【摘要】 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Ad-人TIMP1(hTIMP1),将其感染用同型半胱氨酸(Hcy)刺激的人脐静脉内皮细胞CRL-1730,观察该细胞基质金属蛋白酶(MMP)1、MMP9、TIMP1mRNA的表达变化。方法在大肠杆菌BJ5183中通过同源重组构建重组腺病毒Ad-hTIMP1和对照重组腺病毒Ad-Track。将CRL-1730细胞分为空白对照组、Track对照组和基因治疗组,并根据加入Hcy浓度的不同各分为三个亚组,即空白对照组、生理浓度组和病理浓度组。病毒感染48 h后加入Hcy刺激,6 h后收集各组CRL-1730细胞,RT-PCR法检测CRL-1730细胞中的MMP1、MMP9、TIMP1 mRNA。结果 Ad-hTIMP1和Ad-Track的滴度分别为1.9×1012VP/mL和0.6×1012VP/mL。基因治疗组Ad-hTIMP1感染CRL-1730细胞后,该细胞的TIMP1mRNA表达明显高于空白对照组(P<0.05),即使加入病理浓度的Hcy,CRL-1730细胞中TIMP1mRNA的表达仍处于高水平,而MMP1、MMP9 mRNA的表达明显降低(P均<0.05)。结论成功构建了Ad-hTIMP1和Ad-Track。重组腺病毒Ad-hTIMP1感染CRL-1730细胞后,该细胞中TIMP1 mRNA过表达,可抑制高浓度Hcy刺激造成的MMP1、MMP9mRNA的表达升高,提示其可对抗Hcy对内皮细胞的损伤。更多还原

【Abstract】 Objective To construct recombinant adenoviral vector carrying human TIMP1 and to study the expression of MMP1,MMP9 and TIMP1 mRNA when TIMP1 overexpressed in cultured human umbilical vein endothelial cells CRL-1730 interferred by homocysteine(Hcy).Methods A recombinant adenovirus(Ad-hTIMP1) containing a human TIMP1 cDNA fragment and a control recombinant adenovirus(Ad-Track)was generated by homologous recombination in BJ5183 bacteria.CRL-1730 cells were divided into 3 groups which was blank group,Track group and TIMP1 group.Then they were divided into 3 subgroups due to the concentration of Hcy which was blank group(Hcy 0 mmol/L),physiological concentration group(Hcy 0.01 mmol/L) and pathological concentration group(Hcy 0.1 mmol/L).After transferred by recombinant adenovirus for 48 hours,CRL-1730 cells were stimulated by Hcy,which were collected 6 h later.Then,RT-PCR was used to detect the MMP1,MMP9 and TIMP1 mRNA.Results The titer of Ad-hTIMP1 and Ad-Track were separately 1.9×1012 VP/mL and 0.6×1012 VP/mL.After transferred with Ad-hTIMP1,the expression of TIMP1 mRNA in HUVEC could be increased(P<0.05).After stimulated by Hcy at the concentration of 0.1 mmol/L,the expression of TIMP1 mRNA was still high,while the expression of MMP1 mRNA and MMP9 mRNA decreased(P<0.05).Conclusions A recombinant adenoviral vector carrying human TIMP1 and a control recombinant adenoviral vector is successfully constructed.Overexpression of Ad-hTIMP1 can inhibit the expression of MMP1 and MMP9 mRNA in CRL-1730 stimulated by Hcy,which plays an important role in protecting the endothelial cells.更多还原