基质细胞衍生因子-1α对血清剥夺诱导的心脏干细胞凋亡的影响及相关机制

[Impact and related mechanisms of stromal cell-derived factor-1α on serum deprivation-induced cardiac stem cells apoptosis].

作者信息

Huang Rong, Ma Gen-shan, Pan Xiao-dong, Chen Zhong-pu, Sheng Zu-long, Hu Sheng-da, Yao Yu-yu, Chen Zhong

机构信息

Department of Cardiology, Zhongda Hospital Affiliated Southeast University, Nanjing 210009, China.

Email:

出版信息

Zhonghua Xin Xue Guan Bing Za Zhi. 2013 Oct;41(10):870-5.

PMID:24377895

Abstract

OBJECTIVE

To explore the impact and related mechanisms of stromal cell-derived factor-1α (SDF-1α) on serum deprivation-induced apoptosis of cardiac stem cells (CSCs).

METHODS

CSCs were isolated from adult mouse heart tissue and cultured in vitro. Obtained cells were purified using magnetic-activated cell sorting (MACS) with c-kit magnetic beads. C-kit(+)CSCs were divided into five groups: normal control group, serum deprivation group, serum deprivation+SDF-1α group, serum deprivation+SDF-1α+AMD3100 group, serum deprivation+SDF-1α+LY294002 group. Cell apoptosis was assessed using the DeadEnd Colorimetric TUNEL System and flow cytometry analyses with an Annexin V-FITC Apoptosis Detection Kit. The viability of CSCs was assessed by CCK-8. The protein expression of Bcl-2 and phosphorylated Akt were detected by Western blot. The caspase-3 activity was determined using caspase-3 Colorimetric Assay Kit.

RESULTS

After magnetic separation, more than 85% of cardiosphere derived cells were positive for c-kit expression. Compared with the normal control group, the apoptosis rate of serum deprivation group was significantly increased[(27.03 ± 0.80)% vs. (1.51 ± 0.54)%, P < 0.01], which could be significantly reduced by SDF-1α in a concentration dependent manner and peak effect was seen with 100 ng/ml SDF-1α[(10.67 ± 1.06)% vs. (27.03 ± 0.80)%, P < 0.01]. The expressions of p-Akt and Bcl-2 were significantly increased and the activity of caspase-3 was significantly decreased in serum deprivation+SDF-1α group compared to serum deprivation group (P < 0.01). Further more, the expression of p-Akt and Bcl-2 were significantly decreased and the activity of caspase-3 was increased in both serum deprivation+SDF-1α+AMD3100 group and serum deprivation+SDF-1α+LY294002 group compared to serum deprivation+SDF-1α group (P < 0.01).

CONCLUSIONS

SDF-1α reduces serum deprivation induced CSCs apoptosis via modulating PI3K/Akt signaling pathway.

摘要

目的

探讨基质细胞衍生因子-1α（SDF-1α）对血清剥夺诱导的心脏干细胞（CSCs）凋亡的影响及其相关机制。

方法

从成年小鼠心脏组织中分离CSCs并进行体外培养。使用c-kit磁珠通过磁激活细胞分选（MACS）对获得的细胞进行纯化。将c-kit(+)CSCs分为五组：正常对照组、血清剥夺组、血清剥夺+SDF-1α组、血清剥夺+SDF-1α+AMD3100组、血清剥夺+SDF-1α+LY294002组。使用DeadEnd比色TUNEL系统和Annexin V-FITC凋亡检测试剂盒通过流式细胞术分析评估细胞凋亡。通过CCK-8评估CSCs的活力。通过蛋白质免疫印迹法检测Bcl-2和磷酸化Akt的蛋白表达。使用caspase-3比色检测试剂盒测定caspase-3活性。

结果

磁分离后，超过85%的心脏球衍生细胞c-kit表达呈阳性。与正常对照组相比，血清剥夺组的凋亡率显著增加[（27.03±0.80）%对（1.51±0.54）%，P<0.01]，SDF-1α可呈浓度依赖性显著降低凋亡率，100 ng/ml SDF-1α时达到峰值效应[（10.67±1.06）%对（27.03±0.80）%，P<0.01]。与血清剥夺组相比，血清剥夺+SDF-1α组中p-Akt和Bcl-2的表达显著增加，caspase-3活性显著降低（P<0.01）。此外，与血清剥夺+SDF-1α组相比，血清剥夺+SDF-1α+AMD3100组和血清剥夺+SDF-1α+LY294002组中p-Akt和Bcl-2的表达均显著降低，caspase-3活性增加（P<0.01）。