降钙素基因相关肽对c-kit^+心脏干细胞生存活力的影响

The Influence of CGRP on the Cell Activity of c-kit^+ Cardiac Stem Cells

目的探讨降钙素基因相关肽(CGRP)对缺氧状态下c-kit^+心脏干细胞(c-kit^+CSC)生存活力的影响及其可能的机制。方法体外建立细胞缺氧模型,实验随机分为缺氧细胞组、CGRP+缺氧细胞组、CGRP+CGRP8-37+缺氧细胞组和对照组(不缺氧细胞);通过CCK-8法检测细胞的活力,采用流式细胞仪和线粒体膜电位法来检测细胞凋亡。结果在缺氧状态下,缺氧细胞组细胞增殖活力较对照组下降,CGRP作用缺氧细胞后不同时间点,与缺氧细胞组比较,细胞增殖活力均增加(P<0.05),尤其以30 min和60 min最为明显;与CGRP+缺氧细胞组比较,CGRP+CGRP8-37+缺氧细胞组在30 min和60 min细胞增殖活力明显下降(P<0.05)。流式细胞仪结果显示:与对照组比较,缺氧细胞组早期凋亡率最高(P<0.05);与缺氧细胞组比较,CGRP+缺氧细胞组早期凋亡率降低(P<0.05);与CGRP+缺氧细胞组比较,CGRP+CGRP8-37+缺氧细胞组早期凋亡率增高(P<0.05)。线粒体膜电位结果显示:与缺氧细胞组比较,CGRP+缺氧细胞组红色荧光/绿色荧光比值增高(P<0.05);与CGRP+缺氧细胞组比较,CGRP+CGRP8-37+缺氧细胞组红色荧光/绿色荧光比值降低(P<0.05)。结论 CGRP能够促进缺氧状态下c-kit^+CSC增殖存活,抑制细胞早期凋亡。

Aim To investigate the influence of calcitonin gene-related peptide（ CGRP） on the survival activity of c-kitposcardiac stem cells（ c-kit~＋CSC） in hypoxia,and research its potential mechanism. Methods c-kit＋CSC was obtained by enzyme digestion and immune magnetic bead separation. Then serum-free hypoxia model of c-kit＋CSC was established in vitro. The models were randomly divided into hypoxia group,CGRP ＋ hypoxia group,CGRP ＋ CGRP8-37＋ hypoxia group and control group（ no hypoxia）. The activity of cells was detected by cell counting kit（ CCK-8 kit）,the apoptosis of cells were detected by flow cytometry（ FCM） and mitochondrial membrane potential. Results Under hypoxia condition,compared with the control group,the activity of c-kit＋CSC was significantly decreased in the hypoxia group（ P〈0.05）,but compared with the hypoxia group,the activity of c-kit＋CSC was significantly increased in CGRP ＋hypoxia group at different time points of CGRP effect on hypoxia cells（ P〈0.05）,especially in 30 min and 60 min; When inhibiting the effect of CGRP on hypoxia,compared with CGRP ＋hypoxia group,the activity of c-kit＋CSC was significantly decreased in the CGRP ＋CGRP8-37＋hypoxia group at 30 min and 60 min after hypoxia（ P〈0.05）. In terms of apoptosis,compared with the control group,the early apoptosis rate was the highest in hypoxia group（ P〈0.05）; After CGRP treating c-kit＋CSC in hypoxia,we could find that CGRP ＋hypoxia group had a lower early apoptosis rate（ P〈0.05）,when inhibiting the effect of CGRP on hypoxia,the early apoptosis rate was the highest in CGRP ＋CGRP8-37＋hypoxia group,but there was no significant difference in early apoptosis rate between hypoxia group and CGRP ＋ CGRP8-37＋ hypoxia group（ P〉0. 05）. Mitochondrial membrane potential showed,compared with the hypoxia group,red fluorescence and green fluorescence ratio increased in CGRP ＋hypoxia group（ P〈0.05）,when inhibiting the effect of CGRP on hypoxia,compared with the CGRP ＋hypoxia,the red fluorescence and green fluorescence ratio decreased in CGRP ＋CGRP8-37＋hypoxia group（ P〈0.05）. Conclusion CGRP can promote the proliferation and survival of c-kit＋CSC under hypoxia,and inhibit the early apoptosis of the cell.