| 石瑜 王晓玲 王敏 李传芬 曹秉振.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[J].,2016,16(12):2218-2222 |
| 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 inHuman Vascular Smooth Muscle Cells |
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| DOI： |
| 中文关键词: 慢病毒表达载体 HTRA1 CARASIL 人脑血管平滑肌细胞 |
| 英文关键词: Lentivirus vector HTRA1 CARASIL Human vascular smooth muscle cells |
| 基金项目:国家自然科学基金面上项目（81271318） |
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| 中文摘要: |
| 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/pHelper 1.0/pHelper 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。 |
| 英文摘要: |
| Objective:To construct the lentiviral vector containing HTRA1 as well as its mutant gene(HTRA1-Mut,1091T>C) and
express themin HBVSMCs.Methods:HTRA1 and HTRA1-Mut gene regional fragment were amplified by RT-PCR and then cloned into
the plasmid GV287.Lentiviral plasmid, pHelperl.0, and pHelper2.0 were co-transfected into 293T 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 293T 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
mechanismresearches of HTRA1 gene. |
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