本文選題：CYP4F2 + 基因��； 參考：《中國人民解放軍醫(yī)學院》2017年碩士論文

【摘要】：背景:盡管新型口服抗凝藥已經(jīng)問世,華法林作為一線口服抗凝藥仍然在臨床廣泛應用;由于應用華法林抗凝治療存在嚴重出血并發(fā)癥的風險,因此,如何安全有效的個體化應用華法林抗凝治療仍然是臨床亟待解決的難題。華法林藥物基因組學研究發(fā)現(xiàn),除臨床環(huán)境因素外,華法林代謝和作用通路上的基因遺傳變異很大程度上影響了華法林的療效和副作用。細胞色素P450家族成員CYP4F2具有氧化水解維生素K的功能,因此,編碼基因CYP4F2的基因變異可能通過改變維生素K循環(huán)直接參與凝血因子的活化,進而影響華法林的抗凝效果和出血并發(fā)癥發(fā)生。然而,目前尚缺乏關于CYP4F2變異型與華法林出血并發(fā)癥相關聯(lián)及其功能機制的報道。第一部分:CYP4F2候選基因變異與華法林嚴重出血并發(fā)癥的關聯(lián)性和生物信息功能預測分析:隊列研究目的:篩查并驗證與華法林主要出血并發(fā)癥相關聯(lián)的CYP4F2候選基因變異型。方法:前瞻性分階段連續(xù)募集解放軍總醫(yī)院首次接受華法林抗凝治療的患者,并進行為期至少3個月的隨訪。利用Hapmap和NCBIdsSNP數(shù)據(jù)庫以及Haploview軟件篩查CYP4F2的候選標簽SNPs,通過Snapshot方法對所有入選患者的外周血DNA進行基因分型,利用logistic回歸篩查并驗證與華法林主要出血事件相關聯(lián)的CYP4F2基因變異型。利用生物信息學分析尋找具有潛在生物功能的CYP4F2出血關聯(lián)變異型。結果:本課題組于2008年1月至2011年9月期間和2014年1月至2015年12月期間,分別連續(xù)募集了符合入選標準并完成隨訪的312例和241例華法林抗凝治療患者,前期收集病例作為篩查組,后期收集病例作為驗證組。所有入選患者中包括:房顫410例(74.14%),瓣膜置換術后106例(18.63%);肺栓塞12例(2.17%),靜脈血栓18例(3.25%),其他患者9例(1.63 %)。篩選出并成功行基因分型檢測的CYP4F2標簽SNPs總計8個,經(jīng)logistic回歸分析發(fā)現(xiàn),篩查組中CYP4F2rs3093168 CC基因型攜帶者主要出血事件的發(fā)生風險明顯高于CT+TT 基因型攜帶者(7.79% vs.2.13%, OR:5.39,95% CI:1.28-23.12,p=0.02)。驗證組中,上述相關性依然存在(6.45% vs. 1.68%,OR:7.93,95%CI:1.03-61.06,p=0.04)。在所有病例中,CYP4F2rs3093168CC 基因型攜帶者主要出血事件的發(fā)生風險同樣明顯高于CT和TT基因型攜帶者者7.19%vs.1.93%, OR:5.23,95%CI:1.78-15.39,p=0.003 )。通過連鎖不平衡分析發(fā)現(xiàn),CYP4F2啟動子上游第44,244堿基處存在與rs3093168相連鎖的SNP位點rs2079288。利用 SNPalyze V4.0 軟件分析發(fā)現(xiàn),rs2079288 處于 linc RNA(NONHSAGO25013.2 )編碼區(qū),其功能機制可能與lincRNA通過cis調(diào)控CYP4F2基因表達有關。結論:CYP4F2rs3093168 CC基因型與華法林嚴重出血并發(fā)癥密切相關,生物信息分析發(fā)現(xiàn)其功能機制可能與影響CYP4F2基因表達調(diào)控有關。第二部分:CYP4F2目標基因再測序和生物信息功能分析篩查與華法林嚴重出血并發(fā)癥相關聯(lián)的基因變異型:病例對照研究目的:通過CYP4F2目標基因再測序和生物信息功能分析篩查與華法林嚴重出血并發(fā)癥相關聯(lián)的基因變異型。方法:在前瞻性連續(xù)募集的解放軍總醫(yī)院首次接受華法林抗凝治療的患者中,選取華法林主要出血病例及與之相匹配的未發(fā)生出血病例,對CYP4F2基因進行再測序。對所有基因變異型進行最小等位基因頻率計算及Hardy-Weiberg平衡檢驗。利用logistic回歸分析篩查出的CYP4F2基因變異型與主要出血事件的相關性,并利用生物信息學分析尋找具有潛在生物功能的CYP4F2出血關聯(lián)變異型。結果:再測序分析總計發(fā)現(xiàn)了 64個CYP4F2基因變異型,其中12個為首次發(fā)現(xiàn)的新的基因變異,以上基因變異型均符合Hardy-Weiberg平衡。新發(fā)現(xiàn)的一處位于CYP4F2第3內(nèi)含子區(qū)的插入缺失變異CYP4F2-1705 ins AGAT -1733 del CAGA僅見于嚴重出血病例中,發(fā)生率為22.22%。Mutation Taster軟件分析該插入缺失變異位于第3個內(nèi)含子和外顯子剪接位點下游,并可能通過影響mRNA的剪接從而影響基因表達。結論:CYP4F2-1705 ins AGAT-1733 del CAGA與華法林嚴重出血并發(fā)癥密切相關,生物信息分析發(fā)現(xiàn)其功能機制可能與影響CYP4F2基因轉錄有關。第三部分:華法林嚴重出血并發(fā)癥相關聯(lián)的CYP4F2基因變異型的體外生物功能分析目的:對華法林主要出血相關聯(lián)CYP4F2基因變異型進行體外生物功能分析。方法:募集健康男性志愿者,提取外周全血DNA,對嚴重出血相關聯(lián)基因變異(rs3093168 及 CYP4F2-1705 ins AGAT-1733 del CAGA )進行基因分型,篩選人口統(tǒng)計學和其他遺傳背景相匹配的主要出血相關基因型攜帶者和非攜帶者。提取白細胞mRNA,利用qRT-PCR分析基因變異對CYP4F2基因轉錄的影響;提取血漿,利用CYP4F2 Elisa試劑盒分析基因變異對CYP4F2蛋白表達影響,利用FIXaAssay試劑盒分析基因變異對FIXa活化的影響。利用pIRES-FIX質(zhì)粒構建pIRES-FIX-CYP4F2共轉染質(zhì)粒并酶切鑒定。將pIRES-FIX質(zhì)粒,pIRES-FIX-CYP4F2質(zhì)粒以及空白對照質(zhì)粒分別轉染L02細胞,western-blot檢測CYP4F2及FIX的蛋白表達。利用FIXa檢測試劑盒檢測CYP4F2對FIX羧化功能的影響。結果:rs3093168 CC基因型與TT+CT基因型攜帶者的CYP4F2mRNA表達水平無明顯差異,CYP4F2-1705 ins AGAT-1733 del CAGA插入缺失變異基因型攜帶者較非攜帶者CYP4F2mRNA表達水平明顯升高。rs3093168CC基因型攜帶者及 CYP4F2-1705 ins AGAT-1733 del CAGA rs3093168 基因型攜帶者 CYP4F2蛋白表達水平較對照組均明顯升高。FIXa活性檢測發(fā)現(xiàn),rs3093168 CC基因型攜帶者以及CYP4F2-1705 ins AGAT-1733 del CAGA插入缺失變異攜帶者的FIXa活性較對照組明顯降低。轉染相應質(zhì)粒后,CYP4F2及FIX的蛋白成功過表達。在過表達FIX的L02細胞培養(yǎng)上清和細胞裂解液中,FIXa活性均較對照組顯著升高;同時過表達CYP4F2和FIX后,L02細胞培養(yǎng)上清和細胞裂解液中的FIXa活性較單獨過表達FIX組明顯降低。結論:與華法林主要出血并發(fā)癥相關的rs3093168 CC基因變異型及CYP4F2插入缺失變異可能通過增加CYP4F2蛋白表達,抑制凝血因子活化,導致變異攜帶者服用華法林的出血風險增加。
[Abstract]:Background: Although the new oral anticoagulants have come out, Hua Falin is still widely used as a first-line anticoagulant in the clinic. Because of the risk of severe bleeding complications in the application of Hua Falin anticoagulant therapy, how to apply Hua Falin anticoagulant therapy safely and effectively is still a difficult problem to be solved in clinic. Hua Falin drug is a difficult problem. Genomic studies have found that, in addition to the clinical environmental factors, the genetic variation in Hua Falin's metabolic and action pathways greatly affects the efficacy and side effects of Hua Falin. The cytochrome P450 family member CYP4F2 has the function of oxidative hydrolysis of vitamin K, so the gene mutation of the CYP4F2 gene may be altered by the change of vitamin K Circulation directly participates in the activation of coagulation factors and further affects the anticoagulant effect and bleeding complications of Hua Falin. However, there is still a lack of reports on the association of the CYP4F2 variant with the complications of Hua Falin bleeding and its functional mechanism. Part 1: the association and biology of the variant of the CYP4F2 candidate gene and the complications of Hua Falin's severe bleeding. Information functional prediction analysis: cohort study objective: to screen and verify the CYP4F2 candidate gene variant associated with the major bleeding complications of warfarin. Methods: a prospective, continuous recruitment of warfarin anticoagulants for the first time in the General Hospital of the Liberation Army, and a follow-up of at least 3 months. Use of Hapmap and NCBIdsSNP data. The library and the Haploview software screened the candidate label SNPs for CYP4F2. The Snapshot method was used to genotyping the peripheral blood DNA of all selected patients. The logistic regression was used to screen and verify the CYP4F2 gene variant associated with the major bleeding events of the warfarin. The bioinformatics analysis was used to find the CYP4F2 out of the potential biological function. Results: during the period from January 2008 to September 2011 and January 2014 to December 2015, we collected 312 cases and 241 cases of warfarin anticoagulant treatment, respectively, which met the criteria of admission and completed the follow-up. The early collection cases were selected as the screening group and the latter period was collected as the verification group. There were 410 cases of atrial fibrillation (74.14%), 106 cases (18.63%) after valve replacement, 12 cases of pulmonary embolism (2.17%), 18 cases of venous thrombosis (3.25%) and 9 cases (1.63%) of other patients (1.63%). A total of 8 CYP4F2 tags were selected and successfully detected by genotyping, and the main bleeding events of the CYP4F2rs3093168 CC genotype carriers in the screening group were found to be the main bleeding events in the screening group. The risk of occurrence was significantly higher than that of CT+TT genotype carriers (7.79% vs.2.13%, OR:5.39,95% CI:1.28-23.12, p=0.02). In the verification group, the above correlation still existed (6.45% vs. 1.68%, OR:7.93,95%CI:1.03-61.06, p=0.04). In all cases, the risk of major bleeding events in the CYP4F2rs3093168CC genotype carriers was also significantly higher than that of C. T and TT genotype carriers 7.19%vs.1.93%, OR:5.23,95%CI:1.78-15.39, p=0.003). Through linkage disequilibrium analysis, it was found that the SNP site linked to the rs3093168 phase in the 44244th base of the CYP4F2 promoter was found by the SNPalyze V4.0 software analysis of the rs3093168 phase. The mechanism may be related to the lincRNA regulation of CYP4F2 gene expression through CIS. Conclusion: the CYP4F2rs3093168 CC genotype is closely related to the severe bleeding complications of warfarin. Bioinformatics analysis found that its functional mechanism may be related to the regulation of CYP4F2 gene expression. The second part: CYP4F2 target gene re sequencing and biological information functional analysis. Screening and genetic variants associated with severe warfarin bleeding complications: a case-control study: screening of the gene variants associated with the complications of warfarin bleeding through CYP4F2 target gene sequencing and bioinformatic functional analysis. Methods: warfarin anticoagulants were first accepted in the PLA General Hospital of prospective continuous recruitment. The CYP4F2 gene was re sequenced in the main bleeding cases of warfarin and the non bleeding cases matched with them. The minimum allele frequency and Hardy-Weiberg balance test of all gene variants were performed. The CYP4F2 gene variant was screened by logistic regression and the major bleeding events were screened by logistic regression analysis. Correlation, and using bioinformatics analysis to find the associated variant of CYP4F2 bleeding associated with potential biological function. Results: 64 CYP4F2 gene variants were found by re sequencing analysis, of which 12 were new genetic variants found for the first time, and all of the above variants were in line with Hardy-Weiberg balance. A new site was found in CYP4F2 third. The insertion deletion mutation CYP4F2-1705 ins AGAT -1733 del CAGA in the intron is only in severe bleeding cases, the occurrence rate is 22.22%.Mutation Taster software analysis that the insertion deletion mutation is located downstream of the third introns and exon splicing sites, and may affect the gene expression by affecting the shear splicing of mRNA. Conclusion: CYP4F2-1705 in S AGAT-1733 del CAGA is closely related to the severe bleeding complications of warfarin. Bioinformatics analysis found that its functional mechanism may be related to the impact of CYP4F2 gene transcription. The third part: in vitro biofunctional analysis of the CYP4F2 gene variant associated with the severe hemorrhage complications of warfarin: related to the main bleeding phase of the warfarin CYP4F2 base Methods: in vitro biological function analysis of the variant. Methods: the healthy male volunteers were collected, the peripheral blood DNA was extracted, the gene mutation (rs3093168 and CYP4F2-1705 ins AGAT-1733 del CAGA) of the severe hemorrhage phase was genotyped, and the major haemorrhagic related genotype carriers, which matched the demographic and other genetic scenes, were screened. Non carrier. Extract leukocyte mRNA, use qRT-PCR to analyze the effect of gene mutation on CYP4F2 gene transcription; extract plasma, use CYP4F2 Elisa kit to analyze the effect of gene mutation on the expression of CYP4F2 protein, analyze the effect of gene mutation on FIXa activation by FIXaAssay kit. Use pIRES-FIX plasmid to construct pIRES-FIX-CYP4F2 co transfection. Plasmid and enzyme digestion were identified. PIRES-FIX plasmid, pIRES-FIX-CYP4F2 plasmid and blank control plasmid were transfected into L02 cells respectively. The protein expression of CYP4F2 and FIX was detected by Western-blot. The effect of CYP4F2 on FIX carboxylic function was detected by FIXa detection kit. Results: the expression level of rs3093168 CC genotype and TT+CT genotype carrier. There was no significant difference between the CYP4F2-1705 ins AGAT-1733 del CAGA insertion mutant genotype carriers and the CYP4F2mRNA expression level of the non carriers significantly higher.Rs3093168CC genotype and CYP4F2-1705 ins AGAT-1733. It was found that the rs3093168 CC genotype carriers and the FIXa activity of the CYP4F2-1705 ins AGAT-1733 del CAGA insertion deletion carriers were significantly lower than those of the control group. The proteins of CYP4F2 and FIX were overexpressed successfully after the transfection of the corresponding plasmid. At the same time, after overexpression of CYP4F2 and FIX, the activity of FIXa in L02 cell culture supernatant and cell lysate was significantly lower than that in FIX group. Conclusion: the rs3093168 CC gene variant and CYP4F2 insertion deletion mutation associated with the major bleeding complications of warfarin may be induced by increasing the expression of CYP4F2 protein and inhibiting the activation of coagulation factor. Carriers with warfarin had a higher risk of bleeding.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R973.2

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