation of the plasmid pKS-CT-1, containing the expression cassette consisting of a promoter of rous sarcoma virus (nucleotides RSV 4526-5108 no access M in the Bank of genes GenBank), the peptide signal of nerve growth factor (nucleotides NGF 298-378 no access V00836 in the Bank of genes GenBank), cDNA ST-1 mouse (nucleotides 20-639 no access U18366 in the Bank of genes GenBank) and SV40 polyadenylation signal (nucleotides 2546-2775 no access NC0016691 in the Bank of genes GenBank). The expression cassette of prowess and of the plasmid pKS-CT-1 with BamHI/SalI and ligated with the Shuttle plasmid adenovirus pGY63 on the website HinfI with the formation of plasmid pGY63-CT-1. The obtained plasmid pGY63-CT-1 contains a left inverted terminal repeat adenovirus (ITR), the packaging signal (ps), a part of the gene pIX and tape the expression of CT-1, located between the last two elements. Plasmid pGY63-CT-1 was cotransformation in electrocompetent cells SF800 E.coli with plasmid pXL2689 containing the adenovirus genome for homologous recombination. Correct recombinants were digested with PacI and transfusional in 293 cells (kidney cells from human embryos transformed with DNA of adenovirus 5, the reference number of ATSS CRL-1573) for the production of adenoviruses. Structure AdCT-1 are presented in figure 1. The transformed E. coli strains were included as Deposit in the Spanish type culture collection (SISTER) of 12 September 2001 (E.Coli PKSCT1, CECT No. 5980) at the University of Valencia (Burjasot, Valencia, Spain).

For the production of adenoviruses used 293 cells infected with supernatant containing recombinant adenovirus. The 293 cells were first sown on 6-hole tablets at the confluence of approximately 80%, using 2% DMEM. After several hours, the culture medium was removed and cells were infected with 0.5 µl of the supernatant containing the recombinant adenovirus diluted in 3 ml of DEMEM. Cells were incubated for 1 hour at 37°C, after which the inoculum was removed and added to 4 ml of agar. Tile and cultured for 5-7 days at 37° C. using a Pasteur pipette, the sample of the virus were collected from viral plaques formed in the monolayer of cells; agar cylinder again suspended in 500 μl of DMEM with 2% fetal calf serum and stored at -80°C. identification of recombinant adenovirus 293 cells were sown on 12-hole tablets and infected previously allocated virus in a volume of 250 μl. After detection of cytopathic effect, the cells were collected separately from each hole. Then the cells three times froze and thawed with a view to their destruction and release the maximum amount of viral particles. Cell lysate from each series was centrifuged with a speed of 1500 rpm for 10 minutes. The supernatant containing the virus was used to infect 293 cells, cultured on 6-hole tablets. Once the cells had started to get the rounded shape, the supernatant was collected and tested for the presence of virus by detecting viral DNA and RNA in the specified supernatant. Supernatant with high levels of expression of the virus were selected for amplification with the aim of creating recombinant adenoviruses.

The 293 cells were cultured in 150 mm cups (from 50 to 100 cups) and infected with adenovirus obtained from stock at multiplicity of infection of 10 (10 plaque-forming units, PFU/cell). Cells expressing cytop the political action collected and centrifuged with a speed of 1500 rpm for 10 minutes, re-suspended in 0.1 M Tris buffer (pH 8) and frozen at -80°to further treatment.

Recombinant adenovirus was purified by gradient of cesium chloride. For this purpose, cells were stored at -80°With, again suspended in 0.01 M Tris-buffer and treated with 5% sodium deoxycholate in the ratio of 1:10 (V/V) for 30 minutes. The cells are then destroyed in a pre-chilled manual glass homogenizer to obtain a semi-solid solution. After that, the cell extract was added to a saturated solution of cesium chloride in respect of 5.8 ml of a solution of caesium chloride in 10 ml of cell extract. This solution was received in a special sealed polyallomer tubes Quick-seal, Beckman Instruments, CA, USA). Centrifugation was carried out in a rotor fixed angle Beckman 50 Ti with a speed of 35,000 rpm for 16-20 hours at 4°C. the Band corresponding to the virus, collected by syringe with sterile needle and again centrifuged under the same conditions. The band was extracted and dialyzed against 0.01 M Tris buffer, pH 8, at 4°With, in the form of two independent processes with a duration of 1.5 hours each. Aliquots of virus preparation was placed in a test tube with sterile glycerol (ICN, USA) in 10% (V/V), freezers and refrigerators and kept in liquid nitrogen until further use.

To determine the infectious titer of the purified recombinant adenoviruses performed the test on limiting dilution in 96-well plates. The basis of this test is the study of cytopathic effects that a virus in 293 cells, to determine the maximum decimal dilution of a suspension of the virus, wherein the virus is able to infect 293 cells and multiply in these cells. Cells pre-293 were sown in 96-well plates in an amount of 104cells/well. Then from the wells was removed and cells were infected with adenovirus in the amount of 50 µl/well in serial doubling dilutions. After 6 hours was added 150 μl of fresh DMEM medium and incubated the cells at 37°within a maximum period of 7 days. Upon expiration of the specified period were evaluated by the cytopathic effect of the virus on the cells. To determine the titer of the number of cells experiencing cytopathic effect was multiplied by the maximum dilution that causes a similar effect, and the result was divided by the total measured volume (0.05 ml), which allowed to determine the number of plaque-forming units (PFU) per ml this definition was repeated for each sample at least three times.

2. ST-1, recombinant protein

cDNA encoding CT-1, was obtained from the plasmid pGEM-T/CT-1 split is the group with EcoR1 and cloned in the vector pET28b (Novagen) (pET28b/CT-1). This vector has a sequence that encodes the number of histidine residues (1 kDa) and transmitted in accordance with the cloned cDNA, thus producing a fused protein containing at his aminobenzo terminal his-tag segment with a length of 1 kDa and ST-1, between which is the site of cutting thrombin.

For this protein, the authors present invention used competent bacteria strain BL21 (DE3) (Novagen, Germany, catalog No. 70235), as this strain contains a gene induced RNA polymerase T7, which is a necessary requirement for the subsequent production of the specified protein. Competent bacteria were transformed previously obtained vector pET14b (vector pET-14b company Novagen, catalog No. 69660-3) cDNA cloned ST-1. Transformed bacteria were selected for cultivation in LB medium with ampicillin, since this vector contains the gene for resistance to a specific antibiotic.

To obtain recombinant CT-1 transformed bacteria were grown in LB medium with ampicillin at 37°to achieve an optical density of 0.4 at 600 nm. The expression of recombinant protein was induced IPTG added to a final concentration of 0.5 mm. The result of this treatment is the induction of the lac promoter and then promoter RNA polymerase T7, which contains the vector and regulates the xpressio cloned cDNA. The culture was grown for another 4 hours under the same conditions.

To obtain extracts immediately after growing the bacteria were subjected to centrifugation at 4°C. the Precipitated bacteria again suspended in buffer containing 10 mm Tris/HCl, 10% sucrose, 2 mm 2-mercaptoethanol and protease inhibitors. Bacteria were incubated with lysozyme at 4°C for 30 minutes and homogenized by ultrasound. As a result of this treatment was the destruction of the membranes of the bacteria, which improved the yield of extracted product. Cytosolic extract was obtained by centrifugation of the homogenate at 100000 g for 90 minutes. The obtained protein was checked by analyzing the cytosolic fraction by the method of SDS-PAGE.

Protein His-CT-1 was purified by chromatography of cytosolic extract in 2 ml Nickel column. Then the column was washed and the protein was suirable 1 M imidazole. Pure protein was treated with thrombin and allocated ST-1.

3. Analysis method, Northern blotting to measure the expression of CT-1 in vivo

Gene expression of various cytokines (growth factor hepatocytes, HGF; LIF; oncostatin; CNTF; CT-1) were analyzed in the process of liver regeneration by means of Northern blotting after extraction of mRNA from rat liver. RNA was extracted using thiocyanat guanidine - phenol - chloroform. The analysis Northern blotting was performed according to the earlier description [15], using the expression of 28S as a reference and probes specific for each analyzed gene.

4. Culture of cell lines isolated from hepatocytes

For in vitro cells were used In line hepatocytes isolated from malignant hepatoma in rats. Cells were cultured in modified according Dulbecco environment Needle (DMEM)containing 10% calf serum, 2 mm glutamine, 100 U/ml streptomycin, 100 mg/ml penicillin. Cell cultures were incubated at 37°C in an atmosphere with 5% CO2.

5. Methods of analysis of apoptosis on the basis of cell cycle and the expression of annexin

Cell cycle was analyzed by the method of staining DNA with iodide of propecia. Cells (0,5·106made permeable using 50 µl of 0.1% NP40 solution before staining with 0.5 µl of 50 µg/ml solution of iodide of propecia and 4 KED/ml Gnkazy (set of reagents DNA-Prep Coulter, Coulter). Cells were incubated at 37°C for 20 minutes and examined in cytofluorometry FACScalibur. Cells positive painted iodide of propecia analyzed in the doublet-sensing module (DDM) flow cytometer (FACScalibur, Becton-Dickinson, USA), eliminating duplicates, and use the parameter FL3. Frequency subdiploid cells defines the percentage of the number of cells undergoing apoptosis.

The presence of phosphatidylserine, oriented towards the outside on the shell of the cell, is one of the parameters, which allows to determine the apoptotic cell. Annexin V detects apoptotic cells due to its ability to bind with molecules phosphatidylserine, facing to the outside of the cell membrane at the moment when the cell undergoes apoptosis. Cells (0,5·106once washed in buffer for incubation containing: 140 mm NaCl, 5 mm KCl, 1.2 mm MgCl2, CaCl2and 10 mm HEPES. Cells were incubated in 100 μl of buffer for incubation and 5 μl of the fluorescein-isothiocyanato conjugate associated with annexin V (Annexin-FITC) for 15 minutes at room temperature. The cells are then examined in a flow cytometer FACScalibur using the FL1. The apoptosis index was determined on the basis of the percentage of cells positive for annexin-FITC.

6. Methods of analysis of proteins

The electrophoresis.For protein analysis cells were literally in the buffer for lysis (20 mm Tris, pH 7.5; 150 mm NaCl, 1 mm EGTA, 1 mm EDTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm Na3VO4, 1 μg/ml of lapetina, pepstatin, 10 µg/ml trypsin inhibitor, 1 mm PMSF. The lysate 0,5·106cells are again suspended in the volume ratio of buffer for migration (125 mm Tris-HCl (pH 6.8), 10% sodium dodecyl sulfate, 20% glycerol, 100 mm dithiothreitol, and 0.2% bromophenol blue). Samples of protein extract was heated at 100°St for 5 minutes and subjected to electrophoresis in 10% polyacrylamide gel.

Immunological assays, Western blotting.After electrophoresis proteins were transferred to nitrocellulose membranes in a buffer for transfer (25 mm Tris, 0.2 M glycine, 20% methanol, pH 8.5) under the influence of a current of 300 mA for 1 hour. Transferred proteins were stained with a solution of crimson red to verify a successful migration. Then the membrane was used for immunological analysis of specific proteins. For this purpose, the membrane was blocked in buffer for incubation TBS-T (20 mm Tris, 137 mm NaCl at pH 7.6 and 0.5% tween-20) with 2% BSA (fraction V albumin) for 1 hour. Membranes were incubated with specific antibodies against protein within 2 hours. Then the membrane was washed with buffer TBS-T for 1 hour and again incubated with protein G-HRPO (BIORAD) for 1 hour. The membrane is washed several times in buffer TBS-T, showed chemiluminescence reagents (NEN Life Science Products) and immediately exposed to ultra-sensitive film (Amersham) at a pre-determined periods of time.

Immunoprecipitate.For thus specific protein lysates 106cells were incubated for 18 hours in the presence of specific antibody and 20 μl of protein G-Sepharose at 4°C. the immune complexes were isolated by centrifugation, washed twice with a buffer for lysis and was dissolved in the buffer for migration. Then samples agrawala to 100° With and caused migration by electrophoresis in 10% gels. Immunological analysis of specific proteins was performed by the method of Western blotting.

7. Determination of DNA synthesis. Analyses of proliferation

Cells H-35 were sown in 96-well plates. Cells were cultured without serum for 24 hours and then stimulated CT-1 (50 ng/ml)diluted in serum-free medium DMEM. Cells were incubated with CT-1 for 24 hours and were marked 10 µci/ml of [methyl-3H]thymidine (ICN, Amersham) for 12 hours. The radioactive medium was removed, cells were separated using 100 μl of trypsin at 37°and collected in a 25 µl mixture for scintillation account (Ecolite; ICN). The content of [3H]thymidine were analyzed in a scintillation counter tri-Carb 2900TR (Packard, Meriden, CT).

8. Tests of liver regeneration in vivo after partial hepatectomy (surgical resection 75% of the liver)

Liver regeneration was investigated using rat Fischer (males weighing 180 g). In the surgical resection was removed 75% of the liver and squashed rats at different time periods (1 hour, 3 hours, 6 hours, 10 hours, 24 hours, 48 hours, 3 days, 6 days and 9 days). Then taking samples of liver and divided them into three parts for histological examination (fixation in formalin), immunohistochemistry (fixation in the EAST) and analysis of RNA (freezing in liquid nitrogen). For each and elizaryeva period of time have used at least 4 rats. The parameters analyzed regeneration of the liver was expressed as a percentage of the mass of the liver and the expression of the nuclear antigen of proliferating cells (PCNA), as determined by immunohistochemical method.

8.1. Expression of CT-1 in the process of regeneration of the liver

Gene expression of various cytokines (HGF, LIF, oncostatin, CNTF, CT-1) were analyzed using a model of partial hepatectomy to study their participation in regeneration of the liver. In this study the authors present invention analyzed samples of rat liver obtained at various times after partial hepatectomy (1 hour, 3 hours, 6 hours, 10 hours, 24 hours, 48 hours, 3 days, 6 days and 9 days). Each group consisted of at least 4 animals. In addition, we analyzed the liver of healthy rats not subjected to hepatectomy (control animals). The corresponding mRNA levels of each cytokine were identified by means of Northern blotting. As a result of performing these experiments were done entirely new observation of a significant increase in the mRNA levels of CT-1 after 24 and 48 hours after hepatectomy (figure 2 and 3)corresponding to the maximum proliferation of hepatocytes, as evidenced by the expression of PCNA and absorption of bromosuccinimide (BrdU) hepatocytes immunohistochemical study samples of liver tissue from experimental animals. In addition, the authors of altoadige invention found that increased transcriptional expression of CT-1 was preceded by the maximum expression of HGF, which occurred 10 hours after hepatectomy.

8.2. The effect of CT-1 on liver regeneration after partial hepatectomy

To study the role of CT-1 in the regeneration of liver animal was intravenously injected with adenovirus with ST-1 (AdCT-1) in the dose of 108The FIGHT or adenovirus with the reporter gene LacZ (AdLac-Z) in the same dose as the control sample. After 48 hours produced a surgical resection of 75% of the liver. Then rats were killed at the above time periods. For each time period analyzed at least 4 rats and a maximum of 8 rats.

Introduction AdCT-1 caused an increase in the weight of liver in rats treated with AdCT-1, compared with rats that were injected AdLac-Z, and between these two groups was significant difference in 48 hours, while the maximum proliferation of hepatocytes (as evidenced by the results of the immune staining for PCNA in liver samples obtained from rats after hepatectomy). After 3 and 6 days after hepatectomy, the liver weight in rats treated with AdCT-1, was greater than in control rats, although these time periods, the difference between groups was not statistically significant (figure 4). The results show that the liver, which was affected by ST-1, features : what was ovalis faster regeneration and had a large mass compared with the control animals in the early period after hepatectomy, but ultimately reached the same performance as the liver of control animals under the action of homeostatic mechanisms that regulate the final size of the liver.

9. Analysis of liver regeneration in vivo after extensive hepatectomy (surgical resection >85% of the liver)

To determine whether CT-1 to prevent the death of animals subjected to the almost total hepatectomy was performed experiments using rats Fischer, who in the surgical resection was removed more than 85% of the liver. In these experiments we used two groups of 30 rats each. One group was injected intravenously AdLac-Z and the other group was injected AdCT-1 in the above doses. Surgical resection of this type were made at 48 hours after injection of adenovirus. The number of rats surviving after surgical resection, was 14 rats in the group treated AdLac-Z, and 13 rats in the group that were injected with AdCT-1. In rats controlled the duration of survival after extensive surgical resection.

It was found that in the first hour after hepatectomy infant mortality was 77% in the group treated AdLac-Z, and has not reached 20% in the group treated with AdCT-1. Twenty-four hours after hepatectomy were alive only 7% of rats receiving AdLac-Z, while the survival rate among rats treated with AdCT-1, was 61%; there are shown the difference is statistically significant level. The above percentages remained at the same levels 4 days after surgical resection (figure 5). The data obtained by the authors of the present invention, show that ST-1 prevents mortality due to extensive liver resection.

10. Protective effect of CT-1 against apoptosis/necrosis of hepatocytes in vivo. Tests for rapidly developing liver damage

To evaluate the role of CT-1 in the modulation of liver damage caused by various harmful substances, used Balb/c mice (males weighing 30 g), and the liver was assessed in three models destruction of hepatocytes: (i) lesions caused by intravenous injection of 100 mg/kg of concanavalin And, Con-A (Sigma, St. Louis, MO., USA); (ii) lesion caused by a combination of intravenous TNFα (Peprotech) (0.5 μg/mouse) and intraperitoneal administration of 25 mg of D-galactosamine, TNFα/D-Gal (Sigma); (iii) lesion caused by the intravenous injection of 1.5 μg/mouse antibodies against Fas (Jo2. Pharmingen). Six hours after administration of Con-A, TNFα/D-Gal or antibodies against Fas mice took samples of blood, after which they were killed.

To determine the effect of CT-1 in the liver, the group of mice was injected with saline, group "B" was introduced AdLac-Z (107The COMBAT) and group "C" was introduced AdCT-1 (107The COMBAT). After 48 hours in each group three of the above models were induced time for the giving of the liver. The group of mice that were injected with the serum in saline solution instead of the inductor hepatitis, was also included in this experiment as a negative control group (NC). Each group of animals consisted of 5 mice. After 6 hours investigated the extent of liver damage by 2 parameters: measurement of transaminases (GPT) in serum using automated colorimetric analysis (Technicon RA-1000, Bayer) and measurement of apoptosis by the TUNEL method in the liver samples, fixed in the EAST, using the Kit to detect cell death in situ" (Roche Diagnostics GmbH, Indianapolis, IN, USA).

All mice took blood samples for determination of transaminases, after which the animal was immediately squashed and made the treatment of liver for histology (fixation in formalin) and studies of apoptosis by the TUNEL method (freezing in the EAST).

In the first model of acute liver injury induced by Con-A, it was found that while the mice in the control groups (animals treated with saline or AdLac-Z) observed very high rates of GPT, in animals treated with AdCT-1, transaminase levels were not significantly altered, and the differences between these two groups were highly significant (figa). In the study of the liver tissue TUNEL method was observed in the absence of apoptosis in liver samples from mouse on the th, treated with AdCT-1, compared with extensive areas of necrosis and apoptosis in animals that received serum in saline solution or AdLac-Z before the introduction of Con-A (Fig.7).

In the second model of acute liver injury caused by the introduction of monoclonal antibodies against Fas, it was found that the introduction of AdCT-1 prevents the death of hepatocytes (pigv). Six hours after administration of antibodies against Fas content transaminases in animals treated with AdCT-1, was significantly lower (statistically significant difference)than in animals treated with the serum in saline solution or AdLac-z in Addition, using the TUNEL method and histological study revealed a significant decrease in apoptotic bodies in samples of liver tissue of mice treated with AdCT-1, compared to animals in the control groups.

Hepatoprotective role of CT-1 was also evaluated in the third model of liver injury caused by the combined introduction TNF and D-galactosamine (TNF-α/DGal). According to the results of histological analysis and TUNEL method six hours after liver injury was observed a significant decrease in transaminase levels, and the number of apoptotic hepatocytes of mice treated with AdCT-1, compared with mice in the control groups (figs).

All the obtained data show that CT-1 can protect liver cells from R. the importance of factors causing apoptosis or necrosis of liver cells.

11. Analysis of the impact of CT-1 on cell cycle and survival of hepatocytes isolated from cell lines

Using the cell line In rat liver, the authors of the present invention investigated the biological effects that recombinant CT-1 as cytokines regulator of apoptosis of hepatocytes. For test stimulation cardiotrophin CT-1 cells pre-cultured without serum for 18 hours. Test stimulation of the ST-1 was performed without serum.

The authors of the present invention were first analyzed the effect of CT-1 on the cell cycle the cell line of liver. The cell cycle was determined by staining DNA with iodide of propecia with subsequent analysis by the method of flow cytofluorometry. Apoptosis was induced by removal of serum from the culture medium of the cells for 4 days. The results showed that in the four-day cultivation in these conditions, 86% of the cells N underwent apoptosis. It should be noted that when using the ST-1 at a dose of 50 ng/ml and in the absence of any other additional incentives ST-1 was significantly delayed the start of apoptosis N, which was observed in approximately 52% of the cells (Fig.7).

Similar experiments were performed, cultivating without serum cell culture N for 3 is it and measuring the ability of cells to bind on the surface of purified annexin, associated with FITC (isothiocyanate fluorescein). The binding of annexin-FITC with the surface of the cells N investigated by performing the analysis of flow cytofluorometry. The results confirm that cells cultivated without ST-1, are characterized by approximately 21% are positive for annexin and cells treated with 50 ng/ml CT-1, are characterized by approximately 12% positive response (Fig). Thus, the performed experiments showed that CT-1 used in the dose capable of exerting anti-apoptotic action.

12. Analysis of the impact of CT-1 on cell proliferation

Using the cell line H-35, the authors of the present invention investigated the ability of CT-1 to affect DNA synthesis in hepatocytes. For this purpose, 20,000 cells were sown in a single well of 96-well plates. In order to detect potential stimulation, the cells were pre-cultured without serum for 24 hours. Test stimulation of the ST-1 was performed without serum using ST-1 at a dose of 50 ng/ml within 24 hours. The results showed that cells cultured in the presence of CT-1, are characterized by a higher percentage of DNA synthesis compared to control cells that were not subjected to CT-1 (Fig.9). Thus, these experiments confirm that ST-1 used in the dose able to induce DNA synthesis.

13. Research and analysis of the transmission of signals induced by CT-1 in cell lines derived from hepatocytes

The discovery that ST-1 has anti-apoptotic effect on hepatocytes in vivo and in vitro, allowed the authors of the present invention to investigate ways of signaling involved in stimulation of the receptor ST-1 in hepatocytes. Stimulation of the receptors of the family of cytokines IL-6/LIF causes immediate phosphorylation of transmitters of signals relating to the family of JAK-1. After stimulation, cells N cardiotrophin ST-1 at different time periods, the authors of the present invention performed immunoprecipitation JAK-1 specific antibody (Cell Signaling Technology), selected from a total lysates of these cells. When using specific antibodies to the phosphorylated tyrosines (4G10, Upstate Biotechnology) and perform Western blotting, it was found that CT-1 induces tyrosine phosphorylation of a molecule JAK-1 after 5 minutes, the signal disappears after 60 minutes (figa).

Phosphorylation of STAT-3 is one of the described pathways of activation involved in the signal transmission of the cytokine family, IL-6 by JAK. Activates the specified molecule phosphorylation in some cases due to the induction of cellular differentiation, and in other cases, hypertrophy (myocardiocytes). Using Western blotting and the Torah of the present invention analyzed the lysates N, treated in vitro with 50 ng/ml CT-1 in different periods of time. Using antibodies specific for phosphorylated STAT-3 (Santa Cruz Biotechnology), revealed that CT-1 is able to induce phosphorylation of STAT-3, starting with the 5-th minute after stimulation and causing maximum induction after 30 minutes (pigv).

One way, obviously, determining the inhibition of apoptosis signal is the path PI-3/AKT (phosphatidylinositol-3-kinase/AKT-kinase). Activation of PI-3K induces activation by phosphorylation ACT in serine 475 and threonine 308. Activating the ACT in turn causes phosphorylation of BAD at serine at positions 112 and 136. BAD is a member of Bcl-2 is an important regulator of the survival signal. Inactive BAD forms a dimer protein Bcl-x or Bcl-2, neutralizing their anti-apoptotic activity. Phosphorylation of BAD triggers the release of Bcl-2 or Bcl-x, which inhibit the activation path of apoptosis. Therefore, it is assumed that the phosphorylation of BAD is a method of suppressing ways of activation of apoptosis. The authors of the present invention investigated whether CT-1 to activate the path of survival in cells N. Cells were treated with 50 ng/ml CT-1 in different periods of time, received the cytosolic fraction and was immunoprecipitate ACT using polyclonal antibodies against ACT (Cell Signaling Technology). Then the method of Western-blot the Tinga analyzed the presence of phosphorylated ACT, using a polyclonal antibody, which is specific to the specified form of the ACT, phosphorylated at serine 475 (Cell Signaling Technology). It was found that CT-1 induces phosphorylation sustainable ACT serine 475 after 15 and 30 minutes and disappears after 60 minutes. Thus, CT-1 induces survival signal in line cells hepatocytes (figs).

In summary, we can conclude that CT-1 is able to induce a way of signaling JAK/STAT, and the path of survival PI-3K/AKT. A cascade of signals induced by CT-1 in hepatocytes, explains how ST-1 acts as a cytokine, exerting anti-apoptotic effect by using the path PI-3K/AKT, and possibly as an inducer of proliferation and differentiation of hepatitis by using the path JAK/STAT-3.

14. Research and analysis of the transmission of signals induced by AdCT-1 in models of acute liver failure in vivo

For analysis of protective actions AdCT-1 observed in models of in vivo acute liver injury in rats and mice, were investigated transmission of signals in accordance with data obtained in the study in vitro, apparently, involved in stimulation of the receptor ST-1 hepatitis, with the use of these models in vivo.

As described above in the in vitro study, ST-1 can induce the activation of three main ways, determine what their survival or the inhibition of apoptosis: STAT-3 (transducer signals and activator of transcription), PI-3K (phosphatidylinositol-3-OH-kinase)/akt and Erk1/2 (extracellular regulated kinase).

A. Model extensive hepatectomy in rats

As was shown in the experiments described in example 9 (liver resection >85%), the highest mortality was observed within 1 hour after surgical resection (see figure 5). For this reason, these experiments were repeated in 3 groups of animals, which were injected with different drugs (AdCT-1, AdLac-Z and saline solution), but the rats were killed 1 hour after surgical resection to obtain samples of the liver.

The collected liver samples were divided into three parts for histological examination (fixation in formalin), immunohistochemistry (fixation in the EAST) and protein analysis (freezing in liquid nitrogen). Of the samples, frozen in liquid nitrogen, got homogenates of the liver in the buffer for lysis (20 mm Tris, pH 7.5; 150 mm NaCl; 1 mm EGTA; 1 mm EDTA; 1% Triton X-100; 2.5 mm sodium pyrophosphate; 1 mm Na3VO4the mixture of antiprotease). Method Western blotting using specific antibodies company Cell Signaling Technology (Beverly, Mssachusetts) investigated the transmission of signals in the three groups of rats: treated with AdCT-1, AdLac-Z and saline (S). Used the following antibodies: anti-Stat-3, against phosphorylated Stat-3 (Stat-3-Y-705), against the ACT, against phosphorylated ACT (Akt-Ser-473), anti Erk/2 and against phosphorylated Erk1/2, which simultaneously detect phosphorylated forms of Erk1 (Erk1-Thr-202) and Erk2 (Erk2-Y-204). It was found that the liver of rats treated with AdCT-1, characterized by the phosphorylation of STAT-3, ERK1/2 and ACT in contrast to the rat liver, which was introduced AdLac-Z and physiological solution, where this effect was absent (figa).

On the other hand, in the occurrence of apoptosis in response to many stimuli, including extensive hepatectomy (>85%), participates of caspase-3. For this reason, we studied the activity of caspase-3 using part of the samples in liquid nitrogen, in accordance with the test method proposed by the company (CaspACE, Promega, Madison, Wisconsin). It was found that rat liver, which was previously introduced AdCT-1, are characterized by a lower activity of caspase-3 compared with the liver of rats, which were injected AdLac-Z and saline (Fig. 12B), indicating a lower apoptosis index in the first case.

The foregoing suggests that the protective effect of the AdCT-1 on liver damage in rats caused by the initiation of a cascade of anti-apoptotic signals induced by CT-1, with the consequent reduction in apoptosis, as evidenced by the detected low activity of caspase-3.

b. The model of acute liver injury caused by concanavalin And, in mice

To study the transmission of signals in a given m the Delhi were re-performed experiments on induction, caused by Con-A (see example 10) in 3 experimental groups (AdCT-1, AdLac-Z and saline solution), but at the same time the mice were killed 1 h after the introduction of Con-A. liver Samples obtained at the time of killing the animals were treated in accordance with the above description in example 14a. Western blotting was performed in a similar way and used the same antibodies.

As shown in Fig, introduction AdCT-1 has caused the phosphorylation of akt and ERK 1/2, the main ways of suppression of apoptosis and activation of survival induced by CT-1. Therefore, the results suggest that AdCT-1 protects mice from liver injury induced by Con-A, activating these are the main ways suppression of apoptosis.

Description of the drawings

Figure 1. Schematic illustration of the structure of the adenoviral vector AdCT-1, containing the sequence encoding CT-1. RSV: the promoter of the rous sarcoma virus; NGF: peptide signal of nerve growth factor; ST-1: cDNA ST-1 mouse; SV40: polyadenylation signal of the SV40 virus. Suppressed region E1 and E3 are painted in black color.

Figure 2. Detection method, Northern blotting of mRNA encoding ST-1 in samples of rat liver obtained at different times (h = hours; d = days) after partial hepatectomy. 28S rRNA, used as a control sample.

Figure 3. Graphic expression of CT-1 according to tremani (h = hours; d = days) when performing Northern blot shown in figure 2. Ordinate: arbitrary units of optical density (ST-1/28S).

Figure 4. Percentage weight of the rat liver (on the ordinate) at different periods of time (abscissa: h = hours; d = days) after partial hepatectomy before the introduction of the AdCT-1 or AdLac-Z and after partial hepatectomy.

Figure 5. Percentage survival (on the ordinate) of rats treated with AdCT-1 or AdLac-Z and subjected to hepatectomy (>85%) at 48 hours after administration of these drugs. Abscissa: time (hours) after hepatectomy.

6. The graphical representation of the levels of transaminases GPT in serum (on the ordinate, SOY/ml) and histological picture of the liver tissue (TUNEL method for imaging apoptosis) of 3 models, the induction of a fast-growing hipatia in mice: as a result of the introduction of concanavalin And, Con-A (Piga); injection of antibodies against Fas (pigv) and co-injection of TNFα and D-galactosamine, TNFα/D-Gal (figs). 48 hours prior to induction of hepatitis animals were injected with an adenoviral vector (AdCT-1 or AdLac-Z) or saline (S). Negative control result (NC) obtained in the group of mice that were injected with the serum in saline solution instead of inducing hepatitis substances.

7. The cell cycle analysis of cells N-35 after 1 day (top) and 4 days (bottom) cultivated the I without serum in the absence of CT-1 (C = control sample) and in the presence of CT-1 (CT-1). The selected zone from left to right: cells with DNA less than 2n (apoptotic cells, Apo); cells in phase G0-G1 (resting cells) and cells in the S phase and M (proliferating cells). Ordinate: number of cells. Abscissa: concentration of DNA.

Fig. Analysis of the expression of annexin V in cells N-35 by flow cytometry under cultivation without serum for 3 days in the absence of CT-1 (C = control sample) and in the presence of CT-1 (CT-1). Cells cultured with ST-1, was characterized by approximately 12% apoptotic cells compared with 21% of apoptotic cells detected in the absence of CT-1.

Fig.9. Analysis of the impact of CT-1 on cell proliferation, measured by the introduction of [3H]thymidine. The results indicate percentage increase (ordinate) proliferation of cells treated with CT-1 (CT-1), compared to control cells not exposed to the treatment (C = control sample).

Figure 10. Immunological analysis of phosphorylated signaling proteins (Jak-1-Y, Stat-3-Y-705 and ACT-Ser-475) in lysates of cells N obtained in different periods of time (minutes) after incubation of cells with ST-1.

A) Immunoprecipitated cell lysates with antibodies specific for Jak-1. When performing Western blotting using antibodies specific to the phosphorylated tyrosines, phosphorylation of the molecule Jak-1 was observed after 5 minutes.

C) estern-blotting using antibodies specific to phosphorylated Stat-3 (Stat-3-Y-705), where a positive result is observed after 5 minutes of processing.

(C) Immunoprecipitate cytosolic fractions with antibodies against ACT followed by Western blotting using antibodies specific for phosphorylated ACT Serino 475 (AKT-Ser-475), indicates the detection of induction after 15 and 30 minutes.

11. The structure of the vector pET-14b.

Figa. Western blotting of signaling proteins in the liver of rats treated with AdCT-1, AdLac-Z or saline (S), which then was subjected to resection of more than 85% of the liver and were killed 1 h after resection.

1A) Western blotting using antibodies specific for phosphorylated Stat-3 (Stat-3-Y-705).

1b) Western blotting using antibodies specific for Stat-3, for the quantitative determination of the total content of Stat-3.

Phosphorylation of Stat-3 was detected in rats treated with AdCT-1.

2A) Western blotting using antibodies specific for phosphorylated Erk1 and Erk2 (Erk1-Thr-202 and Erk2-Tyr-204).

2b) Western blotting using antibodies specific for Erk1 and Erk2, for the quantitative determination of the total content of Erk1 and Erk2.

Phosphorylation of ERK1/2 was detected in rats treated with AdCT-1.

3A) Western blotting using antibodies specific to factorily is consistent Akt (Akt-Ser-473).

3b) Western blotting using antibodies specific for Akt, for the quantitative determination of the total content of Akt.

Phosphorylation of Akt was detected in rats treated with AdCT-1.

Figv. The activity of caspase-3 in the liver of rats subjected to extensive liver resection (>85%). Samples obtained from the same group subjected to hepatectomy in rats that were used in the experiments shown in figa. The following chart shows the increased activity of caspase-3 (number of times) compared to animals with a healthy liver.

Fig. Western blotting of signaling proteins in the liver of mice treated accordingly AdCT-1, AdLac-Z or saline (S), before the introduction of the Con-A for the induction of liver injury. Samples were obtained at the time of killing animals 1 hour after induction of liver injury by means of Con-A.

1A) Western blotting using antibodies specific for phosphorylated Akt (Akt-Ser-473).

1b) Western blotting using antibodies specific for Akt, for the quantitative determination of the total content of Akt.

Phosphorylation of Akt was detected in rats treated with AdCT-1.

2A) Western blotting using antibodies specific for phosphorylated Erk1 and Erk2 (Erk1-Thr-202 and Erk2-Tyr-204).

2b) Western blotting using antibodies specific for Erk1 and Erk2, for the quantitative determination of the total content of Erk1 and Erk2.

Phosphorylation of ERK1/2 was detected in rats treated with AdCT-1.

1. Application cardiotrophin-1 (ST-1) or polynucleotide sequence, and expressing the coding ST-1, to obtain a composition that can be used to stimulate the regeneration of liver.

2. Application cardiotrophin-1 (ST-1) or polynucleotide sequence, and expressing the coding ST-1, to obtain a composition that can be used as a hepatoprotective tools.

3. The use according to claim 1 to obtain a composition that can be used to stimulate the regeneration of liver after hepatectomy.

4. The use according to any one of claims 1 and 2 to obtain a composition that can be used for the treatment of acute, subacute, rapidly developing or chronic viral hepatitis, metabolic or toxic etiology.

5. The use according to any one of claims 1 and 2 to obtain a composition that can be used to treat cirrhosis of the liver.

6. The use according to any one of claims 1 and 2 to obtain a composition that can be used to restore the function of the transplanted liver.

7. The use according to any one of claims 1 and 2 to obtain a composition that can be used to treat intrahepatic tumors.

8. The use according to any one of claims 1 and 2 to obtain a composition, which contains the viral vector, bearing polynucleotide sequence, expressing and encoding CT-1.

9. The use of claim 8, wherein the viral vector is an adenovirus.

10. The method of cultivation of hepatocytes, characterized in that the ST-1 or viral vector carrying the polynucleotide sequence encoding and expressing CT-1, enter into the cultural medium, in particular containing serum, as reputationmanager money or money stimulating the proliferation of hepatocytes.



 

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