大鼠骨髓间充质干细胞去血清培养后钙激活中性蛋白酶10基因转录变化

Changes of Calpain-10 Gene Transcription in Bone Marrow Stem Cells Treated by Serum Deprivation in Rats

目的检测血清剥夺是否诱导骨髓间充质干细胞(BMSCs)凋亡,探讨钙激活中性蛋白酶-10(Calpain-10)是否参与血清剥夺诱导的大鼠BMSCs凋亡。方法原代培养的大鼠BMSCs,流式细胞术鉴定细胞表型,去血清培养,应用PI/Annexin-V双染法流式细胞仪计数0、24、48h凋亡细胞的比例,应用荧光定量PCR测定血清剥夺24h诱导凋亡的Calpain-10基因mRNA水平。结果流式细胞术鉴定细胞发现CD29、CD44、CD105表达阳性,CD45、CD14、CD34表达阴性,符合BMSCs特点;血清剥夺后24h早期凋亡细胞比例最高。荧光定量PCR结果示与正常对照组比较血清剥夺诱导凋亡24h Calpain-10基因mRNA水平显著上调(2-ΔΔCT=22.433)。结论血清剥夺可诱导大鼠BMSCs凋亡,并上调Calpain-10基因mRNA水平,Calpain-10可能参与血清剥夺诱导的大鼠BMSCs凋亡。

Objective To determine whether serum deprivation can induce apoptosis of bone marrow stem cells （BMSCs） in rats,and explore whether Calpain-10 involved in the apoptosis of BMSCs.Methods Primary cultured rat BMSCs were incubated.Cells phenotype was identified by flow cytometry. Apoptotic cells were counted by flow cytometry following PI/Annexin-V staining. Fluorescence quantitation polymerase chain reation（PCR） was used to detect Calpain-10 mRNA levels in BMSCs at 24 h after serum deprivation.Result Cell phenotype identification showed positive antigen expressions with CD29,CD44,CD105 and negative antigen expressions with CD45,CD14,CD34, Apoptotic BMSCs of early stage took up the highest proportion of the whole cells at the time point of 24 h after serum deprivation compared with the other 2 time points, 0 h and 48 h. Compared with normal control ,the mRNA levels of Calpain-10 in BMSCs exposed to serum deprivation for 24 h significantly increased（2-ΔΔCT=22.433）.Conclusion Serum deprivation can induce apoptosis of BMSCs, and Calpain-10 may be involved in the apoptosis of rat BMSCs.