Synergistic effect of a tissue kallikrein 1 and tissue inhibitor of matrix metalloproteinase 1 co‑expression vector on the proliferation of rat vascular smooth muscle cells.
Synergistic effect of a tissue kallikrein 1 and tissue inhibitor of matrix metalloproteinase 1 co‑expression vector on the proliferation of rat vascular smooth muscle cells.
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组织激肽释放酶1和基质金属蛋白酶组织抑制剂1共表达载体对大鼠血管平滑肌细胞增殖的协同作用
DOI：
10.3892/mmr.2015.4198
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发表时间：
2015-10
影响因子：
3.4
通讯作者：
Zheng W
中科院分区：
文献类型：
--
作者：
Zhu P;Yu H;Huang S;Xiang H;Li F;Zheng W
组织激肽释放酶1（Tissue kallikrein 1，TK 1）和基质金属蛋白酶组织抑制剂1（tissue inhibitor of matrix metalloproteinase 1，TIMP 1）分别在抑制血管平滑肌细胞（vascular smooth muscle cell，VSMC）增殖和改善血管重塑中起重要作用。假设由腺病毒载体介导的TK 1和TIMP 1基因的组合可以增强或协同作用以增强抑制作用。将携带hTIMP 1 cDNA的启动子mCMV亚克隆到pDC 316-hTK 1中，构建了携带hTK 1和hTIMP 1基因的重组质粒。随后，将双基因质粒和腺病毒骨架质粒包装到HEK 293 A细胞中。基因转录和蛋白表达进行了检查，分别采用逆转录-定量聚合酶链反应（PCR）和蛋白质印迹法。采用细胞计数和甲基噻唑基四唑啉法检测VSMC增殖情况。经PCR、双酶切和测序鉴定，所构建的含hTK 1和hTIMP 1基因的质粒正确。成功构建了hTK 1-hTIMP 1共表达载体Ad-hTK 1-hTIMP 1，并包装入HEK 293 A细胞中。共表达载体转染VSMCs后，hTK 1和hTIMP 1的mRNA转录和蛋白表达均呈浓度依赖性和时间依赖性的高水平表达。结论：与单基因表达载体相比，共表达载体能协同抑制血小板源性生长因子-BB诱导的细胞生长和增殖。
Tissue kallikrein 1 (TK1) and tissue inhibitor of matrix metalloproteinase 1 (TIMP1) are important in inhibiting vascular smooth muscle cell (VSMC) proliferation and improving vascular remodeling, respectively. It was hypothesized that a combination of TK1 and TIMP1 genes, mediated by an adenovirus vector could augment or act in synergy to enhance the inhibitory effects. The promoter, mCMV carrying hTIMP1 cDNA was subcloned into pDC316-hTK1 to construct a recombinant plasmid carrying hTK1 and hTIMP1 genes. Subsequently, the double gene plasmid and adenovirus backbone plasmid were packaged into HEK293A cells. Gene transcription and protein expression were examined, respectively using reverse transcription-quantitative polymerase chain reaction (PCR) and western blotting assays. VSMC proliferation was assessed using cell counting and methyl-thiazolyl-tetrazoliuin methods. The constructed plasmid containing hTK1 and hTIMP1 genes was correctly identified by means of PCR, double digestion and sequencing analysis. The co-expression vector, Ad-hTK1-hTIMP1 was successfully constructed and packaged into HEK293A cells. When VSMCs were transfected with the co-expression vector, the mRNA transcription and protein expression of hTK1 and hTIMP1 exhibited abundant expression in a concentration-dependent and time-dependent manner, independently. In conclusion, the co-expression vector synergistically inhibited the cell growth and proliferation induced by platelet-derived growth factor-BB compared with the single gene vector.