【作者】 周为民；

【导师】 吴浩荣；

【作者基本信息】 苏州大学 ， 外科学， 2010， 博士

【摘要】 研究背景深静脉血栓形成(deep vein thrombosis, DVT)是一种严重的,具有潜在危险的疾病,如未得到适当治疗易发展为血栓形成后综合征、股青肿、股白肿甚至发生肺栓塞而死亡。流行病学研究提示,深静脉血栓形成年发病率为1.60‰～1.82‰。回顾性研究资料报道,静脉血栓栓塞症患者有高达5%-23%的死亡率,即使正规服用抗凝剂的有症状患者,死亡率也有1%-2%。而深静脉血栓形成患者有三分之一发生血栓形成后综合征,肺栓塞患者4%-5%发生肺动脉高压。此外,深静脉血栓形成的治疗效果又不确切。因此,研究深静脉血栓形成的病因、发病机制和治疗至关重要。本研究分两大部分,第一部分探讨静脉血栓形成的病因学,研究亚甲基四氢叶酸还原酶(methylenetetrahydrofolate reductase, MTHFR) C677T和蛋氨酸合成酶还原酶(methionine synthase reductase, MTRR)A66G基因多态性与深静脉血栓形成的关系,试图发现国人静脉血栓形成的易感因子。第二部分为静脉血栓形成基因治疗的体外实验研究,以腺病毒介导的尿激酶型纤溶酶原激活剂(urokinase-type plasminogen activator,uPA)基因转染人脐静脉内皮细胞(human umbilical vein endotheliocyte, HUVEC),研究转uPA基因感染人脐静脉内皮细胞后的蛋白表达以及纤溶活性改变情况,试图探讨uPA基因治疗静脉血栓形成的可行性和可能机制。第一部分MTHFR C677T和MTRR A66G基因多态性与深静脉血栓形成的关系目的:研究亚甲基四氢叶酸还原酶(methylenetetrahydrofolate reductase, MTHFR)基因和蛋氨酸合成酶还原酶(methionine synthase reductase, MTRR)基因的多态性与深静脉血栓形成的关系,探讨深静脉血栓形成的病因学。方法:采用病例对照研究,以101例下肢深静脉血栓形成患者和同期行健康体检的正常人群120例(对照组)的血白细胞为样本,应用等位基因序列特异性引物聚合酶链反应(polymerase chain reaction-sequence specific primer ,PCR-SSP)多态性技术检测两组的MTHFR基因第677位点的多态性和MTRR基因第66位点的多态性,分别比较每两组的基因型和等位基因的分布频率。同时统计下肢深静脉血栓形成的发病部位。结果:MTHFR的677位点CC、CT和TT基因型频率在疾病组中分别为41.58%、25.74%、32.67%,在对照组中分别为58.33%、23.33%、18.33%,MTHFR的677位TT基因型频率与对照组的比较差异有显著性(χ2=7.7,P<0.05)。MTRR的66位点AA、AG和GG基因型频率在疾病组中分别为26.76%、43.66%、29.58%,在对照组中分别为43.57%、44.28%、12.14%,两组的分布频率差异无显著性(P>0.05)。左下肢深静脉血栓形成的发病率为83.2%。结论:MTHFR基因第677位点的TT基因型多态性可能与DVT的发病有关。MTRR基因A66G多态性可能不是DVT的独立遗传危险因素。下肢DVT好发于左下肢。第二部分腺病毒介导uPA基因转染人脐静脉内皮细胞的体外实验研究目的:构建含有人uPA基因的重组腺病毒载体,利用重组腺病毒载体介导人uPA基因转染人脐静脉内皮细胞,从基因、蛋白水平检测目的基因uPA的蛋白表达和纤溶活性,探索静脉血栓形成基因治疗的可行性和可能机制。方法:用PCR将合成的oligo拼接成完整的uPA基因,将合成好的目的基因序列装入pMD-18T载体,经XhoI和SalI酶切将目的基因uPA克隆到含增强绿色荧光蛋白(Enhanced green fluorescent protein, EGFP)的质粒载体pIRES2-EGFP中,并转化至感受态细胞DH5α。以pIRES2-EGFP质粒为模板,扩增带attB1和attB2的uPA-IRES2-EGFP片段。采用pAD/CMV/V5-DEST Gateway Vector载体系统作为载体,该系统是缺失了E1和E3早期区的AD5型腺病毒,可容纳6～8.5 kb的外源基因,外源基因插入到E1区。以Gateway两步重组技术重组腺病毒质粒:BP重组系统将目的片段重组到载体pDONR221上,LR重组系统将目的序列重组到腺病毒质粒载体pAD/CMV/V5-DEST上,测序验证重组质粒的正确性,构建含有人uPA基因的重组腺病毒载体。Pac I将重组腺病毒载体质粒线性化后用脂质体lipofectamine 2000转染293A细胞进行包装并对病毒液进行扩增、纯化和滴度测定。HUVEC细胞培养后实验分3组:ad.uPA转染组,ad.neg空载体对照组和空白对照组(n=3)。分别以重组腺病毒载体介导的人uPA基因、空载体对照和空白对照转染HUVEC细胞,观察转uPA基因对HUVEC细胞的影响,测定最佳感染复数((multiplicity of infection, MOI);采用实时荧光定量聚合酶链反应(real time fluorescence quantitative polymerase chain reaction , FQ-PCR)、免疫印迹(Western Blot)和酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)检测转uPA基因在HUVEC的蛋白表达;比色法检测转uPA基因在HUVEC细胞上清中的的纤溶活性变化。结果:成功克隆了人uPA基因,并将携带EGFP的目的基因uPA克隆至质粒载体pIRES2-EGFP,成功构建了uPA cDNA的质粒载体puPA-IRES2-EGFP,测序结果与Genebank公布的人uPA基因cDNA序列完全一致,同源性为100%,该基因编码的蛋白为人uPA。成功构建了人uPA基因重组腺病毒载体pAD-ZWM-PLAU,重组腺病毒载体pAD-ZWM-PLAU经PacI酶切后用Lipofectamine 2000能有效转染293A细胞,包装成功,获得了人uPA基因重组腺病毒ad.uPA。经大量扩增、纯化后病毒滴度为5.74×1010ifu/ml。本研究制备的人uPA基因重组腺病毒ad.uPA感染HUVEC细胞后最佳MOI=800。以MOI=800感染HUVEC细胞后,FQ-PCR检测ad.uPA组、ad.neg组和control组三组目的基因2 -⊿⊿ CT分别为15.2422、4.5631和1,显示阳性感染病毒细胞中目的基因表达量有明显提高(P<0.01)。Western Blot检测结果示ad.uPA侵染病毒的细胞目的蛋白表达量明显提高,而侵染阴性腺病毒对照组和空白对照组的HUVEC细胞中几乎没有检测到目的蛋白的表达。目的蛋白大小为55 kDa。ELISA检测转基因HUVEC培养上清uPA含量结果ad.uPA转基因组,ad.neg空载体组和空白对照组分别为379.4043±2.46 ng/L,240.0099±1.16 ng/L和256.1024±3.04 ng/L,ad.uPA转基因组uPA蛋白含量明显高于空载体组和空白对照组(P<0.01)。比色法检测转基因HUVEC细胞uPA活性结果ad.uPA转基因组,空载体组和空白对照组分别为68.3062±0.64 IU/106cells/24h,5.1845±0.19 IU/106cells/24h和5.0299±0.12 IU/106cells/24h,ad.uPA转基因组uPA活性明显高于各对照组(P<0.01)。结论:成功构建了EGFP和uPA双表达重组腺病毒载体pAD-ZWM-PLAU,并于HUVEC成功表达,为静脉血栓形成的基因治疗奠定了基础。转uPA基因感染HUVEC细胞后从基因、蛋白水平均可检测到明显的目的基因表达和纤溶活性增高。外源性基因表达的蛋白在在体情况下有否生物学作用,还有待于动物实验来证实。本研究为静脉血栓形成的基因治疗方案的制定进行了有益的探索。更多还原

【Abstract】 Background Deep vein thrombosis (DVT) is a serious disease with potential danger to develop into post-thrombotic syndrome (PTS),phlegmasia cerulea dolens,thrombotic phlegmasia and even pulmonary embolism (PE) which can lead to death if it had not got proper treatment. Epidemiologic study has demonstrated that the morbidity of DVT is 1.6 ~1.82 per 1000 population per year. Retrospective studies report that the mortality rate following venous thromboembolism (VTE) is 5%–23%,although the mortality is 1%-2% in symptomatic patients with adequate anticoagulation. Post-thrombotic syndrome occurs in about one-third of DVT sufferers and pulmonary hypertension in 4%–5% of PE sufferers. Moreover, the therapeutic efficacy of DVT is controversy. Therefore, it is important to study the etiology, pathogenesis and therapeutic methods of DVT. In this study, experiment consists of two parts. In part one, the correlations between genetic polymorphism of methylenetetrahydrofolate reductase (MTHFR) C677T,methionine synthase reductase (MTRR) A66G and lower extremities deep vein thrombosis was studied so that the risk factor for DVT can be found and the etiology of DVT was evaluated. In part two, in vitro experiment of DVT gene therapy was performed. In this experiment, human umbilical vein endotheliocyte (HUVEC) was transfected by adenovirus-mediated Homo sapiens urokinase-type plasminogen activator (uPA) and the protein expression and change of fibrinolytic activity was determined with the aim to explore the feasibility and possible mechanism of DVT gene therapy with uPA. MTRR A66G and lower extremities deep vein thrombosisObjective:To detect the distribution of polymorphism of MTHFR gene and MTRR gene in lower extremities deep vein thrombosis (disease group) and normal controls, and to evaluate the etiology of deep venous thrombosis.Methods:Polymorphism of the 677th site C/T of MTHFR gene and the 66th site A/G of MTRR gene in disease group (n=101) and normal controls(n=120) was detected by polymerase chain reaction-sequence specific primer (PCR-SSP). The patients in the disease group were from the clinical cases which were diagnosed as DVT and the health examination adults were used as the normal control. The morbidity site of DVT was assayed simultaneously.Results:The 677th site of MTHFR gene frequencies of CC,CT and TT genotypes were 41.58%, 25.74% and 32.67% in disease group, while 58.33%, 23.33% and 18.33% in normal controls, respectively.A significant difference was observed in the distribution frequency in two groups(χ2=7.7,P<0.05). The 66th site of MTRR gene frequencies of AA,AG and GG genotypes were 26.76%, 43.66% and 29.58% in disease group, while 43.57%, 4.28% and 12.14% in normal controls.No significant difference was seen in the distribution frequency in two groups (χ2=3.22, P>0.05). Conclusions:The distribution frequency of MTHFR 677TT genotype may have a correlation with morbility of deep vein thrombosis. However, the distribution frequency of MTRR gene seems to have nothing to do with morbility of deep vein thrombosis. Moreover, left lower extremity is the predilection site of deep venous thrombosis.Part TwoThe in vitro experimental study of adenovirus-mediated gene transfer of uPA to infect HUVECObjective:To construct recombinant adenovirus vector expressing with human uPA gene, then to transfect cultured HUVEC in vitro and detect the protein expression andPart OneThe relationship between genetic polymorphism of MTHFR C677T, change of fibrinolytic activity after adenovirus-mediated gene transfer uPA in the level of gene and protein so that the feasibility and possible mechanism of venous thrombosis gene therapy with uPA can be explored.Methods:The synthetical oligo which was amplified with PCR and was then spliced into integrated uPA gene and cloned into pMD﹣18T vector. Next, the aim gene uPA was cloned into plasmid vector pIRES2-EGFP which contains the enhanced green fluorescent protein (EGFP) by Xho I and Sal I restriction enzyme digestion and was transformed into competent cells DH5α. The uPA-IRES2-EGFP fragment containing attB1 and attB2 was amplified from plasmid vector pIRES2-EGFP using polymerase chain reaction. The pAD/CMV/V5-DEST Gateway Vector system was adopted as a vector, which was an AD5 adenovirus absenced of E1 and E3 early region with 6~8.5kb exogenous gene which was inserted into E1 region. The Gateway two-step recombinant technology was taken to construct recombinant adenovirus plasmid. The aim gene fragment was carried to pDONR221 vector through BP recombinant system, and the aim gene sequence was recombined into adenovirus vector pAD/CMV/V5-DEST through the LR recombinant system and sequencing was performed to verify the correctness of recombinant plasmid. The recombinant adenovirus vector plasmid was linearizated using Pac I and then transfected into 293A cells by Lipofectamine 2000. The recombinant plasmid was then packaged, amplified and purified and the titre was measured. For transfection experiment of HUVEC cells, HUVEC cells were infected by adenovirus-mediated uPA gene, empty vector and the blank control, respectively. The multiplicity of infection (MOI) was determined and the protein expression of uPA gene in HUVEC was detected by real time fluorescence quantitative polymerase chain reaction (FQ-PCR), Western Blot and enzyme linked immunosorbent assay (ELISA), respectively. The activity of uPA gene in HUVEC cells was detected with chromatometry.Results : The aim gene uPA with EGFP was cloned into plasmid vector pIRES2-EGFP to construct plasmid vector puPA-IRES2-EGFP of uPA cDNA and sequencing showed its complete homology with Homo sapiens urokinase-type plasminogen activator cDNA published in Genebank. The Homo sapiens urokinase-type plasminogen activator gene recombination adenovirus vector pAD-ZWM-PLAU was successfully reconstructed, which can effectively transfect 293A cells by Lipofectamine 2000. The titer of the recombinant Ad was measured by infection units’method and was 5.74×1010 ifu/ml after generous amplification and purification. The best MOI is 800 when HUVEC cells were infected by the Homo sapiens urokinase-type plasminogen activator gene recombination adenovirus ad.uPA which prepared in our experiment. After HUVEC cells infected by ad.uPA, the aim gene 2 -⊿⊿ CT is 15.2422、4.5631and 1 in ad.uPA group, ad.neg group and control group, respectively. It showed that the expression of interest protein in the ad.uPA infected cells was increased obviously(P<0.01). The expression of aim protein was increased obviously in ad.uPA infected cells, but no expression in the HUVEC cells in ad.neg group and control group by Western Blot assay. The molecular weight of aim protein is 55 kDa. The uPA contents in transfected HUVEC culture supernatant are 379.4043±2.46 ng/L, 240.0099±1.16 ng/L and 256.10±3.04 ng/L in ad.uPA group, ad.neg group and control group by ELISA, respectively. A significant difference was seen in the ad.uPA group compared with ad.neg group and control group(P<0.01). Moreover, the activity of uPA in transgenic HUVEC cells is 68.3062±0.64 IU/106cells/24h, 5.1845±0.19 IU/106cells/24h and 5.0299±0.12 IU/106cells/24h in ad.uPA group, ad.neg group and control group by chromatometry,respectively. A significant difference was seen in ad.uPA group compared with the other groups(P<0.01).Conclusions:The recombinant Ad expressing uPA and EGFP was successfully constructed and could effectively mediate target genes’expression in infected HUVEC cells, which lays foundation of gene therapy for venous thrombosis. The increase of interest gene expression and fibrinolytic activity were detected in gene and protein level after transgenic uPA infected HUVEC cells. However, it should be verified by animal experiment whether the exogenous gene expression protein had biology effect in vivo. Our study showed an important implication for the exploration of ideal gene therapy style for the treatment of venous thrombosis.更多还原