本文選題：慢性乙型肝炎　切入點(diǎn)：維生素D受體　出處：《山西醫(yī)科大學(xué)》2014年碩士論文

【摘要】：實(shí)驗(yàn)?zāi)康?通過(guò)探討維生素D受體的2號(hào)外顯子FokⅠ位點(diǎn)基因多態(tài)性與慢性乙型肝炎患者細(xì)胞免疫功能之間的關(guān)系，以更深入的了解慢性乙型肝炎的發(fā)病機(jī)理，有利于預(yù)測(cè)人感染乙肝病毒之后的臨床轉(zhuǎn)歸，同時(shí)也為改善慢性乙型肝炎的臨床治療方法提供有價(jià)值的理論依據(jù)。 實(shí)驗(yàn)方法 第一部分：收集130例山西醫(yī)科大學(xué)第一臨床醫(yī)院感染科門(mén)診及住院的慢性乙型肝炎患者的靜脈血標(biāo)本2ml(EDTA抗凝)，分裝、標(biāo)記并于-80℃冷凍保存。利用血液DNA提取試劑盒，從抗凝血液中提取基因組DNA。利用紫外分光光度儀及計(jì)算公式測(cè)定基因組DNA濃度，并將樣本稀釋、分裝保存以備用。進(jìn)行PCR特異性擴(kuò)增反應(yīng)獲取目的基因，限制性內(nèi)切酶FokⅠ對(duì)PCR產(chǎn)物進(jìn)行酶切，將酶切產(chǎn)物經(jīng)2%瓊脂糖凝膠電泳，以DL1000Marker為參考，紫外凝膠成像儀下觀察結(jié)果。 第二部分：將90例慢性乙型肝炎患者按照其不同的VDR-FokⅠ位點(diǎn)基因型進(jìn)行分組，分別為FF基因型組30例、Ff基因型組30例及ff基因型組30例，設(shè)正常對(duì)照組20例，將所有研究對(duì)象的EDTA抗凝血2mL于離心機(jī)下2000r/min離心20分鐘,收集血漿于1mL EP管中并凍存。利用ELISA試劑盒定量檢測(cè)IL-17和IL-10的水平，實(shí)驗(yàn)操作嚴(yán)格按照試劑盒說(shuō)明書(shū)進(jìn)行。 實(shí)驗(yàn)結(jié)果 第一部分：慢性乙型肝炎重度組、中度組患者VDR-FokⅠ位點(diǎn)的基因型與對(duì)照組（慢性HBsAg攜帶者）相比，差異有統(tǒng)計(jì)學(xué)意義(χ2＝30.768，P＜0.01；χ2＝22.453，P＜0.01)。慢性乙型肝炎重度組、中度組患者VDR-FokⅠ位點(diǎn)的等位基因F/f分布頻率與對(duì)照組相比，差異有統(tǒng)計(jì)學(xué)意義(χ2＝44.228，P＜0.01；χ2＝31.506，P＜0.01)，提示等位基因F/f分布頻率對(duì)慢性乙型肝炎患者的不同臨床表型存在影響。慢性乙型肝炎重度組、中度組患者f等位基因分布頻率高于對(duì)照組。 第二部分：FF基因型組、Ff基因型組及ff基因型組3組慢性乙型肝炎患者血清中IL-17、IL-10水平均顯著高于正常對(duì)照組（P＜0.01）；Ff基因型組及ff基因型組患者血清中IL-17的水平也顯著高于FF基因型組（P＜0.01），F(xiàn)f基因型組及ff基因型組患者血清中IL-10的水平低于FF基因型組（P＜0.01）。 結(jié)論 慢性乙型肝炎重度組、中度組患者的f等位基因分布頻率高于對(duì)照組（慢性HBsAg攜帶者），提示等位基因f分布頻率的提高可能與慢性乙型肝炎患者的炎癥活動(dòng)存在一定的關(guān)系，推測(cè)VDR-FokⅠ位點(diǎn)的基因多態(tài)性與慢性乙型肝炎的病情發(fā)展程度有一定的關(guān)系，具備f等位基因的慢性乙型肝炎患者其炎癥活動(dòng)的可能性更大。 通過(guò)進(jìn)一步實(shí)驗(yàn)分析，慢性乙型肝炎患者中具備f等位基因的患者,促炎因子IL-17的表達(dá)水平更高，它極可能是通過(guò)影響個(gè)體的細(xì)胞免疫功能，使此類型患者更容易出現(xiàn)免疫清除的免疫應(yīng)答情況，進(jìn)而導(dǎo)致炎癥反應(yīng)的增強(qiáng)，但同時(shí)可能會(huì)導(dǎo)致其肝損傷程度也較重。 綜上，VDR-FokⅠ基因多態(tài)性可能對(duì)慢性乙型肝炎患者的細(xì)胞免疫功能有所影響，，繼而使慢性乙型肝炎患者的臨床表型有所不同。
[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.
絎簩閮ㄥ垎錛欶F鍩哄洜鍨嬬粍,Ff鍩哄洜鍨嬬粍鍙?qiáng)ff鍩哄洜鍨嬬粍3緇勬參鎬т箼鍨嬭倽鐐庢?zhèn)ｈ�呰娓呬腑IL-17,IL-10姘村鉤鍧囨樉钁楅珮?shù)簬姝ｅ父瀵圭収缁?P錛

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