标题：靶向S1PR3基因 shRNA慢病毒表达载体构建及功能鉴定

      作者：王航 1，2，黄浩 3，蔡克银 2，丁世芳 1
    (1.广州军区武汉总医院心血管内科，湖北 武汉 430070；，
2.广州军区武汉总医院干部病房二科，湖北 武汉 430070；
3.深圳市合一康生物科技股份有限公司临床医学中心，广东 深圳 513000)

      卷次： 2016年27卷11期

      【摘要】 目的 构建靶向小鼠 S1PR3基因的 shRNA慢病毒表达载体，并验证其对内皮祖细胞迁移能力的影
响。方法 依照 shRNA设计原则，合成对应的 shRNA序列，将其克隆到PHBLV载体中，使用三载体系统进行病毒
包装，收集制备成功的病毒，并用荧光显微镜法测定病毒滴度，使用病毒感染EPC后，测定各组细胞中S1PR3蛋白
表达情况，选择合适的病毒感染EPC后，测定其对EPC迁移能力的影响。结果 成功将 shRNA序列克隆到慢病毒
表达载体PHBLV-U6-RFP，表达质粒与辅助质粒共转染293细胞后48 h就可以看到细胞中成功表达红色荧光。收
集培养成功病毒感染小鼠EPC，可以不同程度干扰细胞中S1PR3蛋白表达，S1PR3蛋白表达受到抑制后，EPC的迁
移能力明显减弱。结论 我们成功构建了可以有效干扰S1PR3表达的慢病毒载体，并初步观察了其对EPC细胞的
迁移能力的影响，为后续进一步调控S1PR3相关的细胞功能的研究奠定了基础。

      【关键词】 慢病毒；1-磷酸鞘氨醇受体3；内皮祖细胞；细胞迁移

      【中图分类号】 R-332 【文献标识码】 A 【文章编号】 1003—6350（2016）10—1721—06


Construction and function identification of short hairpin RNA lentiviral vector targeting S1PR3.
WANG Hang 1, 2,
HUANG Hao 3, CAI Ke-yin 2, DING Shi-fang 1. 1. Department of Cardiovasology, Wuhan General Hospital of Guangzhou
Military, Wuhan 430070, Hubei, CHINA; 2. Second Department of Geratology, Wuhan General Hospital of Guangzhou Mili-
tary, Wuhan 430070, Hubei, CHINA; 3. Clinical Medical Center, Shenzhen Hornetcorn Biotechnology Co. Ltd., Shenzhen
513000, Guangdong, CHINA

【Abstract】 Objective To construct the short hairpin RNA (shRNA) lentiviral vector targeting sphingo-
sine-1-phosphate receptor 3 (S1PR3) gene and then detect the effect of shRNA on the migration of endothelial precursor
cells (EPCs). Methods Three siRNA interference sequences targeting S1PR3 gene were designed, synthesized and
cloned into the lentivirus vector respectively. Recombinant lentivirus and control were extracted from the 293T cells, and
the viral titer was checked by observing the expression of red fluorescent protein. The lentivirus with the best interferine
effect was detected by Western-blot. Transwell assay was used to detect the migration of EPCs. Results Recombinant
lentivirus with a high title and efficient infection were obtained by the lentivirus vectors system PHBLV-U6-RFP. 48 h
after plasmids and helper plasmids were co-transfected to 293T cells, the expression of red fluorescent protein was ob-
served. Mouse EPCS could be efficiently infected. The expressions of S1PR3 in EPCS were significantly reduced, and
its migration ability was significantly inhibited. Conclusion The recombinant shRNA lentivirus targeting the S1PR3
gene were constructed successfully, and the effect of shRNA on the migration of EPCs was observed, which lays the
groundwork for subsequent study of the role of S1PR3 in the repair process after vascular endothelial injury.

      【Key words】 Lentivirus; Sphingosine-1-phosphate receptor 3 (S1PR3); Endothelial precursor cells (EPCs); Cell
migration
·论 著·
6350.2016.11.001