慢病毒介导的HTRA1基因稳定过表达人脑血管平滑肌细胞株的建立

Establishment of Lentivirus Vector Mediated Expression of HTRA1 Gene in Human Vascular Smooth Muscle Cells

目的:构建并包装针对HTRA1基因以及其1091T>C突变基因(HTRA1-Mut)的过表达慢病毒载体,以及建立稳定表达HTRA1及HTRA1-Mut基因的人脑血管平滑肌细胞(HBVSMC)株。方法:采用RT-PCR方法扩增HTRA1及HTRA1-Mut基因片段并将其连接于GV287载体质粒,采用慢病毒包装三质粒系统(GV287/p Helper 1.0/p Helper 2.0)转染293T细胞,收集富含慢病毒颗粒的细胞上清液并标定病毒滴度,慢病毒感染经培养和鉴定的HBVSMC细胞株。结果:成功构建含HTRA1及HTRA1-Mut基因的慢病毒重组载体,PCR鉴定阳性的克隆进行测序和BLAST比对分析显示与源基因序列一致,并能够有效的感染并在293T细胞中表达。表达载体包装后测定病毒滴度为:2E+8 TU/mL。过表达慢病毒感染后HBVSMC有荧光表达,并且荧光率达80%以上,细胞生长良好传后细胞几乎无死亡现象。结论:成功构建了过表达HTRA1及HTRA1-Mut基因的慢病毒表达载体,得到了较高滴度的病毒悬液,建成了稳定表达HTRA1及HTRA1-Mut基因的HBVSMC细胞株,为进一步探讨HTRA1基因及突变后细胞的功能变化提供了良好的研究工具。

Objective： To construct the lentiviral vector containing HTRA1 as well as its mutant gene（HTRA1-Mut,1091TC） and express them in HBVSMCs. Methods： HTRA1 and HTRA1-Mut gene regional fragment were amplified by RT-PCR and then cloned into the plasmid GV287.Lentiviral plasmid, p Helperl.0, and p Helper2.0 were co-transfected into 293 T cells to obtain recombinant virus. The lentiviral titer was detected. Collected lentivirus was applied to infect human vascular smooth muscle cells. Results： The recombinant lentiviral vectors were constructed and were confirmed by PCR and sequencing analysis. They could efficiently transfect 293 T cells and express in the cells. The lentiviral titer was 2E＋8 TU/ml. HBVSMCs produced fluorescent expression after lentivirus transfection, and HTRA1 gene expressed over 80%. Transfected HBVSMCs grew in order without inducing excessive death rate. Conclusion： HTRA1 and HTRA1-Mut recombined lentiviruses with high viral titer were successfully constructed and packaged, and the HTRA1 and HTRA1-Mut gene could be transfected into HBVSMCs with stable and high expression in infected cells, and these cells might be applied for mechanism researches of HTRA1 gene.