低氧反应元件对低氧复氧状态下心肌细胞转染人血管内皮生长因子_(165)基因表达的调控作用  

Regulation of hypoxic response elements to expression of human vascular endothelial growth factor gene transferred to myocardiocyte under hypoxic and reoxygenation conditions

作　　者：张中明[1] 姜波[1] 董红燕[2] 张宜乾[1] 

机构地区：[1]徐州医学院附属医院胸心外科,江苏221002 [2]徐州医学院神经生物研究中心

出　　处：《中华实验外科杂志》2005年第12期1510-1512,共3页Chinese Journal of Experimental Surgery

基　　金：江苏省教育厅重点资助项目(03JKA310140)

摘　　要：目的研究9拷贝低氧反应元件(HRE)对低氧、复氧心肌细胞转染人血管内皮生长因子(hVEGF165)基因表达的调控作用。方法分离新生SD大鼠心肌细胞进行培养,将在 HEK293T细胞进行包装后获得的腺相关病毒转染培养的心肌细胞。心肌细胞分为8组,采用无血清培养基,分别在低氧(氧浓度5％)、正常氧(氧浓度21％)条件进行培养,取低氧／复氧不同时段的心肌细胞或培养液,采用细胞免疫荧光法、ELISA法分别测定心肌细胞及培养液中hVEGF165蛋白表达情况;逆转录-聚合酶链反应(RT—PCR)法测定心肌细胞hVEGF165 mRNA的表达。结果病毒转染率约为87％;转基因低氧1组(A组)、2组(B组)及复氧1组(E组)培养液中hVEGF165蛋白含量显著升高(P<0．01),细胞免疫荧光hVEGF165蛋白染色呈阳性;RT-PCR结果亦显示,转基因低氧1组(A组)、2组(B组)及复氧1组(E组)可见484 bp目的条带。结论在低氧状态下,9 拷贝HRE作为氧敏感调控开关,可促使心肌细胞转染的hVEGF165基因高度表达,而在复氧(正常氧浓度)状态下,hVEGF165因的表达即行中止。Objective To investigate the effect of nine copies of hypoxic response elements （HRE） on regulating expression of human vascular endothelial growth factor 165 （hVEGF165） gene, transferred to myocardiocyte by recombine adeno-associated virus under hypoxic and reoxygenation conditions in vitro. Methods Myocardiocytes from neonatal S-D rats were isolated and cultured. The rAAV vector packaged with 293T cells was using to transferred cultured myocardiocytes. Myocardiocytes were divided into eight groups, which were cultured with serum-free medium under hypoxic （5%） and normoxic （21% ） condition, respectively. After treatment with different oxygen condition, hVEGF165 protein was quantified from culture medium and also detected from fixed cells by using ELISA assay and immunofluorescence stain;RT-PCR was employed to determine the expression of hVEGF165 mRNA from cultured-cells. Results 87% of viral transferred efficiency was achieved. Immunofluorescence positive cells were observed in group A、B and group E, the levels of hVEGF165 protein were higher in group A.B and group E than those of other groups （P 〈 0.01 ）. RT-PCR revealed that a 484bp strip can be found also in group A, B and E, but unavailable in other groups. Conclusion 9HRE, as an oxygen-associated “switch”, can regulate high expressions of VEGF165 gene under hypoxia, whereas under normoxic conditions the expression of VEGF165 gene was ceased.

关 键 词：低氧反应元件 血管内皮生长因子 心肌细胞 基因调控 基因表达 

分 类 号：R541[医药卫生—心血管疾病]