缺氧、复氧条件下低氧反应元件(HRE)对心肌细胞转染hVEGF_(165)基因表达的调控作用

Regulation of hypoxic response elements to expression of hVEGF_(165) gene transferred to cardiomyocytes under anoxic and normoxic conditions

目的:探讨缺氧、复氧条件下,低氧反应元件(HRE)作为氧条件基因表达控制开关,对心肌细胞转染rAAV-HRE9-hVEGF165基因表达的调控作用。方法:分离新生SD大鼠心肌细胞,采用无血清培养,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。实验共分为8组:Ⅰ组(空白对照组):常氧培养24h(氧浓度21%);Ⅱ组(缺氧对照组):常氧培养16h,缺氧8h(氧浓度1%);Ⅲ组(转基因对照组):常氧培养24h;Ⅳ组(转基因缺氧1组):转基因后缺氧8h;Ⅴ组(复氧1组):常氧培养16h,缺氧8h、复氧4h;Ⅵ组(复氧2组):常氧培养16h,缺氧8h、复氧8h;Ⅶ组(复氧3组):常氧培养16h,缺氧8h、复氧12h;Ⅷ组(转基因缺氧2组):转基因后常氧培养16h,缺氧20h。ELISA法测定培养液VEGF蛋白含量;细胞免疫荧光染色及RT-PCR分别检测细胞内VEGF蛋白及VEGF165mRNA的表达。结果:95%的培养心肌细胞可见自律搏动,cTn-I染色阳性率为86%;病毒转染率约为87%。Ⅳ、Ⅴ、Ⅷ组培养液中VEGF蛋白含量显著高于其它各组(P<0·01),细胞免疫荧光VEGF蛋白染色呈阳性;RT-PCR测定显示,Ⅳ、Ⅴ及Ⅷ组可见484bp目的条带。结论:rAAV-HRE9-hVEGF165可成功地转染原代培养心肌细胞,在缺氧环境下,受HRE的调控,VEGF165mRNA及VEGF165蛋白可有效表达,而在常氧状态下,目的基因的表达及蛋白合成即行中止。

AIM： To investigate effects of hypoxie response elements （HRE） on expression of hVEGF165 gene transferred to myocardiocyte under anoxic and normoxic conditions in vitro. METHODS： Myocardiocytes from neonatal SD rats were isolated and cultured in serum - free medium. The r - AAV vector packaged with 293T cells was used to transfer cultured myocardiocytes. Myocardiocytes were divided into eight groups： Group Ⅰ ： myocardiocytes were cultured for 24 hours under normoxic conditions as control; Group Ⅱ ： cells was cultured under hypoxia for 8 hours; Group Ⅲ ： infected cells were cultured for 24 hours under normoxic conditions; Group Ⅳ： infected cells were cultured under hypoxia for 8 hours ; Group Ⅴ ： infected cells were cultured under hypoxia for 8 hours and normoxia for 4 hours ; Group Ⅵ ： infected cells were cultured under hypoxia for 8 hours and normoxia for 8 hours; Group Ⅶ ： infected cells were cultured under hypoxia for 8 hours and normoxia for 12 hours; Group Ⅷ： infected cells were cultured under hypoxia only for 20 hours, hVEGF165 protein was quantified from culture medium using ELISA, hVEGF165 protein in myocardiocytes was detected with immunofluorescence and expression of hVEGF165 mRNA was also determined by RT- PCR. RESULTS： 95% myocardiocytes showed beating rhythmically with 86% of cTn - I positive staining. 87% virual transferred efficiency was achieved. The contents of hVEGF165 protein in group Ⅳ,Ⅴ and Ⅷ were higher than those in other groups （P 〈 0. 01 ）. Immunofluorescence positive cells were observed in group Ⅳ,Ⅴ and group Ⅷ. RT - PCR revealed that a 484 bp strip was also found in the same groups. CONCLUSION： rAAV- HRE9 -hVEGF165 vector infected cultured myocardiocyte successfully. Under hypoxia,expression of hVEGF165 mRNA and hVEGF165 protein were regulated by HRE, whereas under normoxic conditions the expression of hVEGF165 mRNA and hVEGF165 protein were ceased.