The Study Of Relationship Between The Microsatellite Polymorphism Of Cytotoxic T Lymphocyte Associated Antigen-4 Gene And Inflammatory Bowel Disease

Background and purpose:IBD mainly includes UC and CD, but pathogeny is inexplicit. In recent years, the morbidity of IBD in our country has the tendency to increase year by year. According to the research, these two kinds of diseases are related with heredity, environment (food, intestinal bacterium) immunity factors. The research on traditional epidemiology shows that IBD has genetic susceptibility. Immunity is the most affirmative factor in the research on present IBD morbidity field, but also one of mostly studied factors. There is also an opinion that IBD is namely the autoimmunity disease.In recent years, it is concluded form linkage analysis on gene groups that there are many susceptible gene sites of IBD, respectively located on the 16th (IBD1), the 12th (IBD2), 6th (IBD3) and the 14th chromosome and other chromosomes. Among white people in Europe and America, CARD15/NOD2 has been proved to be the first susceptible gene of CD. But conclusions from researches on the people groups in China and Japan are different. These researches show that the genetic susceptibility of IBD is affected by complex polygene and multi-factor. It is proved that the pathogeny of IBD is related with races and environment and other factors, which sets a theoretical basis for the researches on the discrepancy of pathogenesis among different people groups in different districts.Cellular immunity concentrated on activation of T cell plays a very important role in the pathogenesis of IBD. The clinic pathology materials show that a great number of lymphocytes infiltrating into the lamina propria of IBD pathological changes mucous membrane, and inflammatory cells plays a very important role in the development of course of disease. CTLA-4 is a member of immunoglobulin super family, which has an expression on the surface of activated T lymphocyte, and combines with B7 1 and B7 2 ligands on antigen presentation cells and then forms the second signal of lymphocyte activation. CTLA-4 gene of human beings is located on 2q33, with 3 polymorphism sites. It is proved that polymorphism of CTLA-4 gene is related with autoimmunity diseases, such as Grave disease, 1 type diabetes, myasthenia gravis, rheumatic arthritis and so on. Among others, the correlation with Grave disease and 1 type diabetes is fairly positive, and CTLA-4 has been commonly consented as the susceptible gene of the above-mentioned diseases. But research on correlation with IBO is rare in domestic and foreign countries.The researches on relationship between CTLA-4 gene micro-satellite polymorphism and IBD people of Han nationality in China can be seen in Hubei, Zhejiang, etc, which bring to a positive correlation conclusion.To conclude, selecting 137 IBD casess (123 UC, 14 CD), and 145 normal collators of Han nationality without blood lineage in Heilongjiang Province, adopting specificity allele gene PCR method to check and test 3′untranslated region of CTLA-4 exon 4 including (AT)n repetitive sequence of allele, and Heilongjiang Province and CTLA-4 gene micro-satellite polymorphism to accumulate materials for combining, controlling and researching on the research results of other domestic regions.1. Materials and methods1.1 Materials1.1.1 Subject InvestigatedIBD cases are from Affiliated Hospital of Harbin Medical University and other comprehensive hospital clinic and 137 suffers in hospital (among others: 123 UC, 14 CD). The adopted cases for research are all through the comprehensive diagnosis of clinic, laboratory, endoscope, histopathology and radiology check. Among others: there are 19 cases of males, 58 cases of females. And the average age is 41.25±13.15. The diagnosis criterion is the improved Mendeloff criteria. 145 cases of normal control group are healthy. The selected cases for checking are all people without blood lineage of Han nationality in Heilongjiang Province. Design IBD case-control study questionnaire and of IBD generics study informed consent form.1.1.2 Specimen5ml venous blood in outside cycle, EDTA anticoagulation1.1.3 Methods1. Design IBO case-control study questionnaire (to design census schedule according to different cases of CD, UC)2. Diagnosis criteria of IBD cases follow up the improved Mendeloff criteria.3. Collecting Specimens: extract DNA by protease K-phenol: take 5ml venous blood in outside cycle, EDTA anticoagulation, digest with protease K, and extract gene group DNA by phenol.4. Establish specimen file, and store the in low temperature refrigerator.5. Design of primer: Primer 1: 5'-GCCAGTGATGCTAAAGGTTG-3'Primer 2:5'-AACATACGTGGCTCTATGCA-3'6. Target gene of PCR amplification: the total volume of reaction system of PCR is 25μl, including 15.5μl sterilization double distilled water, 2.5μl 10×PCR buffer solution, the primer on each side is respectively 10pmol, 0.5μldNTPs(10mmol/L), 1U TaqDNA polymerase, 1μl (40~100ng) DNA template. Reaction condition: 94℃pre-degeneration 4 min. The reaction products will be dyed with 12% non-degeneration polyacrylamide gel electrophoresis (PAGE) and 1‰silver nitrate.7. Some products will be sequenced by Biotage Gene Company, and the sequenced specimens are taken as masculine control.8. Analysis of statistics: directly judge and read individual gene type, and confirm group representativity of the studied specimens through balance check by Hardy-Weinberg, and calculate genotypic frequency and allele frequency of diseases suffers group and normal control group. Group comparison will be checked by X2 test and exact probability test. Calculate odds ratio (odds ratio,OR)and its 95% confidence interval (confidence interval,CI); P<0.05 has outstanding meanings of statistics. And calculations will be made in SPSS 12.0 software package.Conclusions:1. 3′untranslated region of CTLA-4 exon 4 IBD suffers of Han nationality in Heilongjiang Province includes (AT)n repetitive sequence, with 20 types of allele.2. Compared with normal control group, and after re-correction by Bonferroni, among UC suffers, only 122bp allele frequency increases obviously, and the difference has meanings of statistics (P=0.0001/Pc=0.0025, OR=11.283, 95%CI: 2.369～49.327).3. Among CD suffers, 118bp, 122bp allele frequency is higher than that of normal control group. But after the correction by Bonferroni, only 122bp allele frequency increases obviously, and the difference has meanings of statistics (P=0.0003/Pc=0.0050, OR=22.115, 95%CI:3.586～108.62).Conclusion:There is an outstanding positive correlation between IBO suffers of Han nationality in Heilongjiang Province and CTLA-4 micro-satellite polymorphism.