The Preliminary Study Of Venous Thrombosis In Etiology And Gene Therapy

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.