携带人金属蛋白酶组织抑制剂1重组腺病毒的构建及在人脐静脉内皮细胞中的表达

Construction of recombinant adenovirus with human tissue inhibitor of metalloproteinase-1 and its expression in human umbilical vein endothelial cells

目的构建携带人金属蛋白酶组织抑制剂1(TIMP1)的重组腺病毒AdV-hTIMP1,感染体外培养的人脐静脉内皮细胞CRL-1730,观察其mRNA表达情况。方法应用AdEasy复制缺陷型腺病毒载体系统在大肠埃希菌BJ5183中通过同源重组构建Ad-hTIMP1和对照Ad-Track。阳离子脂质体法将重组腺病毒转染到人胚胎肾细胞(HEK293T)中进行包装和扩增。纯化后PCR鉴定hTIMP1目的基因,OD260法测定滴度,透射电镜观察腺病毒形态。将人脐静脉内皮细胞CRL-1730分为3组,空白对照组、Track对照组和TIMP1实验组,感染48h后收集细胞,半定量PCR(RT-PCR)法检测内皮细胞中TIMP1mRNA的表达情况。结果重组腺病毒Ad-hTIMP1的滴度为1.9×1012v.p/mL。其感染人脐静脉内皮细胞CRL-1730后,实验组TIMP1mR-NA的表达明显升高(P<0.05)。结论成功构建了携带hTIMP1基因的重组腺病毒Ad-hTIMP1,成功感染了体外培养的人脐静脉内皮细胞CRL-1730,使细胞中hTIMP1过表达,为心血管疾病的基因治疗提供平台。

Objective To construct recombinant adenovirus （AdV） with human tissue inhibitor of metalloproteinase-1 （hTIMP1）, transfect in vitro cultured human umbilical vein endothelial cells（HUVEC） human umbilical vein endothelial cells CRL- 1730 for observing the mRNA expression. Methods The AdEasy system was adopted to create recombinant adenovirus Ad- hTIMP1 and Ad-Track. The adenovirus vector was then packaged and amplified in HEK293 cells. They were purified and then iden- tified by electronic microscope. According to the interference,all the cells were divided into 3 groups：the blank group,Track group and TIMP1 group. Cells were collected after transfection for 48 h. RT-PCR was used to detect the expression of TIMP1 mRNA. Re- sults The titer of Ad-hTIMP1 was 1.9 ×10lz v. p/mL. In transfection with Ad-hTIMPl,the expression of TIMP1 mRNA in the TIMP1 group was obviously increased（P（0.05）. Conclusion The recombinant adenoviral vector carrying hTIMPi was success- fully constructed. The overexpression of Ad-hTIMP1 in human umbilical vein endothelial cells CRL-1730 could be detected, and provides the platform for the gene therapy of cardiovascular diseases.