慢乙肝患者VDR-FokⅠ基因多态性及其对细胞免疫功能影响的研究

发布时间：2018-03-31 00:26

  本文选题：慢性乙型肝炎　切入点：维生素D受体　出处：《山西医科大学》2014年硕士论文

【摘要】：实验目的 通过探讨维生素D受体的2号外显子FokⅠ位点基因多态性与慢性乙型肝炎患者细胞免疫功能之间的关系，以更深入的了解慢性乙型肝炎的发病机理，有利于预测人感染乙肝病毒之后的临床转归，同时也为改善慢性乙型肝炎的临床治疗方法提供有价值的理论依据。 实验方法 第一部分：收集130例山西医科大学第一临床医院感染科门诊及住院的慢性乙型肝炎患者的静脉血标本2ml(EDTA抗凝)，分装、标记并于-80℃冷冻保存。利用血液DNA提取试剂盒，从抗凝血液中提取基因组DNA。利用紫外分光光度仪及计算公式测定基因组DNA浓度，并将样本稀释、分装保存以备用。进行PCR特异性扩增反应获取目的基因，限制性内切酶FokⅠ对PCR产物进行酶切，将酶切产物经2%琼脂糖凝胶电泳，以DL1000Marker为参考，紫外凝胶成像仪下观察结果。 第二部分：将90例慢性乙型肝炎患者按照其不同的VDR-FokⅠ位点基因型进行分组，分别为FF基因型组30例、Ff基因型组30例及ff基因型组30例，设正常对照组20例，将所有研究对象的EDTA抗凝血2mL于离心机下2000r/min离心20分钟,收集血浆于1mL EP管中并冻存。利用ELISA试剂盒定量检测IL-17和IL-10的水平，实验操作严格按照试剂盒说明书进行。 实验结果 第一部分：慢性乙型肝炎重度组、中度组患者VDR-FokⅠ位点的基因型与对照组（慢性HBsAg携带者）相比，差异有统计学意义(χ2＝30.768，P＜0.01；χ2＝22.453，P＜0.01)。慢性乙型肝炎重度组、中度组患者VDR-FokⅠ位点的等位基因F/f分布频率与对照组相比，差异有统计学意义(χ2＝44.228，P＜0.01；χ2＝31.506，P＜0.01)，提示等位基因F/f分布频率对慢性乙型肝炎患者的不同临床表型存在影响。慢性乙型肝炎重度组、中度组患者f等位基因分布频率高于对照组。 第二部分：FF基因型组、Ff基因型组及ff基因型组3组慢性乙型肝炎患者血清中IL-17、IL-10水平均显著高于正常对照组（P＜0.01）；Ff基因型组及ff基因型组患者血清中IL-17的水平也显著高于FF基因型组（P＜0.01），Ff基因型组及ff基因型组患者血清中IL-10的水平低于FF基因型组（P＜0.01）。 结论 慢性乙型肝炎重度组、中度组患者的f等位基因分布频率高于对照组（慢性HBsAg携带者），提示等位基因f分布频率的提高可能与慢性乙型肝炎患者的炎症活动存在一定的关系，推测VDR-FokⅠ位点的基因多态性与慢性乙型肝炎的病情发展程度有一定的关系，具备f等位基因的慢性乙型肝炎患者其炎症活动的可能性更大。 通过进一步实验分析，慢性乙型肝炎患者中具备f等位基因的患者,促炎因子IL-17的表达水平更高，它极可能是通过影响个体的细胞免疫功能，使此类型患者更容易出现免疫清除的免疫应答情况，进而导致炎症反应的增强，但同时可能会导致其肝损伤程度也较重。 综上，VDR-FokⅠ基因多态性可能对慢性乙型肝炎患者的细胞免疫功能有所影响，，继而使慢性乙型肝炎患者的临床表型有所不同。
[Abstract]:Experimental purpose
Significant relationship between cells in patients with gene polymorphism and chronic hepatitis B immune function by Fok I site in exon 2 of the vitamin D receptor, with a deeper understanding of pathogenesis of chronic hepatitis B, is conducive to clinical prediction after people infected with hepatitis B virus outcome, as well as for clinical treatment to improve chronic hepatitis B provide valuable theoretical basis.
Experimental method
The first part: to collect 130 cases of the first clinical hospital of Shanxi Medical University Department of infection in outpatients and inpatients with chronic hepatitis B of the venous blood samples of 2ml (EDTA, packaging, labeling and anticoagulant) at -80 deg.c for cryopreservation. Extraction Kit by DNA from blood, anticoagulated blood extracted genomic DNA. by UV determination of genomic DNA concentration UV spectrophotometer and the calculation formula, and sample dilution, packing and storing to spare. PCR specific amplification reaction to obtain the target gene, restriction endonuclease Fok of PCR products were digested, the digestion products by 2% agarose gel electrophoresis, with DL1000Marker as the reference, observation results of UV transilluminator.
The second part: 90 cases of chronic hepatitis B patients according to the different VDR-Fok I genotype groups were FF genotype Ff genotype group 30 cases, group 30 cases and FF genotype group in 30 cases, with 20 cases of normal control group, all subjects EDTA anticoagulant 2mL in centrifugal machine 2000r/min centrifugal 20 minutes to 1mL EP in plasma was collected and frozen. By using the ELISA kit for quantitative detection of IL-17 and IL-10 level of experimental operation in strict accordance with the kit.
experimental result
The first part: severe chronic hepatitis B group, genotype VDR-Fok patients with moderate group and control group I locus (chronic HBsAg carriers) compared to the difference was statistically significant (x2 = 2 30.768, P < 0.01; x 2 = 22.453, P < 0.01). Severe chronic hepatitis B group, moderate group VDR-Fok I site the allele frequency distribution of F/f compared with the control group, the difference was statistically significant (x2 = 2 44.228, P < 0.01; x 2 = 31.506, P < 0.01). The effects of different clinical phenotypes suggest that F/f allele frequency distribution of chronic hepatitis B patients. Chronic hepatitis B patients with severe group, moderate group the f allele frequency was higher than the control group.
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