诱导整合素α5、β1低表达人主动脉平滑肌细胞株模型的建立

Construction of the model of vascular smooth muscle cells with low expression of integrin a5 and in- tegrin β1

目的构建低表达整合素（ITG）α5、β1人主动脉血管平滑肌细胞（VSMC）模型，为进一步研究ITGα5、β1与VSMC增殖及迁移的关系奠定基础。方法运用RNA干扰技术，通过慢病毒载体，分别构建5组细胞株：抑制表达ITGα5株（si-ITGα5-1、si．ITGα5-2），抑制表达ITGβ1株（si—ITGβ1—1、si—ITGβ1-2）及空siRNA载体细胞株（Con—si），分别通过实时荧光定量聚合酶链反应（FQ—PCR）及Westernblot法检测上述各组细胞ITGcd、ITG[31mRNA及蛋白表达情况。结果两个对照组Con—si（ITGα5mRNA：0．252±0．026，ITGβ1mRNA：0．516±0．056）、Con（未感染的空白对照细胞：O．238±0．021，0．471±0．051）之间ITGα5、ITGβ1mRNA表达量比较，差异无统计学意义（P〉0．05），si—ITGα5—1（0．033±0．004，0．459±0．038）、si—ITGα5-2（0．075±0．009，0．493±0．054）组分别与2个对照组比较，ITG[31mRNA相对表达量降低，差异有统计学意义（P〈0．01）；si—ITGβ1—1（0．241±0．023，0．182±0．021）、si—ITGβ2—2（0．234±0．025，0．114±0．013）组分别与2个对照组比较，mRNA相对表达量降低，差异有统计学意义（P〈0．01）；Westernblot结果示：分子质量150×10 3及138×10 3处分别有ITGα5、ITGβ1条带出现，si—ITGcd-1和si-ITGα5—2的ITGα5条带显著弱于空白各组，si—ITGβ1—1和si—ITGβ1-2条带显著弱于空白各组。结论通过慢病毒载体，成功构建诱导ITGα5、TG[31低表达的VSMC细胞株。

Objective To construct the model of vascular smooth muscle cells with low expression of integrin （ITG） α5, ITGβ1, laying the groundwork for further study on the relationship between ITGα5, ITGβ1 with proliferation and migration of vascular smooth muscle cells （VSMCs）. Methods Five groups of VSMCs were constructed by Lentivirus RNA interference （RNAi） vector, named ITGα5 down - regula- ted line 1 （ si - ITGα5 - 1 ） , ITGα5 down - regulated line 2 （ si - ITGα5 - 2 ）, ITGβ1 down - regulated line 1 （ si - ITGβ1 - 1 ）, ITGβ1 down - regulated line 2 （ si - ITGβ1 - 2 ）, and pRNAT - U6. 2/Leni empty vector （ Con - si）. Real-time fluorescent quantitative polymerase chain reaction （FQ-PCR） and Western blotting were used to detect the changes of ITGα5 and ITGβ1 gene and protein in all the stable transfection ceils. Results There were no significant differences between Con - si group （ ITGα5 mRNA ： 0. 252 ±0. 026; ITGβ1 mRNA： 0. 516 ±0. 056） and Con group （0. 238 ±0. 021, and 0. 471 ±0. 051 respec- tively, P 〉 0. 05 ）. As compared with the control groups, the ITGoL5 mRNA expression in si - ITGα5 - 1 group （ 0. 033 ± 0. 004, 0. 459 ± 0. 038 ）, si - ITGα5 - 2 group （ 0. 075 ±0. 009, 0. 493 ±0. 054 ）, si-ITGβ1-1 group （0.241 ±0.023, 0.182±0.021）, and si-ITGβ1 -2 group （0.234 ±.025, 0. 114 ±0. 013 ） was significantly reduced （ P 〈 0. 05 ）. Western blotting revealed that the VSMCs in si - ITGα5, si - ITGβ1 groups grew slower than the control groups. Conclusion Lentivirus RNAi vectors of ITGα5 and ITGβ1 producing siRNA -ITGα5 and ITGβ1 were constructed successfully. VSMCs lines down -expressing ITGα5 and ITGβ1 were established successfully.