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IL-6 gene promoter region -634C / G polymorphism and diabetic nephropathy study

Author: AnXinHuan
Tutor: SongDianPing
School: Kunming Medical College
Course: Internal Medicine
Keywords: allele specific PCR IL-6 gene polymorphism PCR-RFLP promoter region diabetic nephropathy polymerase chain reaction-restriction fragment length polymorphism trial condition
CLC: R587.2
Type: Master's thesis
Year: 2005
Downloads: 74
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Abstract


Objective To investigate the optimum conditions of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method which detected IL-6 gene promoter region -634C/G polymorphism. Methods The factors influencing polymerase chain reaction-restriction fragment length polymorphism in the site of IL-6 gene promoter region -634C/G polymorphism were studied. Results (1) With Taq polymerase amount increasing , the amplification production increased also, however ,the production didn’t rise when Taq polymerase amount >1.5U ; (2) When each of primers concentration was 0.2umol /L ,output was higher than others and less dimer of primers ; (3) With the concentration of dNTP increasing ,the amplification production increased but amplification production was fixed when dNTP concentration 120 ~200μmol /L; (4) When the concentrations of Mg2+ were 1.5~2.5mmol/L , the results were satisfied,however,the concentration of Mg2+ was 5mmol/L, the amplification production declined ; (5) When PCR amplification production amount was 4μl in enzyme digestion system ,DNA fragments after digestion with BsrBI were identified clearly; (6) Restriction enzyme amount of 2.5U could digest amplificationproduction of 4jil completely; (7) PCR productions could be cut completely if digestion time was more than 9 hours . Conclusion The optimum conditions of PCR method which amplified IL-6 gene promoter region were each of primers concentration of 0.2umol /L > Taq polymerase amount of 1.5U > dNTP concentration of 120u,mol/L> Mg~+ concentration of 2.0mmol/L . The most economic and effective system was 20jj.1 including 4u.l DNA solution and 2.5U enzyme. Enzyme digestion time was more than 9 hours.Objective To investigate the association of IL-6 gene promoter region -643C/G polymorphism with diabetic nephropathy. Methods The polymorphism of IL-6 gene promoter region -634 C/G in 246 unrelated Kunming Han people with type 2 diabetes including 88 cases with normal albuminuria (DNO group) ,93 cases with microalbuminuria (DN1 group), 65 cases with macroalbuminuria (DN2 group) and 101 normal controls (NC group)were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method.The genotype frequency, allele frequency and relative clinical data were compared between groups. Results (l)Age and systolic blood pressure (SBP) in DN1 group were significantly higher than in DNO group; Age diabetic duration systolic blood pressure (SBP) diastolic blood pressure (DBP) the level of 2 hour postprandial plasma insulin in DN2 group were significantly higher than in DNO and DN1 groups , and the body mass index(BMI) was less than in DNO and DN1 groups .the ratio of patients with hypertension was significantly higher than in DNO group. (2)The frequency of IL-6-643G/G genotype in DM (DN0+DN1+DN2) group was significantly higher than in NC group, there was statistical in the difference. However, there were no significant differences in genotype between DNO and NC groups. (3)Although there were statistic differences in genotypefrequen- cies .there were no statistic differences in allele frequencies between DNl and DNO groups. (4)The frequencies of G/G genotype and G allele in DN(DN1+DN2) group were significantly higher than in DNO group; (5)The G/G genotype frequencies and G allele frequencies in DN2 group were significantly higher than in DNl and DNO groups.(6) The urinary albumin excretion rate in G/G genotype group was significantly higher than in C/G and C/C genotype groups, There were no statistic differences in others clinical data in type 2 diabetes with different genotype.(7) Logistic regression analysis showed that IL-6 gene promoter region -643G/G genotype and diabetic duration were risk factors of DN development .Multinomial logistic analysis showed that IL-6-634G/G genotype was risk factor of DN development and progression, and the diabetic duration of over than 5 years was risk factor of DN2 development. Conclusion The IL-6 promoter region -634G/G genotype may be a genetic risk factor of DN development and progression, and its urinary albumin excretion rate was significantly higher than the others; IL-6-634G allele may be one of the risk factors in DN progression.Objective To compare the results of polymorphism of IL-6 gene promoter region -634 C/G in allele specific PCR method with the results in PCR-RFLP method. Methods The polymorphism of IL-6 gene promoter region -634 C/G in 101 subjects whose genotypes were detected by PCR-RFLP method (including 43 cases of normal group and 58 cases of diabetes group) were detected by allele specific PCR method, the results of two methods were tested and verified each other, and the two methods were assessed. Results (1) The results of CGn GG and CC genotypes were identical except one case of CC genotype in two methods. (2) Allele specific PCR method was less time consumption . speedy. Conclusion When detecting known point mutation of certain gene, allele specific PCR method will be given priority over PCR-RFLP method if mismatch of base and primers could prevent primer extension.

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CLC: > Medicine, health > Internal Medicine > Endocrine diseases and metabolic diseases > Islet disease > Diabetic coma and other complications
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