无血清条件对诱导转染pEGFP-C1/Akt的骨髓间充质干细胞凋亡的影响  

Effect of Serum Deprivation-Induced Apoptosis on Mesenchymal Stem Cells Transfected with pEGFP-C1/Akt

作　　者：霍鑫[1] 孔宏亮[2] 刘方峰[3] 孟祥民[4] 刘英[5] 张强[1] 

机构地区：[1]中国医科大学附属第一医院干诊外科,沈阳110001 [2]中国医科大学附属第一医院循环科,沈阳110001 [3]中国医科大学附属第一医院普外科,沈阳110001 [4]黑龙江省双鸭山市中医院外科,双鸭山155100 [5]中国医科大学附属第一医院内分泌科,沈阳110001

出　　处：《中国普外基础与临床杂志》2008年第7期479-483,共5页Chinese Journal of Bases and Clinics In General Surgery

摘　　要：目的探讨无血清条件对诱导pEGFP-C1/Akt转染的骨髓间充质干细胞(MSCs)凋亡的影响。方法实验分实验组(pEGFP-C1/Akt转染MSCs)、抑制组(pEGFP-C1/Akt转染MSCs+PI3K/Akt特异性抑制剂)和对照组(MSCs)。在无血清培养基条件下,分别在4、12、24和36h共4个时间点利用RT-PCR检测Akt及Caspase-3mRNA表达,Western blot检测Akt蛋白表达,免疫组化检测Caspase-3蛋白表达,流式细胞仪检测细胞凋亡率。结果在无血清培养4、12、24和36h后,实验组的Akt mRNA和蛋白的表达均高于抑制组及对照组(P<0.01),Caspase-3mRNA及蛋白的表达水平和细胞凋亡率则均低于抑制组及对照组(P<0.01);抑制组及对照组间Akt和Caspase-3mRNA及蛋白的表达和凋亡率的差异无统计学意义(P>0.05)。结论Akt可能通过PI3K/Akt途径抑制其下游底物Caspase-3的表达,从而抑制无血清诱导的MSCs凋亡。Objective To explore the effect of serum deprivation （SD）-induced apoptosis on mesenchymal stem cells transfected with pEGFP-C1/Akt. Methods The study included experiment group （MSCs transfected with pEGFP-C1/Akt）, inhibit group （MSCs transfected by pEGFP-CI/Akt were inhibited by LY294002 20 μmol/L） and control group （MSCs）. At 4 h, 12 h, 24 h and 36 h after SD culture, the expressions of Akt and Caspase-3 mRNA were determined by RT-PCR. Western blot was used for detecting the expression of Akt protein. Immunohistochemistry was used to analyze the expression of Caspase-3 protein. Apoptosis was determined by flow cytometry. Results At 4 h, 12 h, 24 h and 36 h after SD culture, the expressions of Akt mRNA and protein had significant elevation in experiment group compared with inhibit group and control group, respectively （P（0.01）. The expressions of Caspase-3 mRNA and protein, and the percentage of apoptosis cells were markedly reduced in experiment group compared with inhibit group and control group, respectively （P〈0.01）. Moreover, there were no significant difference between inhibit group and control group about the expressions of Akt, Caspase-3 and the percentage of apoptosis cells. Conclusion Akt can protect MSCs from SD-induced apoptosis via PI3K/Akt and Caspase-3 pathway.

关 键 词：骨髓间充质干细胞 凋亡 天冬氨酸特异性半胱氨酸蛋白酶-3 丝/苏氨酸激酶 

分 类 号：Q813.1[生物学—生物工程]