CAJ | 학술논문

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.
목적 탐토기질세포연생인자-1α(stromal cell-derived factor-1 α,SDF-1α)대체외혈청박탈유도적심장간세포(cadiac stem cells,CSC)조망적억제작용,병탐토기궤제.방법 체외분리배양소서CSC.면역자주분선c-kit+ CSC,순화후분위5조,즉정상대조조、기아조、기아+SDF-1α조(우근거SDF-1α불동농도분위10、50、100、200 ng/ml간예아조)、기아+SDF-1α+ AMD3100조화기아+ SDF-1α+LY294002조.원위말단전이매표기법(TUNEL법)화이류청산형광소/전화병정(Annexin V-FITC/PI)류식쌍표법검측각조CSC조망정황,CCK-8법검측CSC활력,Westem blot법검측항조망단백B세포림파류2(Bcl-2)이급린산화사안산소안산격매(p-Akt)적표체수평,병사용비색법검측반광안산천동안산단백매-3(caspase-3)활성.결과 CSC경자주분선후c-kit양성솔가체85％이상.기아조세포조망솔명현고우정상대조조[(27.03±0.80)％비(1.51±0.54)％,P＜0.01].기아+SDF-1α조각아조세포조망솔균명현저우기아조(P균＜0.01),CSC조망솔재기아+SDF-1α 10、50、100 ng/ml간예조점차강저,이100 ng/ml간예조위최저[(10.67±1.06)％,여기아조비교P＜0.01].기아+SDF-1α 100 ng/ml조p-Akt화Bcl-2적단백표체균명현고우기아조(P균＜0.01),이caspase-3활성칙균명현저우기아조(P균＜0.01).기아+SDF-1α+ AMD3100조화기아+SDF-1 α+LY294002조p-Akt화Bcl-2적단백표체균교기아+SDF-1α 100 ng/ml조저(P균＜0.01),이caspase-3활성균명현고우기아+SDF-1α100 ng/ml조(P균＜0.01).결론 SDF-1α가억제혈청박탈유도적CSC조망,차해작용시통과SDF-1 α/CXCR4축격활PI3K/Akt통로실현적.
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.