RU2507519C1 - Method for prediction of clinical course of acute pancreatitis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: DNA is recovered from peripheral venous blood; polymorphism of a tumour necrosis factor α gene is analysed, and if observing the genotypes -308 AA or -308 GA TNFα, a risk of the late onset of a reactive phase of acute pancreatitis is predicted.

EFFECT: obtaining the new criteria for assessing the time of the reactive phase of acute pancreatitis.

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Description

The invention relates to the field of medical diagnosis, can be used to predict the timing of the onset of the reactive phase of acute pancreatitis.

The problem of acute pancreatitis is one of the most urgent in emergency surgery. This is due not only to the fact that the disease is very common, but also to the fact that it is difficult to predict and diagnose. Acute destructive pancreatitis is primarily aseptic pancreatic necrosis with subsequent inflammatory reaction to the foci of formed necrosis. This disease has a phase course: I phase - enzymatic; II phase - reactive; Phase III - sequestration and melting of dead sites. An important prognostic factor in the course of acute pancreatitis is the duration of the enzymatic phase and, accordingly, the duration of the onset of the reactive phase. During the first phase, the main pathological "program" is implemented - the formation of pancreatic necrosis and the development of endotoxemia, multiple organ failure and endotoxin shock.

It is believed that with a prolonged course of the enzymatic phase and a later onset of the reactive phase, the proinflammatory cytokines are overproduced, the functional reserves of the body are depleted, which causes an increase in multiple organ failure, the development of severe immunodeficiency and, subsequently, a more severe course of acute pancreatitis.

Known patent No. 2310848 (C1) according to the application of the Russian Federation 2006112086/15, 11/20/2007 "Method for predicting the risk of occurrence, clinical course and outcome of acute idiopathic pancreatitis" (GOU VPO "Krasnoyarsk State Medical Academy of the Federal Agency for Health and Social Development"), proposed A method for predicting the risk of occurrence, clinical course and outcome of acute idiopathic pancreatitis by isolating DNA from the patient’s peripheral venous blood, typing lymphocyte DNA polymorphism for further use following mutations in the SPINK1, GSTM1, GSTT1, PRSS1, and CFTR genes. If heterozygous mutations are detected in the SPINK1, PRSS1, and CFTR genes, the development of a destructive form of idiopathic pancreatitis is predicted, when a combination of at least two mutated genes from SPINK1, GSTM1, GSTT1, PRSS1, and CFTR is detected in heterozygous carriers of N8 carrier homozygous or homozygous 34 for homozygous N8 carriers with a heterozygous mutation of R122H of the PRSS1 gene, severe pancreatic necrosis is predicted. The advantage of the invention is that it allows you to select a group of patients with a high risk of developing destructive complications and reasonably recommend that they conduct full-scale specialized intensive care in the early stages, thereby reducing or completely eliminating mortality in patients.

The disadvantage of the prototype is that this method does not allow to predict the timing of the onset of the reactive phase in acute pancreatitis.

It is known that the tumor necrosis factor α - TNFα, which has a pro-inflammatory effect and enhances TNF-dependent apoptosis, has a significant effect on the course of phases 1 and 2 of acute destructive pancreatitis [2, 3].

Tumor necrosis factor α-glycoprotein with a molecular weight of 17 kDa is a product of monocytes / macrophages, endothelial, mast and myeloid cells, neuroglia cells, and in special cases activated T-lymphocytes. The TNFα gene is located on the sixth human chromosome (6p21.3) at the locus encoding the molecules of the main histocompatibility complex of the first (HLA-A, B, C) and second classes (HLA-DP, DQ, DR), between the Ltα and Ltβ genes [4 ].

There are three main directions of TNFα action: cytotoxic, directed to tumor cells; immunomodulatory and pro-inflammatory, caused by the activation of macrophages, neutrophils, eosinophils and endothelial cells; effect on metabolism. TNFα is actively involved in the process of rapidly increasing cell size and initiating apoptosis. In addition to the direct pro-inflammatory effect, TNFα has a wide range of immunoregulatory effects and is involved in the regulation of metabolic processes [5].

The objective of this study is to expand the arsenal of methods for predicting the course of acute pancreatitis according to genetic polymorphism - 308 G / A TNFα.

The technical result of using the invention is to obtain criteria for evaluating the timing of the onset of the acute phase of acute pancreatitis.

In accordance with the task, a method was developed for predicting the course of acute pancreatitis, including the isolation of DNA from peripheral venous blood; analysis of tumor necrosis factor α gene polymorphism; predicting the late onset of the reactive phase of acute pancreatitis in the case of the detection of the -308 AA or -308 GA genotypes.

The novelty and inventive step consists in the fact that the possibility of predicting the timing of the onset of the reactive phase of acute pancreatitis according to polymorphism - 308 G / A TNFα is not known from the prior art.

The method is as follows:

DNA is isolated from samples of peripheral venous blood of patients with acute pancreatitis in 2 stages. At the first stage, 25 ml of lysis buffer containing 320 mM sucrose, 1% Triton X-100, 5 mM MgCl ₂ , 10 mM Tris-HCl (pH 7.6) is added to 4 ml of blood. The resulting mixture was stirred and centrifuged at 4 ° C, 4000 rpm for 20 minutes. After centrifugation, the supernatant is decanted, 4 ml of a solution containing 25 mM EDTA (pH 8.0) and 75 mM NaCl are added to the precipitate and resuspended. Then, 0.4 ml of 10% SDS, 35 μl of proteinase K (10 mg / ml) are added and the sample is incubated at 37 ° C for 16 hours.

At the second stage, DNA is sequentially extracted from the obtained lysate in equal volumes of phenol, phenol-chloroform (1: 1) and chloroform with centrifugation at 4000 rpm for 10 minutes. After each centrifugation, the aqueous phase is selected. DNA is precipitated from solution in two volumes of chilled 96% ethanol. The formed DNA is dissolved in bidistilled, deionized water and stored at -20 ° C.

The isolated DNA is then subjected to polymerase chain reaction using standard oligonucleotide primers (table 1).

The polymorphic locus of tumor necrosis factor α was studied by the method of polymerase chain reaction of DNA synthesis on an IQ5 amplifier (Bio-Rad) using standard oligonucleotide primers and probes (Table 1), followed by analysis of polymorphism by allele discrimination.

A 25 μl reaction mixture includes: 67 mM Tris-HCl (pH 8.8), 2.5 mM MgCl2, 0.1 μg of genomic DNA, 10 pM of each primer, 5 pmol of each probe, 200 μM of dATP, dGTP , dCTP, dTTP and 1 unit of active Taq polymerase.

After denaturation (5 min at 95 ° C), 40 amplification cycles were performed according to the scheme: primer annealing - 1 min at 52 ° C; denaturation - 15 sec at 95 ° C.

Table 1 The structure of primers and probes used for genotyping of the studied DNA marker Gene Polymorphism and its localization in the gene Primer structure Literature F: 5 - GAAATGGAGGCAATAGGTTTTTGAG-3 ' TNFα -308G / A (promoter) R: 5'-GGCCACTGACTGATTTGTGTGTAG-3 ' (T. Kim, 2003) 5'-FAM-CCGTCCTCATGCC-RTQ1-3 ' 5'-ROX-CCGTCCCCATGCC-RTQ1-3 '

The invention is characterized by the following graphic materials:

Figure 1 shows the discrimination of alleles at the locus - 308 G / A TNFα, where • - homozygotes -308AA, ■ - homozygotes -308GG, ▲ - heterozygotes -308GA. Alleles are discriminated by the Tag Man probe method according to the RFU values, where RFU is the relative fluorescence (UVF) level of each probe. A probe with a fluorescent dye ROX corresponds to allele G, a probe with a dye FAM corresponds to allele A.

Figure 2 presents the timing of the onset of the reactive phase of acute pancreatitis depending on the genotypes of the locus -308 G / A TNFα.

In Fig. 1, two bands, vertical and horizontal, divide the graph into four sections: one for each homozygous state, one for the heterozygous state and the section without reaction. Assigning genotypes to unknown samples is determined by plotting the PF for one fluorophore on the x axis relative to PF for another fluorophore on the y axis in the allele discrimination diagram.

If the UVR values of an unknown sample are above the horizontal strip and to the right of the vertical strip, the genotype is heterozygous (GA). If the PFV values of an unknown sample are above the horizontal strip and to the left of the vertical strip, the genotype is homozygous for the G allele (the PF of the G allele is plotted along the y axis). If the UOF values of an unknown sample are below the horizontal strip and to the right of the vertical one, the genotype is homozygous for the A allele (the UOF of the A allele is plotted along the x axis). If the UVR values of an unknown sample are below the horizontal strip and to the left of the vertical, genotype determination is impossible (in this case, an undefined sample is a negative control).

The formation of the database and statistical calculations were carried out using the program "STATISTICA 6.0". Associations of alleles and genotypes of the studied DNA marker with the timing of the onset of the reactive phase of acute pancreatitis were evaluated using a nonparametric method, the Mann-Whitney test. To describe the quantitative indicators considered, the median (Me) and the interquartile range (Q25-Q75) were used [7].

The possibility of using the proposed method for predicting the course of acute pancreatitis is confirmed by the analysis of the observation results of 195 patients with acute pancreatitis. Patients were included in the appropriate group of patients only after a diagnosis of the disease was confirmed, using clinical and laboratory-instrumental examination methods.

The study group included individuals of Russian nationality, who are natives of the Central Black Earth Region of Russia and have no kinship with each other.

It was found that in individuals with the -308 AA and -308 GA TNFα genotypes, the acute phase of acute pancreatitis occurred after 6 days (lower quartile 5 days; upper quartile 7 days), and in patients with the -308 GG TNFα genotype this indicator was 5 days (interquartile range 4.00-6.00 days, p = 0.03) (figure 2).

Thus, the obtained data indicate an important pathogenetic role of the molecular genetic marker -308 G / A tumor necrosis factor α in the development of acute pancreatitis. Markers for the late onset of phase 2 of acute pancreatitis are the genotypes -308 AA and -308 GA TNFα.

Patients with acute pancreatitis, when detecting genetic factors of a later onset of the reactive phase, which are -308 AA or -308 GA TNFα, should fully carry out a set of therapeutic measures (medication, elements of “minor” surgery, extracorporeal detoxification) aimed at reducing the time enzymatic phase, which can improve the treatment results of acute pancreatitis.

Literature

1. Kuznetsov, N.A., Rodoman, G.V., Brontwein, A.G. Treatment of patients with pancreatic necrosis // Surgery. 2004. No. 12. S.22-27.

2. Glotov O.S., Baranov B.C. Genetic polymorphism, multifactorial diseases and longevity // Medical Genetics-2007. - t.6, No. 4 (58) - S.17-29.

3. Sotnichenko, B. A., Salienko, S. V., Markelova, E. V. Destructive pancreatitis: prevention and treatment of purulent-septic complications // Annals of surgical hepatology. 2006. Vol. 11 No. 1. S.67-71.

4. Tsaregorodtseva T.M. Cytokines in gastroenterological pathology // Medical newspaper. - 2005. - No. 63. - S.17-24.

5. Baranov B.C. The human genome and genes of “predisposition” (Introduction to predictive medicine) / B.C. Baranov, E.V. Baranova, T.E. Ivashchenko // St. Petersburg: “Intermedica”, 2000 - 272 p.

6. Polymorphisms of tumor necrosis factor (TNF) α and β genes in Korean patients with psoriasis / T. Kim [et al.] // Arch. Dermatol. Res. - 2003. - V.295. - P.8-13.

7. Rebrova, O.Yu. Statistical analysis of medical data. Application of the STATISTICA application software package [Text] / O.Yu. Rebrova. - M .: Media Sphere, 2006 .-- 305 p.

Claims (1)

A method for predicting the course of acute pancreatitis, including the extraction of DNA from peripheral venous blood, characterized in that the polymorphism of the tumor necrosis factor α gene is analyzed and the risk of late onset of the acute phase of acute pancreatitis is predicted if genotypes -308 AA or -308 GA TNFα are detected.

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