RU2417263C2 - Diagnostic technique for inflammatory process in early rheumatoid arthritis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: RNA is recovered from peripheral blood or synovial liquid. Further, cytokine balance is evaluated by quantitative analysis of interleukin-2 (IL-2), interleukin-4 (IL-4) and interleukin-10 (IL-10) or interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-17 (IL-17), interleukin-1p (IL-1p) cytokines mRNA genes expression, as well as by quantitative analysis of interferon-gamma (IFNG) and a tumour necrosis factor (TNF) by reverse transcription and polymerase chain reaction with recording the accumulation of reaction products by direct fluorescence. The direct fluorescence is used to evaluate the cytokine balance. Further, a pair balance of expression of various cytokines is calculated on the basis a functional interrelation.

EFFECT: use of the invention reduces an evaluation error related to specific properties of the control gene expression.

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Description

FIELD OF THE INVENTION

The proposed method relates to the field of medicine, biology and biotechnology and can be used as an additional method for examining patients in the early diagnosis of inflammatory diseases based on polymerase chain reaction with the registration of the accumulation of reaction products "in real time" (PCR "in real time").

Polymerase chain reaction (PCR) is widely used for amplification of nucleic acids and allows, in particular, to detect the presence and determine the amount of nucleic acid with a specific nucleotide sequence in the RNA sample.

The results of the polymerase chain reaction are analyzed directly during the reaction. To record the results of PCR during the process, it is carried out in a detecting amplifier in the presence of either fluorescent intercalating dyes or fluorescently labeled oligonucleotide primers or samples.

Rheumatoid arthritis (PA) is an autoimmune disease of unknown etiology, characterized by symmetrical erosive arthritis (synovitis) and a wide range of extra-articular (systemic) manifestations. RA is a vivid model of chronic inflammation that allows one to study the fundamental problems of modern medicine related to the pathogenesis and treatment of various human diseases. PA is an extremely common disease affecting approximately 1-2% of the world's population. The cardinal signs of RA include a steadily progressive damage to the joints, leading to disability and a decrease in the life expectancy of patients. Traditionally, RA is considered as a model of antigen-specific activation of CD4 + T-lymphocytes of the Tx1 type, characterized by the predominance of the synthesis of interleukin-2 (IL-2), interferon-γ (IFN-γ) over the production of interleukin-4 (IL-4) [1- 3]. An imbalance of the Th1 / Th2 response enhances the activation of macrophages, dendritic cells, B-lymphocytes, which is accompanied by overproduction of pro-inflammatory cytokines (tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-15 (IL-15), interleukin-17 (IL-17), interleukin-18 (IL-18) and others) and reduced expression of anti-inflammatory cytokines (IL-4, soluble receptor antagonist IL-1, soluble TNF-α receptors) [4-7].

There are several subpopulations of T lymphocytes, including T (1 Tx1) and 2 (Tx2) order helper cells. Each of them emits specific cytokines: Tx1 - IFN-γ, IL-2, TNF-α; Tx2 - IL-4, IL-5, IL-6. The body maintains a certain ratio of Tx1 and Tx2 cells, and accordingly the cytokines secreted by them. With pathologies, this balance may be impaired.

Characteristics of various cytokines

TNF-α. The main source of TNF-α are activated macrophages, in vitro causes lysis of lymphoma cells, sarcoma necrosis, in vivo activate polymorphonuclear leukocytes, and exhibit antiviral activity. Under the influence of TNF-α, the formation of hydrogen peroxide and other free radicals by macrophages and neutrophils increases sharply.

IL-1β. The main source of IL-1β production is monocytes and macrophages of various tissue localization, as well as endotheliocytes, T-lymphocytes and B-lymphocytes, fibroblasts, NK cells, keratinocytes and neutrophils. It is a mediator of acute and chronic inflammation. It performs many important functions, including stimulation and release of neutrophils from the bone marrow; activation of lymphocytes and neutrophils.

IL-2. IL-2 is an important pro-inflammatory cytokine that stimulates the proliferation and differentiation of activated T-lymphocytes into effector T-lymphocytes or cytotoxic T-cells. IL-2 can stimulate large granular lymphocytes, macrophages and B cells. IL-2 is secreted by CD4 + T-lymphocytes, as well as T-cells of some other lymphocyte subpopulations. IL-2 belongs to the group of hematopoietins, it was one of the first to be discovered. In the mid-60s, it was shown that a culture of mitogen or antigen-stimulated lymphocytes accumulates a factor in the supernatant that significantly enhances the proliferation of freshly isolated peripheral blood lymphocytes in vitro. Of particular interest was the fact that the supernatant from the culture of stimulated lymphocytes is capable of supporting proliferation of intact T cells for a long time and ensuring the functional activity of clones of such cells. An active compound in the culture fluid of stimulated lymphocytes was a T-cell cytokine, originally called T-cell growth factor. The main result of the action of IL-2 on resting or stimulated antigen or mitogen cells is to ensure their proliferation. It is this biological activity of IL-2 that defines it as a typical growth factor for cells of the lympho-myeloid complex.

IL-4 belongs to the group of hematopoietins. Previously, it was called B-cell growth factor. The source of IL-4 are mitogen-stimulated T-helpers, mast cells, unidentified bone marrow stroma cells. It is known that IL-4 enhances the expression of histocompatibility class II antigens (MHC II) in resting B cells, as well as the synthesis of IgG and IgE immunoglobulins after stimulation with lipopolysaccharide (LPS), supports the viability and growth of intact T cells, increases the activity of cytotoxic T- lymphocytes, enhances the proliferation of hematopoietic precursors in response to growth factors. In relation to b-cells, IL-4 acts as a co-stimulator of proliferation. So, it does not have any effect on resting B cells, but it is enough to act on them with one of the activation inducers specific for these cells, so that the biological effect of IL-4 is manifested: a sharp increase in the proliferation of this type of cell. Initially, IL-4 was described as a B-cell growth factor I. It subsequently became clear that it is capable of enhancing the proliferation of T cells belonging to different subpopulations. Its effect on T cells, as well as on B cells, appears only after their preliminary activation by an antigen or mitogen. By its nature, IL-4 acts as a pleiotropic regulator, as it interacts with a wide variety of cell types. So, its effect on macrophages is manifested in enhancing the expression of class II molecules of MHC and the Fc receptor for IgG, acquiring antitumor activity against fibrosarcoma, and enhancing antigen-presenting function.

IL-6. Interleukin-6 is produced by activated monocytes or macrophages, endothelial cells, fibroblasts, activated T cells, as well as a number of cells that are not immunocytes. Some effects caused by IL-6 are similar to those observed with IL-1 and TNF. However, the main effect of IL-6 is associated with its participation as a cofactor in the differentiation of B-lymphocytes, their maturation and conversion into plasma cells that secrete immunoglobulins. In addition, IL-6 promotes the expression of the IL-2 receptor on activated immunocytes, and also induces the production of IL-2 by T cells. This cytokine stimulates the proliferation of T-lymphocytes and the reaction of hematopoiesis.

IL-8. Interleukin-8 belongs to the group of chemokines, the main property of which is to provide chemotaxis to the area of inflammation of various types of cells: neutrophils, monocytes, eosinophils, T cells. IL-8 has pronounced pro-inflammatory properties, causing the expression of intercellular adhesion molecules and enhancing the adhesion of neutrophils to endothelial cells and subendothelial matrix proteins, which indicates its main role in mediating the inflammatory process. The producing cells of IL-8 are macrophages, lymphocytes, epithelial cells, fibroblasts, epidermal cells. The main function of IL-8 is to act as a chemoattractant for neutrophils, macrophages, lymphocytes, eosinophils. In addition to this biological effect, IL-8 enhances the adhesive properties of neutrophils, altering the expression of integrins and other compounds with adhesive properties. The properties of IL-8 to cause cell migration and promote their adhesion determine it as an active participant in the acute inflammatory reaction.

IL-10. It is a key regulator of the immune response with immunosuppressive properties. It is synthesized mainly by T-lymphocytes, mast cells and macrophages.

IL-17 is characterized as a pro-inflammatory cytokine, is produced by a subpopulation of Tx17 lymphocytes, and functions as a factor in protecting the body from pathogens. It stimulates the synthesis of IL-6, IL-8, TNF-α, IL-1β, suggest participation in maintaining the number of neutrophils (hypothesis Stark M., 2005).

IFN-γ has antiviral activity. The main property is the regulation of immunity. It is produced mainly by activated T-lymphocytes and NK cells. Main functions: macrophage activation; stimulation of the expression of MHC class II molecules; activation of NK cells; regulation of the synthesis of isotype of immunoglobulins with B-lymphocytes; direct antiviral activity.

RA progression is a dynamically developing process, which is conditionally divided into several stages: early (asymptomatic) stage, characterized by vascular and cellular activation; advanced stage (rapid chronization of inflammation), manifested by impaired angiogenesis, activation of endothelium, cell migration, infiltration of synovial tissue activated with CB4 + T-lymphocytes, formation of rheumatoid factors and immune complexes, synthesis of “pro-inflammatory” cytokines, prostaglandins, collagenase, metalloproteinases; late stage, which is characterized by autonomous "tumor-like processes" induced by chronic inflammation, due to somatic mutation and defects in the apoptosis of synovial cells.

One of the most important problems of modern rheumatology is the establishment of a correct diagnosis of RA at its early stage and the differentiation of this disease from other rheumatic diseases. The diagnosis of RA is primarily based on the clinical picture. Due to the insufficient specificity of the clinical criteria for making an objective diagnosis, it is necessary to conduct additional laboratory studies (determination of antibodies to rheumatoid factor (IgM RF) and autoantibodies to cyclic citrullated peptide (ACCP)), as well as studies the results of which allow a more accurate assessment of the patient’s condition and adjust therapy. One of these studies can be the assessment of the cytokine profile in peripheral blood cells (hereinafter referred to as “PC”) and synovial fluid (hereinafter referred to as “SJ”) using a “real-time” polymerase chain reaction with preliminary formulation of the reverse transcription reaction (hereinafter “RT-PCR” ").

The real-time RT-PCR method is widely used in scientific research, including for quantitative evaluation of the human cytokine genes mRNA expression. At the same time, the level of mRNA expression is estimated relative to the expression of mRNA genes that ensure cell viability (referred to as “housekeeping genes”, control genes, normalization genes, reference genes), using ΔCt (method 1) or ΔΔCt (method 2) [8]. The Ct value is the cycle value automatically determined by the instrument software at the point of intersection with the baseline.

The disadvantages of these methods include the lack of an “ideal control gene” that meets all the requirements (first of all, the constant expression in all organs and tissues). Therefore, in practice, normalization is carried out according to several control genes, the normalization factor is determined, and expression levels in different samples are compared with each other (method 2). The disadvantages of the 2 methods include the need to conduct a full volume of research with all samples, which is extremely inconvenient in conditions of clinically significant application in routine practice.

The problem solved by the proposed invention is to eliminate these drawbacks, to achieve a faster, more reliable and easier diagnosis of the presence of the inflammatory process in early rheumatoid arthritis by analyzing only one biological material.

The proposed method for the diagnosis of inflammatory diseases (an early stage model of rheumatoid arthritis) based on the quantitative determination of cytokine gene transcripts by real-time PCR involves:

- the use of a control gene (hypoxanthine-guanine phosphoribosyltransferase 1 - HPRT1) to a greater extent to assess the quality of the execution of all stages of the reaction than to normalize the determination of the level of expression of mRNA (matrix RNA) of cytokine genes,

- determination of the spectrum of cytokines characterized by increased expression of mRNA in PC and SG,

- determination of the spectrum of cytokines characterized by reduced expression of mRNA in PC and SG (i.e. those cytokines that are most informative for determining the patient's condition),

- the search for functional bonds and bonds that determine the molecular processes of mutual influence in the network of the cytokine cascade, which reflects the development and course of the inflammatory process in early rheumatoid arthritis (hereinafter “pRA”),

- calculation of markers (i.e., the most informative cytokines), taking into account the dependence of the intensity of the inflammatory process with PPA, according to

[IL1] / [IL2] = E il2 Ct2 / E i11 Ct1 ( Formula 1),

Where

[IL1] is the concentration of mRNA of the cytokine gene, characterized by increased expression in PC or SJ;

[IL2] is the concentration of the mRNA of the cytokine gene, characterized by reduced (or constant) expression in PC or SG;

E - amplification efficiency - a value characterizing the growth of the matrix on each amplification cycle (formula 2);

Ct1 and Ct2 are the value of threshold cycles, automatically determined by the device, in the test sample for the corresponding cytokine.

In practice, the amplification efficiency is determined experimentally by the formula

E = 10 ^{- (1 / slope)} (formula 2),

Where

slope is the difference in Ct values at 10-fold dilution of the sample (the angular coefficient of the equation for the linear dependence of Ct on the logarithm (log) of the matrix concentration) (Fig. 1).

Thus, directly in the course of “real-time” commissioning, standardized dependences of the fluorescent radiation intensity on the cycle number of the polymerase chain reaction are obtained (Fig. 2). The software automatically calculates the threshold cycle. These values can be substituted into formula 1.

The technical result achieved by the implementation of the proposed invention consists, firstly, in the possibility of assessing the severity of the inflammatory process with PPA immediately after the reaction; secondly, the calculation does not take into account the Ct value of the normalization gene, which reduces the probability of method error associated with the individual features of the expression of the control gene in this type of tissue; thirdly, for a more accurate assessment of expression levels, the relevance of using several normalization genes is called into question.

Brief Description of the Drawings

Figure 1 shows a graph of the dependence of Ct on the logarithm (log) of the matrix concentration. The angular coefficient is -3.3919. Accordingly, the amplification efficiency is 1.97. Amplification efficiency is determined in a series of experiments with matrix dilution in the concentration range of the linear range of test systems as an average value.

Figure 2 shows the standardized dependences of the fluorescence intensity on the cycle number of the polymerase chain reaction for the entire studied spectrum of cytokines. The threshold cycle value is automatically determined by the device, as the point of intersection of the graph data with the base line (Threshold).

Table 1 shows the amplification efficiency values for all test systems.

Table 2 shows the indicators indicating an inflammatory process.

Table 3 shows the scope of the studies: 116 people were examined, while 101 PC samples and 91 SJ samples were examined.

Fig. 3 shows the ratio of the levels of cytokine expression in peripheral blood, revealed as a result of the study of PC, where the left scale is the ratio of the expression level of IL-10 / IL-4 and IL-10 / IL-2 in patients with early rheumatoid arthritis.

Fig. 4 shows the ratio of the levels of cytokine expression in the synovial fluid, revealed as a result of the study of SJ, where the left scale is the ratio of the level of expression of pro-inflammatory cytokines (TNF-α, IL-6, IL-8, IL-17, IFN-γ, IL-2) and IL-4 in patients with rheumatoid and early rheumatoid arthritis.

Table 4 shows the ratios of statistical indicators of the levels of cytokine expression in the synovial fluid in patients of the comparison group with osteoarthritis, where M and σ are mean values and mean square deviations, respectively.

Table 5 shows the correlation coefficients of C-reactive protein (CRP) and rheumatoid factor (RF) with a ratio of cytokines (according to Spearman).

In the table. Figure 6 shows the indicators of the cytokine profile in patients with weak (patient No.P11 / 12) and pronounced (patient No. 31/32) inflammatory processes.

The implementation of the invention

Stage 1. Taking material

The study material is peripheral blood (PC) or synovial fluid (SJ) in patients with suspected PPA.

The severity of the inflammatory process with PPA can be determined by any of these fluids. The most informative is the SG.

The sampling is carried out in tubes with EDTA (anticoagulant that prevents blood coagulation) at a final concentration of 2 mg / ml.

PCs are taken from a vein in the usual way; SJs are taken from the affected joint with a syringe.

2 stage. Cell isolation

Option 1: mononuclear cells are isolated from PC.

The separation of the cellular elements of the blood is carried out by centrifugation in a density gradient of ficoll-urographin (density 1,077 g / ml). Isolation of lymphocytes is carried out from 1 ml of whole blood. The resulting cells are resuspended in 100 μl phys. solution.

Method for isolating blood lymphocytes from ficoll: Boyum A. Separation of leukocytes from blood and bone marrow //Scand.J.Clin.Lab.Investig. - 1968. - Vol.21. - Suppl. 97. p.1-9

2 options: all cells are precipitated from the SJ.

- add 1 ml of physiological saline to 1 ml of SJ;

- precipitate the cells by centrifugation at 6000 rpm for 10 minutes;

- remove the supernatant,

- the resulting cells are resuspended in 100 μl of saline.

3 stage. Isolation of RNA.

To isolate RNA, the Proba NK kits (manufactured by NPF DNA Technology CJSC) with modification (phenol deproteinization) are used. The method is based on lysis of samples in a 4M solution of guanidine thiocyanate, phenol deproteinization, precipitation of nucleic acids with isopropanol in the presence of a co-precipitant, followed by washing with ethanol and acetone.

The volume of samples after isolation of 50 μl.

There are no differences with respect to PC and SJ.

RNA isolation procedure:

- prepare one tube for the negative control sample - "K-";

- add to each tube with PC and SJ cells, as well as “K-", 300 μl of lyse solution;

- tightly close the lids of the tubes, shake on the vortex for 3-5 s;

- thermostat tubes for 15 min at 65 ° C, precipitate condensate by centrifugation at 13000 rpm for 30 s;

- add 400 μl of saturated phenol, shake the tubes on a vortex for 30 s;

- precipitate droplets from the walls of the tube by centrifugation at 13000 rpm for 30 s;

- add 160 μl of a solution of chloroform: isoamyl alcohol in a ratio of 24: 1, shake the tubes on a vortex for 30 s;

- centrifuge the tubes at 13,000 rpm for 10 min;

- take the supernatant into a new tube;

- add 400 μl of saturated phenol; shake the vortex tubes for 30 s;

- precipitate droplets from the walls of the tube by centrifugation at 13000 rpm for 30 s;

- add 160 μl of a solution of chloroform: isoamyl alcohol in a ratio of 24: 1, shake the tubes on a vortex for 30 s;

- centrifuge the tubes at 13,000 rpm for 10 min;

- add 400 μl of precipitation reagent (isopropanol) and shake the vortex tubes for 3-5 s;

- centrifuge the tubes at 13,000 rpm for 15 min;

- Without touching the pellet, completely remove the supernatant (with a separate tip from each tube);

- add 500 μl of washing solution No. 1 (70% ethanol) to the precipitate and carefully invert each tube 3-5 times;

- centrifuge the tubes at 13,000 rpm for 5 min;

- Without touching the pellet, completely remove the supernatant (with a separate tip from each tube);

- add 300 μl of washing solution No. 2 (acetone) to the precipitate and carefully invert each tube 3-5 times;

- centrifuge the tubes at 13,000 rpm for 5 min;

- Without touching the pellet, completely remove the supernatant (with a separate tip from each tube);

- open the lids of the tubes and dry the pellet at 65 ° C for 5 minutes;

- add to the precipitate 50 μl of buffer for dissolution;

- warm the tubes at 65 ° C for 10 min;

- shake the samples on a vortex to dissolve the material, precipitate droplets by centrifugation of the tubes at 13000 rpm for 30 s.

The RNA preparation is ready for a reverse transcription reaction.

The resulting RNA preparation is recommended to be used immediately for the formulation of the reverse transcription reaction. The RNA preparation can be stored at a temperature of minus 20 ° C for 1 month or at minus 70 ° C for 1 year.

4th stage. Reverse transcription

The reverse transcription reaction is carried out by the reverse transcriptase enzyme in a specific buffer in the presence of messenger RNA (mRNA), deoxynucleotide triphosphates and primers at a temperature of 40 ° C for 60 minutes. Reverse transcriptase inactivation is carried out at 95 ° C for 15 minutes.

During the reaction, the reverse transcriptase enzyme from the RNA matrix (mRNA) synthesizes its complementary DNA (cDNA) by polymerization of deoxynucleotide triphosphates, using primers as a seed for the complementary chain.

Specific oligonucleotides are used as primers for reverse transcription.

The composition of the reaction mixture in vitro during the reverse transcription reaction:

- distilled water treated with DEPC - 0.7 μl;

- reaction buffer * - 4 μl

- a mixture of four types of deoxyribonucleotide triphosphates at a concentration of 25 μM of each deoxyribonucleotide triphosphate - 0.2 μl;

- primers ** (11 specific oligonucleotides) at a concentration of 100 μm each - 0.1 μl;

- reverse transcriptase at a concentration of 500 international units of activity - 1 μl;

- the investigated mRNA sample - 33 μl.

* composition of the reaction buffer: 0.5 M Tris-HCl pH 8.3, 0.75 M KCl, 30 mm MgCl ₂ , 50 mm DTT, 0.1% DEPC.

** primers:

TNF-α 5'-ggg TTC gAg AAg - 3 ';

IL-1β 5'-gTC CAT ggC CAC-3 ';

IL-2 5'-gTT gTT TCA gAT C - 3 ';

IL-4 5 '- CCT TCA CAg gAC - 3';

IL-6 5 '- CAC CAg gCA AgT C - 3';

IL-8 5 '- STS TST TSS ATS - 3';

IL-10 5 '- CAC Agg gAA gAA - 3';

IL-17 5 '- Cgg CAC TTT gCC - 3';

IFN-γ 5 '- Tgg ASA TTC AAg - 3';

HPRT1 5 '- TCC ACA ATS Aag - 3'.

To increase sample volumes after reverse transcription, cDNA was diluted 5 times with deionized water.

5 stage. PCR

Primers and probes for PCR are selected taking into account the structures of exons and introns in such a way as to exclude annealing of cytokines and HPRT1 (control gene) on the genomic DNA matrix. This eliminates the need for an additional step in the processing of nucleic acids with DNAase.

In the standard previously proposed protocols, this step was carried out to remove DNA from the sample, because the reaction may go not on cDNA obtained during the reverse transcription reaction, but on genomic DNA. It was necessary to exclude the reaction to genomic DNA, for example, by removing it with DNAase. The proposed method, due to the specificity of primers for mRNA, eliminates the annealing of the template on genomic DNA; therefore, this step is not necessary.

DNA probes used to detect amplification products of cytokine and HPRT1 genes are labeled with FAM. Amplification reactions of cytokines and HPRT1 are performed in different tubes. To increase the sensitivity and specificity of PCR, a "hot start" was applied, which is ensured by the use of paraffin. The optimum annealing temperature of primers and probes was selected experimentally using the “temperature gradient” mode; for all test systems, the temperature was unified and amounted to 62 ° C.

Amplification is carried out in "real time" in a volume of 35 μl according to the following program: 1 cycle - 80 ° C for 30 sec, 94 ° C for 1 min; 50 cycles - 94 ° С 10 sec., 62 ° С 20 sec., The DT-322 and DT-964 devices manufactured by NPO DNA-Technology CJSC were used. The measurement of fluorescence is carried out on each cycle at a temperature of 62 ° C.

The composition of the reaction mixture in vitro during PCR:

bidistilled water - 25.37 μl

- reaction buffer * - 3.5 μl

- a mixture of four types of deoxyribonucleotide triphosphates at a concentration of 25 μM of each deoxyribonucleotide triphosphate - 0.24 μl

- primer No. 1 ** at a concentration of 100 μm - 0.125 μl

- primer No. 2 ** at a concentration of 100 μm - 0.125 μl

- detecting sample ** at a concentration of 50 μM (reagent labeled with a fluorophore to determine the desired target) - 0.07 μl

- Taq - polymerase at a concentration of 5 international units of activity - 0.5 μl

- the studied cDNA sample obtained during the reverse transcription reaction (see step 4 above) - 5 μl.

* composition of the reaction buffer: 100 mM Tris-HCl (pH 8.8 at 25 ° С), 500 mM KCl, 0.8% P40, 20 mM MgCl2

** primers and detection samples: for TNF-α primer No. 1 -5'- gAT Cgg CCT CCA gAg ggA Ag - 3 '; primer No. 2-5'-ggg TTC gAg AAg ATg ATS - 3 '; detection sample - (FAM) -5'- gCC CTC Tgg CCC AggCAg-3 '- (BHQ1);

IL-1β primer No. 1 5'-gCC CTA AAC AgA TgA AgT - 3 ';

primer No. 2 -5'-gTC CAT ggC CAC AAC AAC - 3 '; detection sample - (FAM) -5'- gCg gCC TgC CTg AAg CCC - 3 '- (BHQ1);

IL-2 primer No. 1 -5'-CCC AAA CTC ACC Agg ATg C - 3 ';

primer No. 2 -5'- gTT gTT TCA gAT CCC TTT Ag-3 '; detection sample - (FAM) - 5'- CAg TTC TgT ggC CTT CTT gg - 3 '- (BHQ1);

IL-4 primer No. 1 -5'-gAC ATS TTT gCT gCC TCC AAg - 3 ';

primer No. 2 -5'-CCT TCA CAg gAC Agg AAT T-3 '; detecting sample - (FAM) -5'- TTT GCC GGA GCA CAG TCG CAG - 3 '- (BHQ1);

IL-6 primer No. 1 -5'- gAA CTC CTT CTC CAC AA - 3 ';

primer No. 2 -5'-CAC CAg gCA AgT CTC CTC - 3 '; detection sample (FAM) -5'- GCC TGC TGC CTT CCC TGC C - 3'- (BHQ1);

IL-8 primer No. 1 -5 '- TgC AgC TCT gTg TgA Ag - 3'; primer No. 2 - 5 '- CTC TCT TCC ATS AgA AAg CTT - 3'; detection sample - (FAM) - 5'- gCg Cag TgT ggT CCA CTC TCA - 3'- (BHQ1);

IL-10 primer No. 1 -5'- CTT gCT ggA ggA CTT TAA gg-3 '; primer No. 2 -5'- CAC Agg gAA gAA ATS gAT gA - 3 '; detecting sample - (FAM) 5'-CCT ggA ggA ggT gAT gCC C-3 '- (BHQ1);

IL-17 primer No. 1 -5'- Tgg gAA gAC CTC ATT ggT - 3 '; primer No. 2 5'-Cgg CAC TTT gCC TCC CAg A-3 '; detecting probe (FAM) - 5'- CCA CgA AAT CCA ggA TgC CC - 3'- (BHQ1);

IFN-γ primer No. 1 -5'- gAA gAA TTg gAA AgA ggA gAg - 3 '; primer No. 2 5'-Tgg ASA TTC AAg TCA gTT AC - 3 '; detecting sample - (FAM) -5'- gTC TCC ACA CTC TTT Tgg AT -3 '- (BHQ1);

HPRT1 primer No. 1 5'-ggA CTA ATT ATg gAC Agg ACT g - 3 '; primer No. 2 -5'- TCC ACA ATS AAg ASA TTS - 3 '; detecting sample - (FAM) -5'- CAG CCC CCC TTG AGC ACA CAG - 3 '- (BHQ1).

The tubes are placed in a detecting amplifier (a device that allows PCR and fluorescence emission in a test tube to be simultaneously recorded), for example, a DT-96 detecting amplifier (manufactured by NPF DNA-Technology CJSC, Russia), and PCR is performed with the results recorded "in real time ".

Stage 6: analysis of the results

As shown in Fig. 2, for each PC or SJ sample, a set of Ct values is obtained (the cycle value automatically determined by the instrument software at the point of intersection with the baseline) for each cytokine. These Ct values are substituted into formula 1:

[IL1] / [IL2] = E il2 Ct2 / E il1 Ct1

The amplification efficiency was determined experimentally (Table 1). For various test systems, it was 1.96-1.98, with the exception of IL-4 (1.92).

During the study, it was found that with rRA, the expression of pro-inflammatory cytokines mRNA (TNF-α, IL-1β, IL-6, IL-8 and IL-17), as well as immunosuppressive IL-10, and anti-inflammatory mRNA expression can be increased IL-4. The most informative for assessing the inflammatory process with PPA is SG (option 2). The following indicators (Table 2) in PC and SJ can be a predictor of the inflammatory process:

option 1 for PC: interleukin - 10 / interleukin - 2> 1.5

interleukin - 10 / interleukin - 4> 1.0

option 2 for SJ: interleukin - 2 / interleukin - 4> 10

interferon-γ / interleukin - 4> 300

tumor necrosis factor-α / interleukin - 4> 2000

interleukin - 1β / interleukin - 4> 5,000

interleukin - 6 / interleukin - 4> 10

interleukin - 8 / interleukin - 4> 10000

interleukin - 17 / interleukin - 4> 5

The principal difference of the proposed method is that there is no need to use the normalization gene.

Examples of the proposed method

116 people were examined, 101 PC samples and 91 SJ samples were studied (Table 3). 4 research groups were formed:

- group 1 included conditionally healthy patients (control). At the time of the examination, infectious, respiratory viral, autoimmune, allergic and other diseases were excluded. The size of the group was 25 people. Due to the difficulty of taking SJ from this group, only PC was taken into the study.

- group 2 included patients diagnosed with rheumatoid arthritis (RA). The size of the group was 49 people. The study was taken SJ and PC.

- group 3 included patients with a diagnosis of early rheumatoid arthritis (rRA). The size of the group was 27 people. The study was taken SJ and PC.

- group 4 included patients with a diagnosis of osteoarthrosis (OA). The size of the group was 15 people. Only SJ was taken into the study. This group was chosen as a comparison group, since taking material in this case was possible, as well as due to the fact that this disease is a chronic non-inflammatory joint disease in contrast to rheumatoid arthritis.

The results of a PC study are presented in Fig. 3. The ratio of the expression level of IL-10 / IL-4 and IL-10 / IL-2 in early rheumatoid arthritis was significantly higher (p = 0.0063, p = 0.00009, respectively) than in the control group. Figure 3 shows the medians in the groups, as well as the 25% and 75% quartiles for the control group. The average value (M) in the control group IL-10 / IL-4 was 0.4, the standard deviation (σ) was 0.2. The general formula (Table 2) for all ratios was calculated as

Interleukin-1 / interleukin-2≥M + 3σ (formula 3).

The mean value (M) in the control group IL-10 / IL-2 was 0.4, and the standard deviation (σ) was 0.3.

Thus, the calculations according to formula 3 in the control group IL-10 / IL-4 = 1, IL-10 / IL-2 = 1.3. In the presence of an inflammatory process, these indicators will be higher.

The results of the SJ study are presented in Fig. 4. The ratio of the expression level of pro-inflammatory cytokines (TNF-α, IL-6, IL-8, IL-17, IFN-γ, IL-2) and IL-4 in rheumatoid and early rheumatoid arthritis was significantly higher than in the comparison group with osteoarthritis . Figure 4 shows the medians in the groups, as well as the 25% and 75% quartiles for the control group and the rheumatoid arthritis group. The mean value (M) and standard deviation (σ) in the control group are presented in table 4. Based on them, the claims were deduced.

Comparison of the obtained cytokine profile with the activity of the inflammatory process with pRA.

To assess the activity of the inflammatory process with rRA, a laboratory marker of inflammation such as C-reactive protein (CRP) was chosen. As an immunological marker of RA, IgM rheumatoid factor (RF) was used. The level of CRP and RF was measured by a highly sensitive method using a BN-100 laser nephelometer (Dade Behring, USA), units of mg / L.

Moderate and good positive correlations of these indicators with indicators of cytokine ratios were obtained (Table 5).

Let's look at two examples.

Patient No. P11 / 12. Male, 54 years old. The number of affected joints is 14. At the time of the examination, CRP in the blood was 0.67, in the SJ 0.26 mg / l; RF in blood and SJ 0.1 mg / l, which indicates a low activity of the inflammatory process. The ratio of cytokines (table 6) also indicate a low activity of the inflammatory process at the moment.

Patient No. P31 / 32. Woman, age 59. The number of affected joints 1. At the time of the examination, CRP in the blood of 20.88, in the SZ 19.28 mg / l; RF in the blood 247.2; SJ 324.7 mg / l, which indicates a high activity of the inflammatory process. The ratio of cytokines (table 6) also indicate a high activity of the inflammatory process at the moment.

Information sources

1. Dolhain R.J.E.M., van der Heiden A.N., ter Haar N.T. et al. Shift toward T lymphocytes with a T helper 1 cytokine-secretion profile in the joints of patient with rheumatoid arthritis // Arthritis Rheum. - 1996. - Vol. 39. - P. 1961-1969.

2. Morita Y., Yamamura M., Kawashima M. et al. Flow cytometric singl-cell analisis of cytokine production by CD4 + T cell in synovial tissue and periferal blood from patients with rheumatoid arthritis // Arthritis Rheum. - 1998 .-- Vol.41. - P.1669-1676.

3. Simon A.K., Seipelt E., Sieper J. Divergent T-cell cytokine patterns in iflammatory arthritis // Proc Nati Acad Sci USA. - 1994 .-- Vol. 91. - P.8562-8566.

4. Abbas A.K. Murphy K.M., Sher A. Functional diversity of T helper lymphocytes. // Nature. - 1996. - Vol. 383. - P.787-793.

5. Miosses P., van den Berg W. Th1 / Th2 cytokine balance inarthritis // Arthritis Rheum. - 1997 .-- Vol. 40. - P.2105-2115.

6. Van Roon J.A., van Roy J. L., Duits A. et al. Proinflammatory cytokine production and cartilage damage due to rheumatoid synovial T-helper-1 activition is inhibited by interleukin-4.// Ann Rheum Dis. - 1995 .-- Vol. 54. - P.836-840.

7. Nasonov E.L., Ghukasyan D.A., Nasonova M.B. Immunopathology of rheumatoid arthritis and osteoporosis: new evidence. // Osteoporosis and osteopathy. - 2000. - No. 2. - S. 4-7.

8. Vandesompele J., De Preter K., Pattyn F. et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.// Genome Biology. - 2002 .-- Vol. 3 (7).

Claims (2)

1. A method for diagnosing the inflammatory process in early rheumatoid arthritis, which consists in isolating mononuclear cells from peripheral blood, from which RNA is isolated, based on a quantitative determination of the mRNA expression of the cytokine genes Iiterleukin-2 (IL-2), interleukin-4 (IL- 4) and interleukin-10 (IL-10) by reverse transcription and polymerase chain reaction with registration of the accumulation of reaction products by fluorescence directly during the reaction, the balance of cytokines is estimated by the formula
[IL '] / [IL''] = E IL''^Ct''/ E IL' ^ Ct ',
where [IL '] is the concentration of mRNA of the cytokine gene, characterized by increased expression;
[IL ''] is the concentration of mRNA of the cytokine gene, characterized by reduced expression;
E - amplification efficiency - a value characterizing the growth of the matrix on each amplification cycle;
Ct 'and Ct''is the value of the threshold cycles for the corresponding cytokine, and the presence of an inflammatory process is observed upon receipt of the following balances:
IL-10 / IL-2>1.5;
IL-10 / IL-4> 1.0. 2. A method for diagnosing the inflammatory process in early rheumatoid arthritis, which consists in the fact that all the cells from which RNA is isolated from the synovial fluid cells are precipitated based on a quantitative determination of the mRNA expression of the interleukin-2 (IL-2) and interleukin-4 genes ( IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-17 (IL-17), interleukin-1β (IL-1β), as well as based on the quantitative determination of interferon-gamma (IFNG) and tumor necrosis factor (TNF) by reverse transcription and polymerase chain reaction from register iey accumulation of reaction products on flourestsentsii directly during the reaction is evaluated by the formula cytokine balance
[IL '] / [IL''] = E IL''^Ct''/ E IL' ^ Ct ',
where [IL '] is the concentration of mRNA of the cytokine gene, characterized by increased expression;
[IL ''] is the concentration of mRNA of the cytokine gene, characterized by reduced expression;
E - amplification efficiency - a value characterizing the growth of the matrix on each amplification cycle;
Ct 'and Ct''is the value of the threshold cycles for the corresponding cytokine, and the presence of an inflammatory process is observed upon receipt of the following balances:
IL - 2 / IL - 4>10;
IFNG / IL - 4>300;
TNF / IL - 4>2000;
IL - 1β / IL - 4>5000;
IL - 6 / IL - 4>10;
IL - 8 / IL - 4>10000;
IL - 17 / IL - 4> 5.

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