Study Of Gene Polymorphism And Serum Levels Of Il-10,tnf-α And The Their Relationship With The Etiopathogenesis Of Type 2 Diabetes Mellitus

ObjectiveEntering 21st century, people's living standards and medical treatment technic are moving to a new step. While at the same time, hospitalization rates for senile disease and chronic disease are also increasing. As one of those diseases, diabetes has become more prominent. Therefore, the scientific research of diabetes has caused widespread concern all over the world.More and more researches support the hypothesis that type 2 diabetes is a "chronic low-grade inflammatory state". Numerous clinical studies have shown that often type 2 diabetes are accompanied by changes in the concentration of many kinds of cytokines. Inflammatory markers can predict the occurrence and prognosis of type 2 diabetes. Experimental evidence also has proved that anti-inflammatory treatment or reducing the serum levels of certain inflammatory factors can improve glucose metabolism in patients with type 2 diabetes.Among lots of cytokines, the more concern were showed for IL-10 and TNF-α. IL-10 is secreted by activated monocytes-macrophages and lymphocytes and it can inhibit the production of cytokines, so it has a wide range of anti-inflammatory effect and a strong inhibition of immunity. In recent years, people has made great progress on the research for cytokine IL-10 in type 2 diabetes pathogenesis and found that the changes of its serum levels are closely linked to type 2 diabetes. Animal experiments showed that when the rats suffered type 2 diabetes, IL-10 significantly reduced. While after the injection of IL-10 expression plasmid, the incidence of diabetes reduced. Studies for human body have also found that low levels of serum IL-10 are closely linked to high blood sugar, high glycated hemoglobin (HbA1c) are closely linked. There are similar reports that IL-10 inhibits high blood sugar levels of type 2 diabetes and the serum levels of other inflammatory factors, so it may have some protective effect on the incidence of type 2 diabetes. Therefore, type 2 diabetes may be a cytokine-mediated inflammatory response or an innate immunologic disease. But the molecular and genetic mechanism of type 2 diabetes is still not clear.And the research reports of the relation between IL-10 gene promoter -592 polymorphism and type 2 diabetes also is relatively less and inconclusive.At the same time, TNF-α(tumor necrosis factor-α) as an important inflammatory factor is involved in a variety of immune and inflammatory pathological process. So, it is speculated that TNF-αmay have Promotive effect on the pathological progress of type 2 diabetes. In recent years, the study of TNF-αin type 2 diabetic complications in the pathogenesis of vascular diseases has made great progress. First, from serum levels, it was found in patients with type 2 diabetes serum levels of TNF-αwere significantly higher than that of the normal control group. A large number of experiments have described the role TNF-αacts in insulin resistance (Insulin resistance, IR) of the pathological process. Recent discovery showed that animal and human adipose tissue, muscle tissue can also secrete TNF-αand its levels were high in the tissue of the obesity and diabetes patients.After combining with the corresponding receptors on cell surface,it can affect insulin receptor phosphorylation and kinase activity levels, interfere with the signal transduction of insulin postreceptor, inhibit glucose uptake of adipocyte so as to make blood glucose and lipid metabolic abnormal. The most common complications of diabetes are microvascular and macrovascular diseases, but the pathogenesis has not been fully demonstrated.Since then, people also began to study the relation between TNF-α-308 gene polymorphism and type 2 diabetes mellitus. Since 1993 when firstly it is reported that there is G / A variant in TNF-αgene promoter region 308, domestic and foreign scholars have carried out a series of studies on the relations between TNF-α-308 gene polymorphism and insulin resistance (IR) among type 2 diabetes mellitus. But in different groups, the obtained results are different.With the development of the molecular biology and molecular genetics, a number of genes and gene regions with diabetes susceptibility have been successfully located. Meanwhile, with the deeply penetration in genetic mechanism of type 2 diabetes, it is discovered that there is great genetic heterogeneity with significant regional and national differences in type 2 diabetes. Studies in different regions and different groups the genetic susceptibility to type 2 diabetes mellitus and related factors in type 2 diabetes becomes a hot issue.This study was designed to measure the serum concentration of cytokines IL-10, TNF-αin the clinical samples so as to analyse the pathogenesy of type 2 diabetes and its various risk factors for vascular complications as well as to analyse the relationship between TNF-α-308G / A, IL-10 -592A / C polymorphism and glucose metabolism disorder of type 2 diabetes mellitus. It is hoped that this attempt of studing cytokine serum level and genotype in type 2 diabetes may provide a theoretical basis for the mechanism.Methods1 Cytokine IL-10 serum concentration and IL-10-592A / C gene polymorphism with type 2 diabetes224 cases of patients with type 2 diabetes mellitus (DM group) were new admissions to the second devision of endocrinology in Tangshan workers hospital from November 2008 to April 2009.They were in line with 1999 WHO diabetes diagnostic criteria and were excluded acute or chronic infection, acute concurrent disease, cardiovascular disease and malignant tumors and autoimmune diseases.275 cases of normal control group (NC group) were healthy persons from the health checkup medical center. The numerus of their fasting plasma glucoses were <6.1 mmo / L and postprandial 2h blood glucose were <7.8 mmol / L. They had no acute or chronic infections. And they had no heart, liver, lung, kidney and other diseases, with no family history of diabetes, no history of autoimmune diseases and long-term oral medication.Before these paitents and healthy people entered the groups, informed consent was taken. People among the selected groups had no direct blood relationship between them, no other genetic diseases and sex, age were matched. The difference of age and sex among diabetic group and control group was not statistically significant.Selected patients and people from normal control group were drawed 4ml venous blood on the following morning while they were with empty stomach. Respectively, venous blood were put into two test tubes. One tube was centrifugated 10 minutes at 4000rpm after natural coagulation. Taking the supernatant and placed it in -80℃.Using cryopreservation enzyme-linked immunosorbent assay (ELISA) method to measure the serum concentrations of IL-10. Another tube was with EDTA anticoagulant. Genomic DNA was extracted through DNA extraction kit and it is stored at 4℃.With Rsa I as restriction enzymes, polymerase chain reaction-restriction fragment polymorphisms assay(PCR-RFLP) was applied to analyse IL-10 promoter region -592 gene polymorphism of DNA samples. T test andχ2 test were used by the statistical software SPSS13.0 to compare the different numbers between two groups in order to find whether the difference was significant.2 Relationship between the serum concentration of cytokine TNF-αand type 2 diabetes mellitus as well as its relationship with vasculopathyThis part of the study was to investigate the relationship between TNF-αand type 2 diabetes mellitus by observing serum content of the different levels of the type 2 diabetic group and normal control group, to analyse the relation of type 2 diabetes with vascular complications by observing the TNF-αserum levels among the different vascular lesions groups of type 2 diabetes mellitus. At the same time,analysis of various biochemical parameters between different groups was taken.(1) According to whether there was microvascular disease, type 2 diabetic subjects were divided into 107 cases of microvascular complications group (MiVC) and 117 cases of non- microvascular complications group (Non-MiVC).(2) According to whether there was macrovascular disease, type 2 diabetic subjects were divided into 120 cases of group with macrovascular disease(macrovascular complications, MaVC) and 104 cases of group not with macrovascular disease (Non-MaVC). Subjects fasted after 22:00 on the eve of blood collection and Next Morning they were drawed 4ml elbow venous blood which was centrifugated 10 minutes at 4000rpm after natural coagulation. Supernatant was taken and placed in -80℃cryopreservation. Using enzyme-linked immunosorbent assay (ELISA) to measured the serum levels of TNF-α. T test was used by the statistical software SPSS13.0 to compare the serum levels of TNF-αin different groups so as to find whether there were significant differences.3 TNF-α-308G / A gene polymorphism with type 2 diabetes pathogenesisThis part of study was devised to detect the different distribution of TNF-α-308G / A polymorphism in T2DM group the normal control group so as to explore the potential relationship with pathogenesis of type 2 diabetes. 4ml blood was collected with EDTA anticoagulant and using DNA extraction kit to extract genomic DNA which was stored at 4℃temperature. Using Nco I enzyme as the restriction endonuclease and through polymerase chain reaction-restriction fragment polymorphisms assay (PCR-RFLP) to analyse TNF-αpromoter -308 site gene polymorphism of DNA samples.Results1 Cytokine IL-10 serum concentration and IL-10-592A / C gene polymorphism with type 2 diabetes pathogenesisThe average serum level of IL-10 in type 2 diabetes group was 21.12±5.17 pg / mL while control group was 42.35±6.30 pg / mL. The difference between the two groups was statistically significant (P<0.01). The analysis of relations of type 2 diabetes susceptibility with IL -10-592 genotype and gene frequency showed that comparisons of IL-10-592 genotype and allele frequencies in type 2 diabetes group and control group were significant different (P <0.01). 2 Cytokines TNF-αserum concentration with type 2 diabetes mellitus and its relationship with vascular lesionThe different comparison of biochemical indicators of blood lipids between groups shows that:type 2 diabetes and vascular lesions groups compared with the control group, BMI, TC, TG, LDL-C were significantly higher than those of the normal control group, while HDL-C significantly lower than the normal control group; microangiopathy group and non-microangiopathy group, BMI, TC, TG, LDL-C were significantly higher than the normal control group, while HDL-C significantly lower than the normal control group; macroangiopathy group and the non-macroangiopathy group, BMI, TC, TG, LDL-C were significantly higher than the normal control group, while HDL-C significantly lower than the normal control group; macroangiopathy group and microangiopathy group, BMI, TC , LDL-C were significantly higher than that of microvascular disease group, while HDL-C significantly lower than that of microvascular lesions.TNF-αserum levels in different vascular lesions groups of type 2 diabetes and control showed: type 2 diabetes patient group serum levels of TNF-αwas 56.26±11.34pg/mL, the control group was 28.97±8.89pg/mL, the non-microangiopathy group was 49.73±10.35 pg / mL, microangiopathy group was 59.56±12.41 pg / mL, non- macroangiopathy group was 48.53±11.25pg/mL, macroangiopathy group was 61.76±9.47pg/mL. Compared with normal control group, serum levels of each group with type 2 diabetes were significantly higher(P<0.01); compared with non-microangiopathy group, serum levels of microangiopathy were significantly higher(P<0.01); compared with non-macrovascular disease group, serum level of TNF-αin macrovascular group were significantly higher (P<0.01); while there was no significant difference between macrovascular disease group and microvascular disease group (P> 0.05).3 TNF-α-308G/A gene polymorphism with type 2 diabetes pathogenesis Of GA+AA genotype frequency in T2DM group and the Control group, no significant difference was found between the two groups (P>0.05); of G, A allele frequency between the two groups there was no significant difference (P> 0.05), that is , according to genotype frequencies and gene frequencies of SNP-308 polymorphic location between the two groups, no statistically significant was showed (P> 0.05).Conclusion1 The difference of IL-10 and TNF-αserum levels between diabetic group and normal control group supports the hypothesis that type 2 diabetes mellitus is a "chronic low-grade inflammatory state" and indicats that its pathogenesis may be related to many inflammatory cytokines. Among them IL-10 can inhibit cytokine production, being with a wide range of anti-inflammatory effect and a strong immune inhibition.So the decrease in serum levels of IL-10 may have contributed to the development of "low-grade chronic inflammatory state". Thus IL-10 loses the protective effect in type 2 diabetes. Through analysis of the qualified cases in Tangshan area, this study showed that it may exist a certain correlation between the IL-10-592 polymorphism and susceptibility to type 2 diabetes and that IL-10 gene polymorphism may be one of the factors with susceptibility to type 2 diabetes and that it is also a closely related rehabilitation for the prognosis of type 2 diabetes.2 TNF-α,as an important inflammatory factor, for the difference serum levels between diabetic group and normal control group, may be involved in the pathogenesis of type 2 diabetes. The significantly higher levels of TNF-αin T2DM macroangiopathy group than that of not macroangiopathy group as well as the microangiopathy group than not microangiopathy group indicats that TNF-αis a risk factors in the vascular complications of type 2 diabetes. Its effect may be acted through insulin resistance for the promotion of type 2 diabetes mellitus as well as the occurrence and development of vascular lesions.3 But for the study of genotype TNF-α-308, no significant difference was found. reference documents showed different views for the findings of genetic polymorphisms,of which some people get positive results. Considering that type 2 diabetes after all is a multi-gene-related disease and its etiology is related to racial heredity, environment, regional and many other factors. The results of this study is only from the perspective of inflammatory factors and restricted to the crowd of Tangshan area. If expand the scope of the crowd and increase the sample size, the results obtained will be more persuasive.