CGRP修饰大鼠MSCs对血管平滑肌细胞增殖及表型转化的影响

Effects of rat mesenchymal stem cells modified by CGRP on proliferation and phenotype transformation of vascular smooth muscle cells in vitro

目的:探讨降钙素基因相关肽(calcitonin gene-related peptide,CGRP)修饰的大鼠间充质干细胞(mesenchymal stem cells,MSCs)在体外对大鼠血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖和表型转化的影响及其机制。方法:分离、培养及鉴定大鼠MSCs和VSMCs。CGRP重组慢病毒(Lv-CGRP-EGFP)转染MSCs后,real-time PCR和ELISA法检测MSCs中CGRP的表达。MSCs-CGRP与VSMCs共培养后MTT、台盼蓝染色及划痕实验评价VSMCs增殖、迁移能力及细胞存活率。Western blotting法检测VSMCs中α-平滑肌肌动蛋白(alphasmooth muscle actin,α-SMA)和骨桥蛋白(osteopontin,OPN)的表达水平。结果:与MSCs组和空载病毒转染组(MSCs-EGFP)比较,CGRP修饰的MSCs(MSCs-CGRP组)中CGRP的mRNA和蛋白表达水平增高(均P<0.01)。MTT法和划痕实验显示,与MSCs组和MSCs-EGFP组比较,MSCs-CGRP组中VSMCs的增殖和迁移能力明显减弱(P<0.05),而且台盼蓝染色显示,各组细胞存活率均大于90%。Western blotting结果显示,与MSCs-CGRP共培养的VSMCs中α-SMA较MSCs组和MSCs-EGFP组表达增加,OPN的表达减少(均P<0.05)。结论:CGRP修饰的MSCs能分泌CGRP蛋白,并且能抑制VSMCs的增殖和迁移,其机制可能通过抑制VSMCs的表型从收缩表型向合成表型转化有关。

AIM：To explore the effects of rat mesenchymal stem cells （MSCs） modified by calcitonin gene-related peptide （CGRP） on the proliferation and phenotype transformation of rat vascular smooth muscle cells （VSMCs） in vitro. METHODS：Rat MSCs and VSMCs were isolated, cultured and identified. The MSCs were infected by lentivirus which carried genes encoding enhanced green fluorescent protein （EGFP） and CGRP. The expression levels of CGRP in CGRP-modified MSCs were detected using real-time PCR and enzyme-linked immunosorbent assay （ELISA）. The prolife-ration and migratory abilities of VSMCs were evaluated by MTT assay, Trypan blue staining and scratch test. The expression levels of α-smooth muscle actin （α-SMA） and osteopontin （OPN） were assessed by Western blotting. RESULTS：Compared with MSCs and MSCs-EGFP groups, the expression levels of CGRP in MSCs-CGRP group were markedly increased （P〈0.01）. The results of MTT assay and scratch test demonstrated that the proliferation and migratory abilities of VSMCs in MSCs-CGRP group were significantly inhibited compared with MSCs and MSCs-EGFP groups （P〈0.05）. Furthermore, cell viability was 〉90% shown by Typan blue staining. Western blotting showed that the expression of α-SMA was increased and that of OPN was decreased in MSCs-CGRP group compared with MSCs and MSCs-EGFP groups （P〈005）. CONCLUSION：CGRP-modified MSCs could secrete CGRP protein and inhibit the proliferation and migration of VSMCs, which may be associated with deterring the phenotype transformation from contractile type to synthetic type. These results lay a foundation for gene therapy in vivo.