The Relationship Between Ischemic Cerebralvascular Disease And Hyperhomocysteinema And 5, 10-methylenetetrahydofolate Reductase Gene

In the last decade, a good many of studies have shown that moderately rised homocysteine(Hcy)concentrations which is caused by genetic or environmental factors or a combination of both factors are an independent risk factor for BI. The oxidation of homocysteine promotes the oxidation of low-density lipoprotein cholesterol,which causes injury to vascular endothelial cells and leads to endothelial dysfunction.Homocysteine also promotes vascular smooth muscle cell growth, and increases I platelet thromboxand A2 production,platelet aggregation and factor V activity.Moderate and intermediate hyperhomoxysteinemia is believed to be associated with BI. Methylenetetrahydrofolate reductase (MTHFR) is the key enzyme in the metabolism of Hey. The catalytic domain of this enzyme is believed to locate in the N-terminal.And C677T polymorphism, located in the N-terminal of MTHFR, is believed to be the most common genetic defect that result in the hyperhomoxysteinemia. Now, more and more researchers have paid attention to the correlation between MTHFR gene mutation and HHcy on BI etiology. Our current research work investigated these three factors and elucidated their relevance with clinical and basic methods.Objectives: (1)To clone the human MTHFR(N)gene and constructed GST/ MTHFR(N) fusion expressing vector and provide a basis for investigation of MTHFR(N) protein structure and function; (2)To study the changes of endothelial dysfunction in cultured HUVEC with Hey in different concentration; (3) TO observe whether the folic acid (FA) could preventhyperhomocysteinemia-induced endothelial dysfunction; (4)To test whether C677T and A1298C restriction fragment length polymorphisms(RFLPs) in the MTHFR gene were associated with HHcy and BI and whether they were genetic susceptibility markers of BI.Methods: (1)To amplify the MTHFR(N) coding region. The PCR product was cloned into pGEM-T easy plasmid and sequenced, then subcloned into vector pGEX-4T-2. The MTHFR(N) protein was expressed in E coli as fusion protein with glutathione S-transferase (GST) induced by IPTG. (2)HUVECs were cultured to the third generation. Then HUVECs were divided into 8 groups and cultured with Hey in different concentration for 20 hours. By means of MTT, HE, TUNEL and FCM to observe the cell cycle and endothelial dysfunction. (3)The MTHFR polymorphisms of 191 patients from Xi'an area were studied by PCR-RFLP analyzing methods. We compared C677T and A1298C allele frequency in case and control groups.Results: (1) We successfully constructed GST/ MTHFR(N) fusion expressing vector and expressed it in E coli. (2)After HUVECs were exposed to Hey in different concentration for 20 hours, the survival rate and the apoptosis rate of the cell was decreased significantly(P<0.05) in a dose-dependent manner; the ration of arrested cell in G1 was increased; the folic acid (FA) could protect the endothelial dysfunction by hyperhomocysteinemia (P < 0.05) . (3)There was a close relationship between the C677T mutation and high levels of Hcy( P<0.01). There was also a close relationship between C677T polymorphism and the presence of BI( P<0.05). In multivariate Logistic regression analysis, 677T gene mutation and Hey were all associated with the BI, with an OR of 1.870 and 1.031, respectively.Conclusions: Our work provide a basis for investigation of MTHFR(N) protein structure and function. We proved that HHcy could damage the endothelial cells and FA can resistant this toxicity. HHcy and C677T polymorphism were risk factors of BI.These research may play important rolesin the investigation of the the genetics of BI and of the cells toxicity and gene toxicity of Hey. Meanwhile, our work provide a theoretical warranty for the clinical use of FA in HHcy and ischemic cerebrovascular diseases.