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背景和目的前期研究表明人组织激肽释放酶1(hTK1)基因过表达具有部分抑制血管平滑肌细胞(VSMC)增殖和改善血管再狭窄的作用,但联合基质金属蛋白酶组织抑制物1(TIMP1)对VSMC增殖是否具有协同效应,目前尚不明了。本研究构建含双基因hTK1和hTIMP1的重组腺病毒载体,并观察其在大鼠VSMC中的表达,以及对VSMC生长增殖影响。方法采用限制性内切酶BglⅡ和SalⅠ,酶切含启动子CMV和hTIMP1基因片段的重组穿梭质粒,经PCR、连接、热激、转化等亚克隆至pDC316-hTK1质粒中,构建含双基因hTK1和hTIMP1的重组穿梭质粒。将骨架质粒和穿梭质粒于293A细胞包装重组腺病毒载体。感染大鼠VSMC后,采用RealtimePCR法、Western blot法检测目的基因转录、蛋白表达。细胞计数法和四甲基偶氮唑盐法(MTT)测定细胞增殖。结果经PCR、酶切和测序法证实,含双基因(hTK1和hTIMP1)的重组病毒穿梭质构建正确。在293A细胞中成功包装出重组腺病毒载体Ad-hTK1-hTIMP1。感染VSMC后,双基因(hTK1和hTIMP1)的转录水平表达呈现随感染复数(50~150 MOI)和时间(1~3d)依赖性地增加,目的蛋白(hTK1和hTIMP1)亦呈现浓度依赖性地高表达。与单基因载体(Ad-hTK1或Ad-hTIMP1)相比较,双基因载体可明显抑制血小板源性生长因子(PDGF)诱导的大鼠VSMC增殖(P<0.01),其峰值抑制率为45.7%。结论首次成功构建并包装重组腺病毒双基因载体Ad-hTK1-hTIMP1。感染细胞后,其目的基因和蛋白均呈现独立高表达,并具有协同抑制VSMC增殖的效应,为血管增殖性疾病的多基因干预治疗实验提供一个新工具。
BACKGROUND & OBJECTIVE Previous studies have shown that the overexpression of human tissue kallikrein 1 (hTK1) gene partially inhibits the proliferation of vascular smooth muscle cells (VSMCs) and improves vascular restenosis. However, the combination of TIMP1 It is not clear if VSMC proliferation has a synergistic effect. In this study, a recombinant adenovirus vector containing double genes hTK1 and hTIMP1 was constructed and its expression in rat VSMC was observed as well as its effect on the proliferation and proliferation of VSMC. Methods Recombinant shuttle plasmids containing promoter CMV and hTIMP1 were digested with restriction endonucleases Bgl Ⅱ and Sal Ⅰ and subcloned into pDC316-hTK1 plasmid by PCR, ligation, heat shock and transformation. And hTIMP1 recombinant shuttle plasmid. The backbone and shuttle plasmids were packaged in 293A cells with recombinant adenovirus vector. After infection of rat VSMC, Realtime PCR method and Western blot were used to detect the transcription and protein expression of the target gene. Cell proliferation was measured by cytometry and MTT assay. Results PCR, restriction enzyme digestion and sequencing confirmed that the recombinant shuttle virus containing double genes (hTK1 and hTIMP1) was constructed correctly. The recombinant adenovirus Ad-hTK1-hTIMP1 was successfully packaged in 293A cells. After infected with VSMC, the transcriptional levels of hTK1 and hTIMP1 were increased with the increase of infection (50-150 MOI) and time (1-3 days), and the expression of hTK1 and hTIMP1 in a concentration-dependent manner High expression. Compared with the single gene vector (Ad-hTK1 or Ad-hTIMP1), the double gene vector significantly inhibited the proliferation of rat VSMC induced by platelet-derived growth factor (PDGF) (P <0.01), and the peak inhibition rate was 45.7%. Conclusion The recombinant adenovirus double gene vector Ad-hTK1-hTIMP1 was successfully constructed and packaged. Infected cells, the target gene and protein showed an independent high expression, and synergistically inhibit the proliferation of VSMC effect, multi-gene therapy for vascular proliferative disease treatment experiment provides a new tool.