Method for diagnosis of ehrlich's carcinoma

FIELD: medicine and biochemistry.

SUBSTANCE: claimed method includes blood lymphocyte treatment with labeled fluorescein isothiocyanate of pea lectin isoform obtained by extraction of milled pea seeds with sodium chloride solution, purifying by undercooling, dialysis and isoform isolating by column chromatography on Sephadex, and elution with glucose-containing solution, and when number of labeled lymphocyte is lower than 50 % in contrast to normal one Ehrlich's carcinoma is diagnosed.

EFFECT: diagnosis method of increased specificity.

1 tbl, 3 dwg

 

The invention relates to medicine, in particular to methods of diagnosis of cancer.

One of the main problems of Oncology is that no one can find the flaws in the immune system of cancer patients, which make possible the growth of tumors in the body. There is a paradoxical situation when the immune system of cancer patients does not differ from healthy, but in the body are growing tumor.

On the background of tumor growth in immune system doesn't change, such as those that develop in immunodeficiency States, when depressed, entire groups of immunocompetent cells or when there is disappearance of any cell receptors. The changes that occur in lymphocytes of the body on the background of tumor growth, affect glycosylation of proteins (receptors) in their cell membranes. In this case, the immunocompetent cells lose their ability to respond only to the tumor cells, whereas the response to infectious agents fully preserved, i.e. does not develop immunodeficiency.

Currently, all methods of diagnosis of cancer blood test is based on measuring the level of tumor associated antigens. Opukholeobrazovanie antigens are patterns, which are normally presented in the tissues of the body the ISM, but their number in normal tissues or slightly, or the location of their tissues is that normal in the blood, they do not fall. As a result of development of oncological process increases the number of tumor cells and increases the body the number of tumor associated antigens that are expressed on the surface of these cells. When death and / or peeling from the surface of these tumor antigens are starting to get into the blood. Thus, this group of methods of diagnostics uses patterns that are on tumor tissues and completely ignores the changes that can occur in the body in response to the growing tumor.

There is a method of diagnosing cancer by determining the content of small lymphocytes (diameter <7.5 μm) in the blood of cancer patients (Viganello and other "Decrease in the content of small lymphocytes in the blood of patients with malignant bone tumours. Oncology issues. 1987, volume 33, No. 9, p.15-21).

As the authors state, reducing them to 40-75% compared with healthy people is observed on the background of growth of tumors.

However, this method has a great subjective and depends on the qualification of specialists. Thus, the diameter of the cells may vary depending on the isotonicity of the solution, the rate of fixation of smears, optizone sizes of cells eyepiece-micrometer MOU-1, etc. Due to the high subjectivity of the method he has not found wide application.

The objective of the invention is to increase the specificity of diagnosis of cancer and preventing its subjectivity.

The problem is solved by the method lies in the fact that the blood lymphocytes treated with labeled fluorescent dye - fluoresceinisothiocyanate isoform of pea lectin, and by reducing the number of labeled lymphocytes by more than 50% with respect to the norm judge the presence of cancer.

The method is as follows.

Lectin peas allocate the method of affinity chromatography on Sephadex G-100. Selection isoforms of pea lectin with high affinity binding to sugars is a necessary step to improve the specificity of diagnosis, because otherwise with the surface of the cells contacted with the lectin isoforms that when laundering cells from unbound lectin was also removed (due to their low binding constants) with the cell surface and this would lead to a reduction in the percentage of stained cells.

For this purpose, seeds of peas grind in the mill into flour with grain sizes less than 0.1 mm, Then pour 4 times by weight the amount of 0.5% of NaCl and infused for 3 hours at 4°C. the Extract is acidified to a pH of 4.6 with 5N HCl and centrifuged DL the removal of sediment at 1500 g for 30 minutes

Solid ammonium sulfate (NH4)2SO4added to the supernatant to achieve a 60% saturated solution. Precipitated precipitated protein is collected by centrifugation at 3000 g for 30 minutes the Precipitate is dissolved in a minimal volume of water and deleteroute strictly at 4°against M of NaCl in M phosphate buffer (pH 7.2). Protein precipitated precipitated during dialysis, is also removed by centrifugation at 3000 g for 30 minutes

The supernatant is applied on a column of Sephadex G-100 (26×100 cm). Unbound protein elute 4 l M of NaCl in M phosphate buffer (pH 7.2). Lectin peas elute M of NaCl in M phosphate buffer (pH 7.2) containing glucose from 0.01 to M, with a step increase in glucose concentrations 0.01, 0.05 and M. Suirvey lectin collected in three separate fraction, which is subjected to dialysis against M of NaCl in M phosphate buffer (pH 7.2). Protein is sterilized by filtration using 0.22μ Millipore filter.

Next, perform the tagging isoforms lectin fluoresceinisothiocyanate. Tagging isoforms lectin this fluorescent dye is necessary because it is fluoresceinisothiocyanate fluorescent from the laser light source, which is used in a flow cytometer.

This device combines together two block - optical device with a laser light source and power the second computer. Cells of the analyzed sample are moving in a thin glass capillary, a laser beam shines through the capillary and the cell that it can move. In different directions along the laser beam are numerous detectors, which measure the passage of light through the cell, its dispersion on the object, reflection, fluorescence, etc.

A computer with a special program evaluates the received information and displays the optical parameters of each, held by capillary cells, determining the level of fluorescence of each of them that allows you to measure the amount of lectin, contacting the cell and, consequently, the level of sugars on the surface of the analyzed cells that are available for binding with the lectins.

The original solution of lectin stored in M NaCl in M phosphate buffer (pH 7.2) with addition of sodium azide to a final concentration of 0.1% at a concentration of 8 mg of protein per ml For labeling take 0.5 ml of the solution and bring the same buffer volume to 2.5 ml of This volume applied to the column PD-10, balanced 0.1M carbonate buffer pH 9.3. Protein elute 3 ml of carbonate buffer. Thus obtained protein solution with a concentration in the range of 0.5-2 mg/ml 1 mg fluoresceinisothiocyanate dissolved in 1 ml of dimethyl sulfoxide and added to the protein solution at the rate of 25 µl per 1 ml of buffer. The solution peremeci is try and leave overnight at 4° C. after incubation, the solution was added 100 μl of 0.5M solution of ammonium chloride and Stavelot in the same terms if 4°With 2 hours. Then lectin translate in the source buffer (M of NaCl in M phosphate buffer (pH 7.2) with addition of sodium azide to a final concentration of 0.1%) using a column PD-10, balanced M of NaCl in M phosphate buffer (pH 7.2) with addition of sodium azide to a final concentration of 0.1%.

Diagnosis of cancer at the transplantable Ehrlich carcinoma

Transplantable tumor models allow us to quickly explore the changes that occur in the body on the background of tumor growth. To do this, take the animals one line, weight, sex, age, and part of them perejivaut the tumor and part is used as a control. Most of the tumors after two to three weeks to reach considerable size, while animals without tumors are adequate control on the changes in the body that occur on tumor growth.

On the growth of Ehrlich carcinoma in the serum of mice appear for this tumor factors that accelerate its growth in in vivo studies. However, the nature of these changes remains unknown, as it is not possible to detect any tumor-specific factors. We have made the assumption that this phenomenon is associated with changes in the glycosylation of elkow blood and membranes of lymphocytes. To confirm this assumption was studied by changing the binding of the labeled lectin isoforms peas with blood leukocytes on the growth of Ehrlich carcinoma. Selected isoforms lectin has the ability to contact the glucoside and mannose, including part of the polysaccharides in the glycosylation of proteins.

The method is carried out using male mice F1(CBAxC57BL/6), mice of C57BL/6 males. Each group used 10 animals. Cells of Ehrlich carcinoma transplanted intramuscularly in a dose of 106cells in 0.2 ml of RPMI-1640 medium in the mouse. Lymphocytes isolated from the peripheral blood of mice on day 15 after transplantation of the tumor by a standard method in the density gradient 1.093. Each experiment is repeated at least 3 times.

Determination of the expression of surface markers on the surface of lymphocytes carried out using labeled fluoresceinisothiocyanate lectins. Evaluation of the binding specificity of pectins from the cell surface to confirm the inhibition of their binding adding sugars. The results take into account the method of running cytofluorometry on a flow cytometer FACScan (Becton Dickinson, USA). Gate (window) cell population set based on the combination of forward and side light scattering and size of cells. This approach completely eliminates subjecti the significant factor in the analysis, so the measurements are performed by the detectors and their readings are entirely determined by the optical properties of the analyzed object. When taking into account the results of counting 10,000 events in gate. Statistical processing of the material is done using a software package WINMDI 2.8.

Investigated the binding of lectins to the surface of peripheral blood lymphocytes intact mice and mice on the 20th day of Ehrlich carcinoma. Table 1 shows the averaged data for all series of experiments with different visible tumor volume from a minimum of less than 1 mm3up to 5 cm3.

The figure 1 shows the distribution of cells in the blood lymphocytes of healthy mice. The abscissa axis shows the distribution of cells of peripheral blood lymphocytes depending on their front light scattering, and the ordinate axis shows the light scattering of cells depending on their lateral light scattering. Lymphocytes form a dense cloud, which indicates the homogeneity of the considered cells. The selected frame (polygon) in figure 1 is called the gate, it shows that the population of lymphocytes in the blood of mice, which are studied for linking them with lectins. The analyzed cells have similar optical characteristics as to control animals and animals-carriers of tumour, which also excludes the subject is wny factor in the research.

The figure 2 presents a histogram of the distribution of normal blood leukocytes before painting pea lectin (histogram 1) and after painting FITZ-labeled lectin peas (histogram 2). Therefore, after incubation with the lectin shift the histogram to the right along the x-axis, indicating that the increase in fluorescence of the cells due to binding with them labeled lectin isoforms. The percentage of connected cells is to 91.2%. The ordinate axis shows the number of cells that have the appropriate level of fluorescence.

Figure 3 shows histograms of lymphocytes in the peripheral blood of animals-carriers of tumour to their color isoform of pea lectin and after painting. Just as in the case of normal lymphocytes, the color of the pea lectin causes a shift of the histogram to the right. Only 21.3% of cells are in the same color, whereas in the control of such cells to 91.2%. Thus, the percentage of labeled cells decreased by 69,9% (91,2-21,3=69,9).

Thus, the color labeled lectin isoform pea peripheral blood lymphocytes of mice showed that despite the growth of tumors, a decrease in the availability of sugar (glycoside and mannose) for binding to lectins on the cell surface. The changes of glycosylation of membrane proteins can cause damage to the invasive properties of the cells and the reduction of binding them to the surface of tumor cells, that can lead to failure of antitumor immunity.

Allocated in accordance with the invention isoform of pea lectin can be used for the diagnosis of cancer (see table).

Table 1
The average percentage of labeled cells in animals without tumorsThe average percentage of labeled cells in animals with tumorsThe reduction in the percentage of labeled cells on the background of tumor growth
(in %)(in %)(in %)
89+1031+958+9

A method for diagnosing carcinoma, Ehrlich by studies of blood lymphocytes, characterized in that the blood lymphocytes treated with labeled fluoresceinisothiocyanate isoform of pea lectin obtained by extraction of the crushed seeds of peas solution of sodium chloride, purification presidenial, dialysis and allocation isoforms column chromatography on Sephadex and elution with a solution containing glucose, and by reducing the number of labeled lymphocytes by more than 50% with respect to the norm diagnosed with Ehrlich carcinoma.



 

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