【作者】 郑宏宇；

【导师】 赵学凌；

【作者基本信息】 昆明医科大学 ， 外科学， 2012， 博士

【摘要】 本课题就整合素基因家族(Integrin family, ITG family)在深静脉血栓(Deep Venous Thrombosis, DVT)形成中作用,对DVT模型大鼠静脉壁组织、模型兔和人血细胞中与深静脉血栓密切相关的ITG基因家族成员mRNA的表达变化和作用作出初步研究。目的：1.以钳夹大鼠股静脉方法构建DVT大鼠模型,采集大鼠不同血栓形成状态下的股静脉壁组织,提取mRNA后用cDNA基因芯片筛选与DVT形成密切相关,呈差异表达的关键基因。2.摸索建立深静脉血栓兔模型方法,彩色多普勒超声确定血栓形成状态,在不同状态下同期抽取血液,提取mRNA并对在大鼠模型中所筛出的整合素基因家组成员Integrin αV和Integrin β1等在不同血栓形成状态下的表达变化进行检测。3.对不同分组的人血液样本,提取mRNA检测整合素基因Integrin aV和Integrin β3等在人血中mRNA的表达变化,探讨不同血栓形成状态下基因的表达变化与DVT的相关性。方法：1.60只SD大鼠随机分为对照组(10只)和模型组(50只),对模型组采用采用蚊式钳钳夹双侧股静脉联合石膏制动双下肢构建大鼠DVT模型。不同时间点(造模后2.5h和25h)处死大鼠,解剖并游离股静脉、观察血栓的发生,造模后2.5h随机选取10只处死,纳入血栓形成前组,25h后处死余下40只大鼠,根据血栓形成与否,进而分为血栓形成组和血栓不形成组,取大鼠股静脉组织以Trizol一步法提取mRNA,查阅PubMed和GeneCard数据库,以cDNA基因芯片检测与DVT形成相关基因表达变化,对检测结果通过FC倍数分析法筛选显著差异表达的基因。2.采用小切口股静脉近心端结扎法构建DVT兔模型,10只造模兔和5只空白对照兔,分别于造模后1d、2d、3d、8d以血管彩色多普勒超声检查确定DVT形成状态,同期采集不同血栓形成状态下的血样,用RNeasy Protect Animal Blood Kit试剂盒提取兔血mRNA, PCR扩增目的基因,琼脂糖凝胶电泳检测其表达变化,以Bio-Rad自带半定量分析软件Quantity one4.6分析电泳结果,经统计分析作出结论。3.采集人血并依据诊断分为DVT组、血栓未形成组和健康对照组,用Paxgene blood RNA kit试剂盒方法提取mRNA,对动物模型中呈差异表达的Integrin αV和Integrin03以及无差异表达的整合素基因家族成员在不同组的表达变化进行验证,测序证实PCR所扩增基因准确,并以real-time PCR定量检测,通过统计学统计分析不同组间差异。对于临床一般资料、血细胞检验、血生化检验、凝血指标和彩色多普勒超声检查等的结果进行搜集、统计,结合整合素基因表达检测结果进行综合分析。结果：1.DVT大鼠模型中Integrin aV和Integrin β3基因在血栓形成组表达量较血栓形成前组、血栓不形成组和空白对照组增高大于3.2倍。2.小切口暴露股静脉近端并结扎的DVT兔模型造模方法术后DVT形成率高,术后彩色多普勒超声可准确判断血栓形成、不形成、消退和血栓再通等不同状态。3.DVT兔模型于造模后2d原血栓形成静脉出现血流信号,血栓有部分再通。4.DVT兔模型中Integrin aV在血栓形成后的表达较造模前及假手术组呈显著增高。5.PCR电泳检测半定量分析及Real-time PCR定量检测Integrin aV和Integrin (33在血栓组的表达显著高于血栓未形成组和健康对照组,血栓未形成组的表达量与健康对照组无差别；其余整合素家族基因在各组间的表达未表现出差异性。结论：1.大鼠DVT形成后股静脉壁组织中整合素基因Integrin aV和Integrin (33存在高表达。2.从兔及人血液样本中提取mRNA检测基因表达变化具有可操作性和可靠性。3.采用小切口股静脉近端结扎法可以成功构建DVT兔模型,同时可以利用彩色多普勒超声实时监测DVT兔模型股静脉血栓的不同形成状态。4.DVT兔模型在深静脉血栓形成早期(<3天)就已经开始有血栓再通。5.整合素基因Integrin αV, Integrin β3在人DVT形成后呈现高表达,结合Integrin αVβ3在冠心病和肿瘤血管生成中的研究,aVβ3可能在DVT的形成、消退和再通过程中参与了炎性细胞募集和新生血管再通血栓的过程。更多还原

【Abstract】 Through experiments on DVT rat model, DVT rabbit model and human blood, initial research was made in ITG family members who have close relationship with DVT.Objective:1. Establish DVT rat model, gather rat femoral vein tissue in different thrombosis states, then extract mRNA and screen differencial expressed key genes that have close relationship with DVT by cDNA gene chips.2. Explor the DVT rabbit model built method and try to detect model blood vessels with color Dopplor ultrasonography; gather blood samples in different thrombosis states comfirmed with ultrasound, then extract mRNA and detect the ITG genes Integrin aV, Integrin β1and etc. expression changes on different thrombosis status, which screened in rat DVT model.3. Gather blood samples and detect the expression changes of ITG members like Integrin aV and Integrin P3and etc. in DVT group, non-thrombosis group and healthy control, then explor the relationship of gene expression changes and thrombosis states.Method:1.60SD rats were randemely divided into control group (n=10) and model group (n=50). Model was built by clamp both femoral veins with masquito clamp in combination with plaster. Kill model rats in different time (2.5h and25h after model built), dissect and make femoral vein free, observing thrombosis incidence, then divided the model group into pre-thrombosis group (2.5h after model built), thrombosis group (25h after model built) and non-thrombosis group(25h after model built). Gather rat vein tissue and extract mRNA with Trizol one-step method, then search data in PubMed and GeneCard and detect the expression changes of genes that have close relationship with DVT by cDNA gene chips screen, screening significant differential expressed genes by FC mulitiple analysis method.2. Establish DVT rabbit model by small incision and ligation in proximal part method, then comfirm thrombosis state by color Dopplor ultrasonography and gather blood sample with coincident state. Extract mRNA from rabbit blood with RNeasy Protect Animal Blood Kit, then amplificate object genes and the expression changes were detected by agarose gel electrophoresis, semiquantitative analyzing electrophoresis result by Bio-Rad software Quantity one4.6and making conclusion through statistical analysis.3. Gather human blood samples and divided into DVT group, non-thrombosis group and healthy control group according to diagnosis. Extracte mRNA with Paxgene blood RNA kit, validate Integrin αv and Integrin P3genes that differential expressioned in previous animal models, verify PCR result by sequencing, and quantitatively detected by real-time PCR, then analyze the differences among the groups. Gather clinical data including the results of complete blood count, blood biochemistry, blood coagulation, color Doppler ultrasonography and etc, and then make statistical analysis.Result:1. The expressions of gene Integrin aV and Integrin β3were remarkablely higher in thrombosis group than those in pre-thrombosis, non-thrombosis and control group (>3.2folds) in rat model.2. Small incision expose and ligation of femoral vein in proximal part has a high DVT incidence, and rabbit blood flow, thrombosis formation, regression and thrombosis recanalization can be monitored by color Doppler ultrasonography.3. There is blood flow signals illuminate thrombosis reanalization2days after model built.4. The expression of Integrin aV mRNA was highly elevated after thrombosis than that before model built and sham operation group in rabbit model. 5. Gene expression changes of Integrin aV and Integrin β33were detected by PCR amplificate and electrophoresis, as well as real-time PCR. The gene expressions in DVT group were higher than non-thrombosis group and control group, and there is no difference between non-thrombosis and control group. The rest ITG family members have no expression difference among three groups.Conclusion:1. ITG genes Integrin aV and Integrin β33have a high expression in femoral vein tissues when rat DVT formation.2. Extracting mRNA from rabbit and human blood has maneuverability and accuracy.3. DVT rabbit model can be successfully built by small incision and femoral vein ligation in proximal part, and color Doppler ultrasonography can be used for real time monitoring femoral vein thrombosis states in rabbit model.4. Recanalization has been stared from early period (<3d) of thrombosis in DVT rabbit model.5. αVβ3may play role in recruit inflammatory cells and thrombosis recanalization.更多还原