Onlineby, method thereof and pharmaceutical composition containing onlineby

 

(57) Abstract:

The invention relates to medicine, namely to the field of preparative biochemistry, and for onlinebingo, method of its production and pharmaceutical compositions containing onlinebig. The invention includes a purified and isolated human protein ongoingly produced erythroblastopenia cells with antineoplastic activity against cell lines of breast carcinoma (F7) and carcione uterus Heha and proliferative effect on normal fibroblasts foreskin man. Its production method is incubation of human erythroblastopenia cells (K-562) environments, the collection of conditioned cell media, isolation and purification of human onlinebingo. The pharmaceutical composition includes onlineby and a pharmaceutically acceptable carrier. The technical result consists in obtaining a new factor to stimulate the growth of normal cells. 3 S. and 4 C.p. f-crystals, 45 ill., 9 table.

The invention relates mainly to cytokines. In more detail, the present invention relates to a new cytokine with broad antitumor activity.

The balance between cell growth-stimulants and narushenie balance this growth regulatory cytokines.

Describes several cytokines, which stimuliruyut growth of cells spyhole and normal cells. They include, for example, epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), insulin-like growth factors (IGF), interleukins (IL), colony stimulating factor (CSF), and transforming growth factors (TGF - a and TGF-).

In contrast, other cytokines selectively inhibit the growth of certain tumor cells. Such cytokines include, for example, interferons (IFR), lymphotoxin (LT), tumor necrosis factor (TNF), oncostatin M, amphiregulin, interleukin-I (IL-I), interleukin-6 (IL-6) and TGF -.

Such growth promoters and cytokine inhibiting growth, can be differentiated on the basis of their origin, their specificity towards tumor objects, their physico-chemical properties and primary structure. Thus, critically important for understanding cell growth, including the growth of tumors, is the identification and characterization of regulatory cytokines growth.

In one embodiment, the present invention proposes a new structure. This new composition of matter, called Onlinebingo, outstanding human erythroblastopenia cells is Eulaton sodium) and demonstrates various antitumor activity.

In one embodiment, the present invention proposes a method of obtaining a new human cytokine called Onlinebig. The method includes insulatio human erythroblastopenia cells, inducing the production Onlinebingo and collect the resulting cell supernatants.

Another variant of the present invention provides a method of purification of human Onlinebingo. This method includes the stage of ultrafiltration received cell supernatants containing human Onlinebig. Then dialysis ultrafiltrating of supernatants containing human Onlinebig. Then for the purification of human Onlinebuy spend chromatography (DEAE Affigel blue chromatography), electrophoresis in polyacrylamide gel with sodium dodecyl sulfate (SDA-PAGE) and high performance liquid chromatography on reversed phase.

In another embodiment, the present invention proposes a new immunomodulator for the activation of lymphocytes, monocytes and neuropile for destruction of tumor cells. New immunomodulator will vkluchaet human Onlinebig. A new growth factor to stimulate the growth of normal cells. This growth factor includes human Onlinebingo and processing of tumor cells, including the introduction of effective doses of Onlinegiving.

In order to understand in detail the invention with the above features, advantages and objectives of the invention (as well as others which will be clear below), a more detailed description of the invention, briefly summarized above, refers to a specific variants of the invention, illustrated by the attached drawings. These drawings form a part of this description. However, it should be noted that the attached drawings illustrate preferred options, but do not limit the scope of the invention. The invention consists of equally effective equivalent options.

Fig. 1 shows that air supernatant cells K-562 inhibit the growth of MCF-7 cells.

Fig. 2 depicts a standard bioizluchenie for Onlinegiving.

Fig. 3 shows the production of Onlinebingo cells K-562 in the presence and absence of serum.

Fig. 4 depicts the effects forblog ether on the induction of Onlinegiving.

Fig. 5 shows the effects of ultrafiltration on the activity of Onlinegiving.

Fig. 6 is a characteristic Onlinebuy by the method of gel permeation chromatography.

Fig. 8 depicts SPS-PAGE analysis of Onlinegiving.

Fig. 9 shows binding elution activity Onlinebingo from the column with DEAE Affigel blue (top figure) and the column Q-separate (lower figure).

Fig. 10 illustrates a dose-dependent antiproliferative effects Onlinebingo and TNF.

Fig. 11 illustrates a dose-dependent proliferative effects Onlinebingo on normal human foreskin fibroblasts.

Fig. 12 shows the influence of Onlinebingo on L-929 cells from mice treated with actinomycin D.

Fig. 13 shows a Northern blot analysis for TNF and LT cells K-562 raw and processed forbalului esters (phorbol esters).

Fig. 14 gives a comparison of the effects of inhibition of growth Onlinebuy (figure a) and oncostatin (figure B) cells (A-375 human melanoma.

Fig. 15 shows the effect of inhibiting the growth of oncostatin M and IL-6 in normal fibroblasts.

Fig. 16 shows the effect of IL-6 on cell MCF-7 tumors in the mammary gland of man.

Fig. 17 depicts the effect of antibodies against interferon-on activity Onlinebuy against the MCF-7 cell tumor of the breast of man.

Fig. 18 shows and is sunok) in human foreskin fibroblasts.

Fig. 19 shows the growth rate of cells MCF-6 tumors in the mammary gland of a person in the absence and in the presence of Onlinegiving.

Fig. 20 shows the influence of time-keeping MCF-7 cells on Onlinebig.

Fig. 21 shows a comparison of the effect Onlinebingo with TNF on the morphology of the cells MCF-7 tumors in the mammary gland of man.

Various cell lines, including cell lines connective tissues of mouse L-929 (CCL1), K - 562 (CCL-243), U-937 (CRL - 1543), HL-60 (CCL - 240), Raji (CCL - 86), Jurkat (CRL 8163), BT 20 (HTB - 19), MCF-7 (HTB - 22), SK-BR-3 (HTB - 30), ZR-75-1 (CRL - 1500), RPMI 7951 (HTB - 66), A 375 (CRL-1619), A-431 (CRL - 1555), ME-1-80 (HTB 83), OVCAR-3 (HTB - 161), HeLa (CCL-2), Hep-2 (HB - 8065), and NIH 3T3 (CRL - 1618)

received from American Type Culture collection (Rockvillee, MD). TNF-resistant cells NIH 3T3 distinguish, as described K. Totpal, R. LaPushin, H. N. Ananthaswamy and B. B. Aggatwal and Lymphokine Cytokine Res. 11110(1991) 359 - 367. The cells were tested for Mycoplasma contamination using an experimental dataset based on DNA, purchased from Gen-Probe (San Diego, CA).

All cell cultures contain in a state of continuous growth in a weak stream. Some cultures will subcultured twice a week. Cells grow normally in medium RPMI 1640 with glutamine (2 mm), penicillin (10 units/ml), streptomycin (100 μg/ml) and serum embryo calf (10%) of ultrabestia person produce activity inhibitory growth cell line MCF-7 tumors in the mammary gland of man. Because of the ability to inhibit the growth of tumor cells and no effect on normal cells this activity is called "Onlineby".

For producing and inducing Onlinebuy grown cell line K-562 erythroblastic person in the medium RPMI-1640 containing 10% serum embryo calf with glutamine (2 mm), penicillin (100% units/ml) and streptomycin (100 µg/ml). Cells are harvested by centrifugation when the contents of cells/ml reaches 0,8106the cells are washed once with medium without serum and transferred to the medium RPMI-1640 containing no serum and containing glutamine, penicillin and streptomycin. For the production of Onlinebingo 1106such cells/ml incubated for 48 hours in T-flask (Falcon) in stationary conditions of cultivation in medium RPMI-1640 without serum and then treated turbolover ester (phorbol ester), 100 ng/ml, for 48 hours at 37oC. After this air cell supernatant collected by centrifugation, filtered through a 0,22 μm filter (Falcon) and stored at 4oC until the next acquisition phase characteristics. For concentration Wednesday conditioners is then subjected to dialysis 20 mm Tris, pH 8.0.

Inhibitory activity Onlinebingo on tumor cell growth is checked by three independent methods, see figure 1. These methods include (I) the counting of cells on hemocytometer after dyeing trienoyl blue, (2) method using the crystal violet dye and (3) method with the inclusion of tritium-containing thymidine. All three methods clearly shows that Onlineby inhibits the growth of MCF-cells, and the degree of inhibition depends on the dose. MCF cell line is used as a model to develop a method of biological verification Onlinebuy due to its convenience and sensitivity. Discovered that inhibition of Onlinebingo include tritium-containing thymidine is a highly sensitive method of detection of this cytokine.

Biological study Onlinebuy includes incubation 5103cells on the bottom of the cuvette with 96 cells in 0.1 ml of RPMI-1640 medium with 10% FCS overnight at 37oC CO2incubator. Then the medium is removed, the total final volume of 0.1 ml diluted in two, four, eight and so on again and continue the incubation for 24 hours at 37oC. during the last 6 hours add tritium-containing thymidine (0,5 Cl/0.05 ml/ ACAC,3 mm) for 30 minutes at 37oC. Cells are harvested using the device PHD Cambridge cell harvester, and determine the radioactivity of the cells, using a beta-counter. Data are expressed in % relative viability, which is defined as the number of disintegrations per minute (dpm) of cells in the presence of Onlinebingo divided by dpm is enabled in the presence only of the environment, multiplied by 100. The number of Onlinebingo required for inhibition of the viability of 50% is defined as one unit of the cytokine. As shown in Fig. 1, Onlinebig exerts an inhibitory effect on tumor cell growth in a dose-dependent, as illustrated by all three methods.

As shown in Fig. 2, within 24 hours, you can observe a pure dose-dependent response of MCF-7 Onlinebig. The reciprocal of the dilution of the sample required to achieve 50% inhibition of thymidine incorporation, defined as one unit of Onlinegiving.

As shown in Fig. 3, checked producing Onlinebingo in the absence of serum. The cultivation conditions without serum was used because of the difficulty of purification of proteins from samples containing serum. These results clearly show that the cells K-562 emit Onlineby even otsuchi checked ionophor calcium, of concanavalin a, phytohemagglutinin and Herbology ether. Fig. 4 shows that Herbology ether can increase the production Onlinebingo. Thus, Herbology ether can be used to optimize production Onlinebuy of cells K-562. When cells are incubated in the presence forblog ether (100 ng/ml), see approximately 4-fold increase in production Onlinebingo. Optimal induction of Onlinebingo see, when cells incourt with turbolover ether for 48 hours (table 1) and the density of cells is 1 to 106cells per ml of medium (table II) (table. I-VIII, see the end of the description).

K-562 cells (1106/ml) were cultured in medium (RPMI-1640 without serum or in the presence of forblog ether (100 ng/ml), or in his absence at 37oC CO2incubator during different time, then collect conditioned medium by centrifugation. Samples tested for MCF-7 cells when diluted in 2, 4, 8 and so on time, as described in Materials and Methods.

As shown in Fig. 5, for purification and characterization of Onlinebingo, cell-conditioned medium concentrated by ultrafiltration using a membrane PM-10 off molekulardiagnostik in the remaining fraction or concentrate (C) are proportionally higher, than the activity of standard buffer (the source material, SM). The results indicate that the activity Onlinebingo is stored and concentrated. These results also indicate that the molecular weight Onlinebingo, higher than 10,000.

In the experiment by gel permeation liquid chromatography anchored proteins (Fig. 6, a and b) sample Onlinebuy contribute in a column of Superose-6 (Pharmacia), balancing pre-saline solution with phosphate buffer containing 0.1% bovine serum albumin and 0.01% of sodium azide. The column is washed at room temperature with a flow rate of 0.5 ml/min and the volume of each fraction of 0.5 ml. Column calibrated by standard molecular weights (Schwarz/Mann, Cambridge, MA).

The standards include the following Apoferritin (480 kDa), alpha amylase (20 kDa), gamma globulin (160 kDa), bovine serum albumin (67 kDa), ovalbumin (45 kDa), chymotrypsinogen (24 kDa) and cytochrom C (of 12.4 kDa).

It is evident from Fig. 6 it is clear that Onlineby has a molecular weight of approximately 25 kD. Molecular weight Onlinebuy check by way of gel permeation liquid chromatography anchored proteins on a column of Superose - 6 adenocarinoma conditions. The results of gel filtration using calanog the WMD volume, and the second peak, corresponding to an average molecular weight of approximately 25 kDa (Fig. 6b).

In experiments on electrophoresis in polyacrylamide gel with sodium dodecyl sulfate (SDS-PAGE), shown in Fig. 7 and 8, a 15% polyacrylamide gel takes place mainly according to U. K. Laemmli (Nature 227 (1970) 680-685 and proteins become visually observable when the hair is dyed silver. For preparative gel electrophoresis and elution of activity before fixing and tinting part of the gel is cut with a razor into layers (more than 40), elute in a test tube, 50 mm solution of ammonium bicarbonate by diffusion during the night, the fraction is subjected to dialysis 20 mm Tris, pH 8.0, and then examined for biological activity.

As shown in Fig. 7, for additional confirmation of molecular weight Onlinebingo, perform SDS-PAGE analysis. After electrophoresis the gel is cut into layers, elute in 50 mm ammonium bicarbonate overnight and examined for activity Onlinebingo. More than 50% activity Onlinebuy emit in the range of molecular weight of about 30 kDa. Less than 10% of the activity found in the area near the front of the dye.

As shown in Fig. 8, a second pass biologycorner weight of about 28 kDa.

As shown in Fig. 9 (a and b), checks the linking Onlinebingo with the anion exchange resin and elution from resin. One column (1 cm x 5 cm) fill anion exchange resin Q-separate and then balance 20 mm Tris, pH 8.0 (equilibrating buffer). Sample Onlinebuy after dialysis relatively balanced buffer is loaded into the column with a flow rate of 0.5 ml/minute. The column is rinsed equilibrating buffer and then spend a gradient elution with a solution of NaCl (0-1 M). Different factions analyze the concentration of protein and biological activity.

DEAE Affigel Blue chromatography: the second column (1 cm x 5 cm) fill resin DEAE Affigel Blue and then balance 20 mm Tris, pH 8.0 (equilibrating buffer). Sample Onlinebuy previously, balanced on the buffer by dialysis, loaded into the column with a flow rate of 0.5 ml/min Column rinsed equilibrating buffer and then spend a gradient elution with a solution of NaCl (0-1 M). The different fractions analyzed for concentration of proteins and biological activity.

Fig. 9a and 9b show that Onlineby bound and can be eluted from the anion-exchange resin. Both resins (Q-sepharose and DEAE affigel blue) actively what edstam 0.2 M NaCl in 20 mm Tris - the buffer and Q-sepharose - 0.5 M NaCl.

Onlineby inhibits the growth of various tumor cells.

(Table III) Antiproliferation activity Onlinebuy tested through the inclusion of tritium-containing thymidine.

Tumor cells (5000/per cell) incubated with TNF (0.2 ág/ml) or Onlinebingo (induced turbolover ether and concentrated) within 72 hours at 37oC and then determine the relative cell viability (%) thymidine incorporation as described previously.

As shown in Fig. 10 (A-D), checked the action Onlinebingo, dose-dependent, some are listed in table VII cell lines and compared it with TNF. From the results shown in table VII and Fig. 10, it is clear that in addition to MCF-7 Onlineby can inhibit the growth of several different types of leukemias, melanomas, carcinomas and Hepatol. The growth of rat cells also inhibited. Thus, it is clear that in contrast to interferon of Onlineby is not specific for samples.

As shown in Fig. 11 and 12, to study the growth of cells placed overnight in 0.1 ml of medium (RPMI-1640 with 10% FBS) in a cuvette with 96 cells (FaIcon). Then the medium is removed and produce a series of dilutions of 0.1 and cells, crystal violet staining dye, according to the procedure described in B. B. Aggarwal, Human Iymphotoxin, Meth. of Enzymol., 116:441-448 (1985), included here as a quote. A method of testing the viability of cells with staining dye correlates with the number of cells defined by selection using a solution of trypsin and counted under a microscope using hemocytometer. The percentage of relative cell viability is calculated as the optical density in the presence of the test sample divided by the optical density in the absence of the test sample (Wednesday) and multiplied by 100. For LT and TNF conducting a study of cytotoxicity similar to the study of growth inhibition, except that 20 103cells L-929 treated with actinomycin D (1 µg/ml) in parallel with the cytokine within 24 hours.

Study the growth of cells. Studies stimulate the growth of cells was performed mainly according to the procedure described in Vilcek, etc., J. Exp, Med. 163:632-643 (1986). In short, for research to stimulate the growth of confluent cells using diploid foreskin fibroblasts human at level passage 12-16 (approximately meet caku) placed in 0.1 ml of medium (RPMI-1640 + 10% FBS) in a ditch on 96 wells (FaIcon). After incubation overnight in CO2-incubator at 37oC Wednesday removed and conduct a series of dilutions of cytokine in 0.2 ml volume. After five days of incubation, the medium is decanted and stained cells of the crystal violet dye. All measurements are performed three times. The percentage of relative cell viability count, as indicated in experiments on the study of growth inhibition.

For experiments to enable (3H) TdR fibroblasts were cultured and treated with cytokines for 5 days. In the last 24 hours for each cell type tritium-containing thymidine (of 6.7 Ci/mmol, New England Nuclear, Boston, MA), with 0.5 Ci/ cell. After that, remove the culture medium, the cells are washed twice in saline solution with phosphate buffer and separate the cells by adding trypsin solution (0,5%) with EDTA (5.3 mm). Then collect the cell suspension using PDH-collector cell (Cambridge Techno-Cogy Inc. Watertown, MA) and are lysed by washing with distilled water. Measure associated with the filter radioactivity in a liquid scintillation counter (model 1600 TR, Packard Co., Meriden, CT). The incorporation of thymidine in fibroblasts defined by this method correlates with cell growth. When radiation inhibiting the growth of tumor leokimvideo.

As shown in Fig. 11, it is clear that Onlineby stimulates the growth of normal fibroblasts foreskin man. When the growth inhibition of tumor cells Onlineby increases the proliferation of normal fibroblasts foreskin man.

As shown in Fig. 12, similar to TNF and LT Onlineby provides cytolysis of actinomycin D in the processing of L-929 cells. Antitumor activity Onlinebuy against cells L-929 cannot be neutralized neither anti-LT or anti-TNF antibodies (table IV). In the study of EL ISA drugs Onlinebuy not detect significant amounts of TNF or LT (table V). Table III shows that some types of tumor cells (for example, SK-BR-3, HeLa, A-431, OYCAR-3, A365 and RPMI-7951), resistant to TNF/LT, acutely sensitive to Onlinebingo. Onlineby also can be distinguished from TNF/LT on the basis that, as detected cell lines, selected for its resistance to TNF/LT (NIH 3T3-LTR and BT-20 TNFR), sensitive to Onlinebingo (table III).

As the source of Onlinebingo use free serum medium conditioned cells K-562 and determine the level of various cytokines standard ways EL ISA (R & D system). ND not determined.

TNF and LT are the product of motorsin and TNF inhibit the growth of MCF-7 cells, but this requires great concentration of TNF and LT (10000 u/ml).

For additional confirmation that Onlineby is neither TNF or LT, hold Noethern blot analysis to search for genes for each cytokine. When performing Northern blot analysis of culture cells K-562, processed and unprocessed turbolover ether, seeded at a density of 1106cells/ml in flask 75 cm, incubated with an activator of protein kinase C for 24 hours and then harvested by centrifugation. All RNA extracted from the cells by means of guanidinoacetate-chloroform, described in Chirgwin, etc., Biochem. 18: 5294-5299 (1979) and Maniatis, etc., Molecular Cleoning 188-209 (1982). Usually get RNA ratio of optical absorption 260 nm/280 nm more than 1.9 and the release of approximately 100 ág RNA in 20106cells.

For electrophoresis fractionary 20 µg RNA 0.8% galagarza containing 2.2 M formaldehyde at 75-100 B for about 3 hours. Then on the gel are treated water diethylpyrocarbonate, when 68oC for 1 hour and then transferred to RNA to Hybond nylon membrane (Amersham Corp., Arlington Heights, IL).

After transfer (3 hours) the filter is washed twice with a solution SSC (SSC: 0.15 M sodium chloride, and 0.15 tion is carried out at 65oC for 1 hour in buffer containing 7% SDS, 500 mm sodium phosphate, 1 mm EDTA, pH 7,2 (hybridization buffer). Then the filters hybridized through samples of TNF or LT link (specific activity 2108the number of pulses per minute /ág DNA). After hybridization the membrane was washed several times with 65oC in hybridization buffer containing DNA salmon sperm (200 μg/ml). Filters impose Kodak XAR-5 film at 70oC for 1-3 days. Carry out the procedure of consecutive cycles of pre-hybridization, hybridization, washing and releasing filter. Was shown equal to the load lines when checking gels after coloring with the use of ethidium bromide, and when re-hybridization of the same filter at the expense of the cDNA for actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Spend densitometry of the bands by scanning filter for counting of radioactivity, using blot analyzer Betascope 603 (Betagen Corp., Waltham, MA), or by scanning autoradiogram to determine the optical density, using a scanning densitometer (Helena Laboratories Inc. Beaumont, TX).

As shown in Fig. 13, for neither LT nor for NTF is not detected the mRNA expression in cells K-562. In addition, gelfiltration and SDS-PAGE the experimental is provided in Fig. 19, the tumor cells human breast grow rapidly in culture medium. However, when the culture add Onlineby, growth of these cells is not supervised. To determine the time-keeping required for inhibition of growth of these cells, the cells were subjected to Onlinebingo within different time. As shown in Fig. 20, the action Onlinebingo on inhibition of growth ends when the cytokine is removed from the environment. These results suggest the need for the constant presence of Onlinegiving.

As shown in Fig. 21, after treatment Onlinebingo and comparison with NTF check the morphology of MCF-7 cells. The results show differences in the ways in which TNF and Ongoingly inhibit the growth of tumor cells of the breast. TNF induces the environment of the cells, which leads to their isolation from the Cup, while Onlineby induces the expansion or swelling of the cells. The latter may be the result of Onlinebingo on the permeability of cells. Onlineby also inhibits colony formation of tumor cells in the breast of man.

The study of the stability of Onlinegiving. Onlineby treated with an organic solvent (acetonitrile, or locname solvents (NaOH, the ammonium hydroxide) or detergents (Sds, Tween 10) for two hours at room temperature, then subjected to dialysis 20 mm Tris-HCl, pH 8.0 overnight in a cold room and examined for biological activity (tables VI and VIII).

For experiments to test thermostability of Onlineby incubated at different temperatures for different time and immediately determine biological activity. Found that the biological activity of Onlinebingo remains constant up to 80oC for 60 minutes, but during curing at 100oC for 30 minutes there is a 50% loss of activity (table VI).

In table VII Onlineby treated with pronasol E. trypsin, chymotrypsin and Y8 Staph protease is then analyzed for biological activity. The results show that the activity Onlinebuy can completely destroy pronasol E, assuming, therefore, that this protein. Found that this protein. Found that its activity is partially sensitive to treatment with trypsin and completely resistant to the action of chymotrypsin and Y8 protease.

Desoksiribonukleaza also has no effect on the activity of Onlinegiving.

Onlineaustralia by adding 10% serum and examined for activity Onlinebingo.

The stability of Onlinebingo to the action of detergents. Sample Onlinebuy treated with different concentrations of SDS, the negatively charged detergent or Tween 20, neutral detergent for 2 hours, dialist and then examined for biological activity. As a control, using bovine serum albumin treated with the same detergent. The results of these experiments are shown in table V. When processing protein SDS or Tween 20 not observed the loss of biological activity. In the case of SDS clearly shows an increase in the biological activity of Onlinebingo dose-dependent. When using Tween 20 see a slight increase in activity.

Samples Onlinebuy treated at room temperature by different agents within 1 hour in 20 mm Tris-buffer, pH 8.0, dialist during the night and then examined for residual biological activity, S and P denote the fraction of the supernatant and precipitate.

The stability of onlinebingo to the action of reducing agents. Onlineby treated with various concentrations of dithiothreitol for 2 hours, then cialiswhat and examined for biological activity. The results of these experiments are shown in table IX. It is clear that the act is and 100 mm DTT.

Samples Onlinebuy treated with various agents, dialist and then examined for biological activity.

Onlineby can be concentrated trichloroacetic acid and ammonium sulfate. Onlineby treated with solutions of various concentrations of TCA or NH4SO4, centrifuged, re-suspended, dialist and then examined for biological activity. The results presented in table IX indicate that the activity Onlinebuy can be precipitated or 5% TCA, or 70% ammonium sulfate (SAS). Thus, these results also suggest the protein nature of Onlinegiving.

Amphiregulin is glikoproteinom, which are separated from the MCF-7 cells treated turbolover ether, and which inhibits the growth of cells 4431. Amphiregulin has an apparent molecular weight of 22kDa. And Ongoingly, and amphiregulin inhibit the growth of tumor cells. However, Onlinebig not produce processed turbolover ether cells MCF-7. Secondly, even though and Ongoingly, and amphiregulin demonstrate antiproliferative activity relative to A431 cells (table III), amphiregulin, unlike Onlinebingo, relatively inactive human melanoma (A375), adenocarcinoma say is, celoveceskih lymphoblastoid cells (CEM), human EBY-transformed B cells, human epidermal carcinoma of the larynx (Hep 2), endothelial cells of the heart bovine embryo (CRL-1395), rat BAL B/3T3 cells and mink lung (CCL-64). Similarly, Onlinebingo amphiregulin stimulates the proliferation of human fibroblasts. In addition to human fibroblasts amphiregulin also stimulates the growth of some tumor cells, including tumor cells of the human pituitary gland (CRL 7386), cells of human ovarian carcinoma (HTB 77), the kidney cells of African green monkeys (BSC-1) cells and rat kidney (NRK). However, the molecular weight Onlinebuy also significantly different from the molecular weight amphiregulin, Mature form of the latter is the protein length is 84 amino acids.

Onlineby as resistant to acid and alkaline conditions in the pH range from 2.0 to 10.0 (table VIII). Reported some cytokines, which are stable at pH 2, including IFN-, IFN-, IL-2, IL-4, IL-8, CSF-1, GM-CSF, TGF -,Oncostatin M and amphiregulin. Found that Oncostatin M, epiregulin, CSF-1 and IL-4 are stable to alkaline conditions (pH 12). Now factor in the promotion of lipolysis (LPF), a protein with a molecular weight of approximately 6kDa, highlighted ipsio, pronate the RNase, DNA and oxidation periodata. LPF can be precipitated trichloroacetic acid (10%) without loss of biological activity. It is found that Onlineby resistant to heat up to 80oC for 30 minutes and at 100oC only partially loses its activity (table VI). Processing Onlinebuy 10% (W/W) trypsin for 24 hours at 37oC leads to partial loss of biological activity. This is similar to that observed for CSF-1, GM-CSF and LT. Also found biological sustainability activity Onlinebuy to 0.5% STS.

Onlineby differs from the defeat of the tumor TKF), cytokine identified from hybridoma macrophage-monocyte person, in which TKF is the main protein (pH 8-9.0) has an apparent molecular weight of 56 kDa, determined by gelfiltration, and can be eluted with Concanavalin a-sepharose 0.4 M - methylmagnesium.

Oncostatin M is a cytokine selected from cells U-937, processed turbolover ether, and inhibits the growth of cell lines of human melanoma A375 studies with the inclusion of thymidine. Estimulando cells U-937 does not Express a gene or do not distinguish this activity. Peripheral blood T lymphocytes of human activated fitage the m weight of 28kDa, a particular method SDS-PAGE and 18 kDa by the method of gel filtration. He synergistically with TGF -,but not with interferone. It is shown that Oncostatin M inhibits proliferation of neuroblastoma cell 10 HTB, cell lung carcinoma A-549 as well as A375 cells and melanoma SKMEL-28; however, did not inhibit cell proliferation of L-929. In contrast, Onlinebig is produced by cells K-562 (not reproduced cells U-937) in the presence and in the absence of forblog ether, and acts on cells L-929. In addition, in contrast to Onlinebingo, Oncostatin M is a relatively weak inhibitor of A375 cells (Fig. 14).

Transforming - growth factor (TGF -) is a cytokine that is a glycosilated with a molecular weight of 25 kDa on SDS-PAGE, and inhibits the growth of some types of cells of epithelial and mesenchymal nature, including cells of the vascular endothelium human T and B lymphocytes, TGF - inhibits actokine way cells A-549, MCF-7. However, unlike Onlinebingo, TGF - is produced by a variety of cells, including platelets, connective tissue and lymphocytes, and requires acidic activation before checking activity. In addition, TGF-has a molecular weight of 12.5 kDa on SDS-PAGE when the recovery condition immunological research "sandwich" enzyme used to verify the presence of known cytokines (TNF, LT, IL - 1, IL - 6 and IL - 8) in preparations Onlinebingo. Use the standard procedure in the presence of feeder. A monoclonal antibody specific for various cytokines, put on a plate for micrometrology and leave overnight for immobilization of antibodies. Then pipette transfer the samples into the cells and record the presence of any of the cytokine by the immobilized antibody. After washing the unbound protein sample in a cell type associated enzyme polyclonal antibody, specific for the cytokine, and give to join the cytokine that has been associated with the first incubation. After washing to remove unbound vzaimodeistvie "polyclonal antibody-enzyme reagent to cells add substrate solution, develops staining is proportional to the amount of bound in the first stage of the cytokine. In parallel with the audited samples prepared a series of cells with a known standard concentrations of cytokines. Write down the curve of optical density on the concentration of the cytokine in these standard drugs, comparing the optical density of the samples to the standard curve. Then calculate the concentration of the cytokine in unknown samples (that is AMI, has a molecular weight of 17 kDa and inhibits growth of tumor cell lines, including ovarian carcinoma, A375 melanoma, K-562 and some cell lines of breast tumors. However, Onlinebig differs from that of interleukin-1 in the source of its receipt, molecular weight and specificity relative to tumors. Crude preparations Onlinebuy tested for the presence of IL-1 by the method of EL ISA. The results presented in table V, demonstrate the absence of protein IL-1 preparations Onlinebingo.

Interleukin-6 is a cytokine produced by cells of various types in response to various stimulation and has a molecular weight of 26 kDa on SDS-PAGE. It is characteristic that IL - 6 is produced by normal human fibroblasts, U-937 cell lines human melanoma A375, RPMI-7951 and so on IL-6 inhibits the growth of cell lines of myeloid leukemia and breast carcinoma. Judging by the source of receipt, the method of induction and specificity relative to tumor cells, it is clear that IL-6 is a cytokine that is different from Onlinebingo. In addition, typically use cells MCF-7 as labels for Onlinebingo, insensitive to the action of interleukin-6 (Fig. 16 ). Also unlike Onlinebanking with a molecular weight of 20-25 kDa on SDS-PAGE, is produced by T-lymphocytes upon activation by various mitogens and inhibits the growth of some tumor cell lines. This cytokine is highly sensitive to acid pH values. Onlineby differs from interferon-in relation to source, method of induction and pH stability. -Interferon is also different from Onlinebingo in relation to its effect on normal human fibroblasts. Onlineby stimulates proliferation, while-interferon inhibits induced TNF proliferation of fibroblasts, but not proliferation, caused by Onlinebingo (Fig. 18). In addition, antibody-interferon not reduce activity Onlinebingo, and increase it (Fig.17).

Thus, the present invention provides a novel cytokine that demonstrate various antitumor activity. Cytokine of Onlineby outstanding human erythroblastopenia cells and has a molecular weight of approximately 28 kDa on SDS-PAGE. It turns out that the production of Onlinebingo increases in the presence of forblog ether. It turns out that Onlineby resistant to the action of various substances in a wide range of pH and high temperatures.

It is clear that due to the activity of pouch neoplastic diseases, including carcinoma and lymphoma. Onlineby you can give to people or other animals as part of a pharmaceutical composition that contains a pharmaceutically suitable carrier. Due to their broad antitumor activity of Onlineby will be useful in the prevention of recurrence of neoplastic diseases. In addition, introduced Onlinebingo in the body is the carrier of tumor cells can prolong the life of the patient. On the other hand, tumor cells can be treated with Onlinebingo in vitro, for example, treating and cleansing the bone marrow, containing tumor cells. These described methods of treatment of tumor cells is well known in the practice of hemoterapia cancer and, therefore, a specialist without additional experiments can determine the appropriate dose and rate of administration Onlinebingo.

Onlineby can also be useful as a new immunomodulator. Onlineby activates lymphocytes, monocytes and neuropile for destruction of tumor cells. In addition to this, Onlinebig may be therapeutically useful as a growth factor. Onlineby stimulates the growth of normal cells.

In conclusion, it is clear that the present invention disclosed here adapted for the OS is blasti of the present invention, you can make certain changes in the method and devices. It is clear that change is possible, and, in addition, it is assumed that each element or stage on any of their claims is to be understood as a reference to all equivalent elements or stages to achieve basically the same results substantially equivalent manner. Means to disseminate widely the invention, in whichever form not applied its principles. In addition, among other integral parts of the present invention is well adapted to perform certain tasks, and obtain the mentioned results and benefits.

1. Purified and isolated human protein onlineby allocated erythroblastopenia cells (K-562), with antineoplastic activity against cell lines of breast carcinoma (MCF-7) and carcinoma of the cervix (cervical carcinoma Heha) and proliferative effect on normal fibroblasts foreskin man, having a molecular weight of 28000 YES in SDS-PAGE, stable at pH range from 2 to 8 and at a temperature of from 4 to 100oC.

2. Pharmaceutical composition having antineoplastic activity, characterized in that it contains the economic onlinebuy under item 1, including the incubation of human erythroblastopenia cells (K-562) environments, the collection of conditioned cell media, isolation and purification of human onlinebingo.

4. The method according to p. 3, characterized in that the cells incubated in the medium containing no serum.

5. The method according to p. 3, characterized in that the cells incubated in medium RPMI 1640.

6. The method according to p. 3, characterized in that it further includes increasing the production of human onlinebuy processing turbolover ether erythroblastopenia human cells.

7. The method according to p. 3, characterized in that the cleaning onlinebuy carried out by ultrafiltration conditioned cell media, dialysis, chromatography on DEAE-Appigale blue, polyacrylamide gel electrophoresis with sodium dodecyl sulfate.

 

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2 ex

FIELD: medicine, cardiology.

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FIELD: biotechnology, molecular biology, medicine, genetic engineering, pharmacy.

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22 cl, 19 dwg, 18 tbl, 117 ex

FIELD: medicine, pharmaceutical industry and technology, pharmacy.

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EFFECT: valuable medicinal properties of composition.

5 ex

FIELD: medicine, pharmacy.

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FIELD: biology, medicine, pharmacology.

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