【摘要】 目的:利用预先铺被琼脂糖的方法抑制骨髓间充质干细胞贴壁,在体外模拟细胞离体后的悬浮状态,从而诱导细胞发生失巢凋亡,初步探讨半胱氨酸天冬氨酸蛋白酶3抑制剂在骨髓间充质干细胞失巢凋亡过程中的作用。方法:实验于2005-04/2006-01在华中科技大学同济医学院医学实验技术公共平台细胞工程实验室以及华中科技大学同济医学院附属协和医院骨科实验室完成。取体质量约150g的SPF级大鼠,雌雄不限,颈椎脱臼法处死。用DMEM培养液冲洗股骨和胫骨髓腔,应用全骨髓贴壁法进行原代培养。细胞铺满整个培养瓶底面80%时,进行传代接种培养,实验选用传第2代细胞。收集细胞随机分为3组:诱导凋亡组、抑制凋亡组、对照组。诱导凋亡组进行实验前,将无菌培养皿预先铺被已消毒的琼脂糖溶液,并待其凝固;抑制凋亡组除预处理培养皿外,在植入细胞的同时,加入半胱氨酸天冬氨酸蛋白酶抑制剂;对照组无任何干预措施。于试验开始后2,6,12,24h分别收集三组细胞,应用荧光比色法和蛋白质印迹法检测各样本半胱氨酸天冬氨酸蛋白酶3活性和表达水平的变化;借助流式细胞仪分析骨髓间充质干细胞失巢凋亡率的改变,实验重复两次。结果:①在对照组和抑制凋亡组中,半胱氨酸天冬氨酸蛋白酶3的活性维持在较低的水平(P>0.05),两组间差异无显著性;诱导凋亡组中,半胱氨酸天冬氨酸蛋白酶3的活性随着诱导持续时间的延长而明显增加,由2h的9.22上升到24h的22.74;诱导凋亡组与其他两组比较,差异具有显著性(P<0.001)。②对照组半胱天冬酶3蛋白的表达水平稳定;抑制凋亡组半胱天冬酶3蛋白的表达维持在低水平略有升高趋势,两组间无显著性差异。诱导凋亡组半胱氨酸天冬氨酸蛋白酶3蛋白的表达水平逐渐升高,12h达高峰;与其他两组比较,具有显著性差异(P<0.01)。③诱导凋亡组的细胞凋亡率随着诱导持续时间的延长而明显增加,与其他两组比较,具有显著性差异(P<0.001);抑制凋亡组和正常贴壁组,细胞凋亡率维持在较低的水平且无明显差异。结论:半胱氨酸天冬氨酸蛋白酶3抑制剂通过有效抑制半胱氨酸天冬氨酸蛋白酶3的活性和表达,可以明显降低细胞失巢凋亡率,起到保护骨髓间充质干细胞的作用。更多还原

【Abstract】 AIM:To prevent mesenchymal stem cells (MSCs) from adherence by pre-intervention with agarose,model cell suspension in vitro,which can induce anoikis of cells,and explore primarily the effect of caspase-3 inhibitor on anoikis of MSCs.METHODS:The experiment was performed at the Medical Experimental Technological Public Platform Cell Engineering Laboratory,Tongji Medical College,Huazhong University of Science and Technology and Laboratory of Department of Orthopaedics,Union Hospital Affiliated to Tongji Medical College,Huazhong University of Science and Technology from April 2005 to January 2006.SPF rats,of either sex,with the body mass of about 150 g were selected and killed by cervical dislocation.Cavities of femur and tibia were washed with DMEM medium.Primary culture was performed with entire marrow adherent method.Passage inoculation culture was done when cells spread out in 80% bottom of culture bottle.The 2nd generation cells were applied.The cultured MSCs were randomly divided into 3 groups:induced group,inhibitor group and control group.Sterile petri dish was spread with disinfectant agarose solutions for coagulation before experiment in the induced group.Except pretreatment,caspase inhibitor was added in the inhibitor group when implanting cells.Control group did not receive any intervention.Cells in the three groups were collected at hours 2,6,12 and 24,respectively.Activity and expression of caspase-3 were measured with fluorescent chromatometry and Western blotting.Change of anoikis rate of MSCs was detected with flow cytometry,and the experiment was done twice.RESULTS:①The caspase-3 activity was low in the control group and inhibitor group (P > 0.05),and there was no significant difference between the two groups.Caspase-3 activity increased significantly from 9.22 at hour 2 to 22.74 at hour 24 with the prolongation of induction in the induced group.There was significant difference among induced group with the other two groups (P < 0.001).②Caspase-3 expressed stably in the control group.Expression of caspase-3 was low and had the tendency to increase in the inhibitor group.There was no significant difference between control group and inhibitor group.The caspase-3 level increased gradually in the induced group and reached the peak at hour 12.Compared with the other two groups,it showed significant difference (P < 0.01).③Apoptotic rate in the induced group increased obviously with the prolongation of induction.Compared with the other two groups,it showed significant difference (P < 0.001).Apoptotic rate was low between inhibitor group and normal group,which showed insignificant difference.CONCLUSION:Caspase-3 inhibitor can reduce markedly the anoikis rate by effectively inhibiting the activity and expression of caspase-3 and play an important role in protecting MSCs.更多还原