降钙素基因相关肽调节间充质干细胞存活对大鼠颈动脉损伤后血管修复的作用

Effect of calcitonin gene-related peptide on mesenchymal stem cell survival and vascular repair in carotid artery injury rats

目的探讨降钙素基因相关肽(CGRP)调节间充质干细胞(MSC)存活和对大鼠颈动脉损伤后血管修复的作用及其可能机制。方法分离培养获得大鼠MSC,分为CGRP组和对照组,均给予过氧化氢(H2O2)处理,前者再同时加入CGRP处理,以流式细胞仪检测MSC的存活和凋亡情况,并采用Western印迹法检测磷酸化糖原合成酶激酶-3β(pGSK3β)和β-链蛋白(β-catenin)的表达情况。构建大鼠颈动脉球囊损伤模型,分为MSC-CGRP组、MSC组和对照组,前两组分别给予MSC-CGRP(以携带CGRP的重组慢病毒载体pLen-CGRP-EGFP转染细胞)和MSC(未携带CGRP的重组慢病毒载体pLen-EGFP转染细胞)移植治疗,采用Western印迹法检测血管局部CGRP、pGSK3β和β-catenin的表达情况,免疫荧光染色检测损伤血管CD31的表达,苏木精-伊红(H-E)染色检测损伤血管新生内膜、中膜面积。结果 H2O2处理8、24h时,CGRP组MSC的早期凋亡率、晚期凋亡/死亡细胞比例均显著低于对照组同时间点(P值均<0.05),细胞存活率均显著高于对照组同时间点(P值均<0.05)。H2O2处理后8h,CGRP组的pGSK3β和β-catenin蛋白表达水平均显著高于对照组(P值均<0.05);应用CGRP阻断剂CGRP8-37后(CGRP+CGRP8-37组)均显著低于CGRP组(P值均<0.05),而与对照组的差异无统计学意义(P值均>0.05)。大鼠颈动脉损伤模型中,细胞移植后28d,对照组、MSC组的新生内膜与中膜面积比均显著高于MSC-CGRP组(P值均<0.05),MSC组与对照间的差异无统计学意义(P>0.05)。细胞移植后3d,MSC组血管局部pGSK3β、β-catenin表达水平均显著高于对照组(P值均<0.05);MSC-CGRP组均显著低于MSC组(P值均<0.05),与对照组间的差异均无统计学意义(P值均>0.05)。结论 CGRP可能通过激活GSK3β/β-catenin信号通路,促进移植的MSC存活,并增强MSC对损伤血管的修复作用,为防治血管增殖性疾病提供治疗思路。

Objective To investigate the effect of calcitonin gene-related peptide （CGRP） on the survival of mesenchymal stem cells （MSCs） and vascular repair after carotid artery injury in rats and its possible mechanism. Methods MSCs were harvested by isolating culture and divided into CGRP group and control group. The cells were treated with H2O2 in both groups and then the cells in CGRP group were treated with CGRP. The survival and apoptosis of MSCs were analyzed by flow cytometry, and the protein expression of phosphorylated glycogen synthetase kinase 36 （pGSK3） and β-catenin were detected by Western blotting analysis. Carotid artery injury was induced and the injured rats were divided into MSC group and control group and treated with pLen- CGRP-EGFP and pLen-EGFP, respectively. The protein expression of CGRP, pGSK3β and β-catenin in the injured carotid were measured by Western blotting analysis. The expression of CD31 was observed by munofluorescence staining and the area ratio of neointima to tunica media was detected by H-E ＇staining. Results On 8 h and 24 h after H2O2 treatment, early apoptosis rate and ratio of late apoptosis to cell death of MSCs in CGRP group were significantly lower than those in control group （all P〈0.05）, while cell survival rate in CGRP group was significantly higher than that in control group （both P〈0.05）. The protein expressions of pGSK3β and βcatenin in CGRP group were significantly higher than those in control group at 8 h after HzO2 treatment （both P〈 0.05）. The protein expression were significantly decreased after application of CGRP 8-37, a blocker of CGRP, as compared with CGRP group （both P〈0.05）, but there were no significant differences between CGRP＋ CGRP 8-37 and control group （both P〈0.05）. In the rats of carotid artery injury model, the area ratio of neointima to tunica media was significantly higher in control and MSC groups than those in MSC-CGRP group （both P〈0.05）, but no statistical difference was found between MSC and control groups （P 〉 0. 05 ） on day 28 after cell transplantation. Furthermore, the protein expressions of pGSK3β and β-catenin in the carotid artery were significantly higher in MSC group than those in control group and MSC-CGRP group （all P〈0.05） on day 3 after cell transplantation, but no statistical difference was found between MSC-CGRP group and control group （both P〉 0.05）. Conclusion CGRP may promote the survival of transplanted MSCs and enhance its effect on vascular repair after arterial injury through the activation of GSK3β/β-catenin signaling pathway, which provides new ways for the prevention and treatment of vascular hyperplastic diseases.