基质细胞衍生因子-1α对小鼠心脏干细胞凋亡的影响及其机制

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

目的 探讨基质细胞衍生因子-1α（stromal cell-derived factor-1 α,SDF-1α）对体外血清剥夺诱导的心脏干细胞（cadiac stem cells,CSC）凋亡的抑制作用,并探讨其机制.方法 体外分离培养小鼠CSC.免疫磁珠分选c-kit＋ CSC,纯化后分为5组,即正常对照组、饥饿组、饥饿＋SDF-1α组（又根据SDF-1α不同浓度分为10、50、100、200 ng/ml干预亚组）、饥饿＋SDF-1α＋ AMD3100组和饥饿＋ SDF-1α＋LY294002组.原位末端转移酶标记法（TUNEL法）和异硫氰酸荧光素/碘化丙啶（Annexin V-FITC/PI）流式双标法检测各组CSC凋亡情况,CCK-8法检测CSC活力,Westem blot法检测抗凋亡蛋白B细胞淋巴瘤2（Bcl-2）以及磷酸化丝氨酸苏氨酸激酶（p-Akt）的表达水平,并使用比色法检测半胱氨酸天冬氨酸蛋白酶-3（caspase-3）活性.结果 CSC经磁珠分选后c-kit阳性率可达85％以上.饥饿组细胞凋亡率明显高于正常对照组[（27.03±0.80）％比（1.51±0.54）％,P＜0.01].饥饿＋SDF-1α组各亚组细胞凋亡率均明显低于饥饿组（P均＜0.01）,CSC凋亡率在饥饿＋SDF-1α 10、50、100 ng/ml干预组渐次降低,以100 ng/ml干预组为最低[（10.67±1.06）％,与饥饿组比较P＜0.01].饥饿＋SDF-1α 100 ng/ml组p-Akt和Bcl-2的蛋白表达均明显高于饥饿组（P均＜0.01）,而caspase-3活性则均明显低于饥饿组（P均＜0.01）.饥饿＋SDF-1α＋ AMD3100组和饥饿＋SDF-1 α＋LY294002组p-Akt和Bcl-2的蛋白表达均较饥饿＋SDF-1α 100 ng/ml组低（P均＜0.01）,而caspase-3活性均明显高于饥饿＋SDF-1α100 ng/ml组（P均＜0.01）.结论 SDF-1α可抑制血清剥夺诱导的CSC凋亡,且该作用是通过SDF-1 α/CXCR4轴激活PI3K/Akt通路实现的.

Objective To explore the impact and related mechanisms of stromal cell-derived factor-1α （SDF-1 α） on serum deprivation-induced apoptosis of cadiac stem cells （CSCs）.Methods CSCs were isolated from adult mouse heart tissue and cultured in vitro.Obtained cells were purified using magneticactivated 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-1 α 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-1 α in a concentration dependent manner and peak effect was seen with 100 ng/ml SDF-1 α[（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-1 α 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.