缺氧环境下大鼠骨髓间充质干细胞的凋亡及Fas、Fas-L的表达  

APOPTOSIS AND EXPRESSION OF FAS AND FAS-L PROTEINS IN RAT BONE MESENCHYMAL STEM CELLS UNDER HYPOXIA CONDITIONS

作　　者：孔宏亮[1] 刘宁宁[2] 张海鹏[3] 池志红[4] 齐国先[1] 

机构地区：[1]中国医科大学附属第一医院心脏内科,辽宁沈阳110001 [2]中国医科大学附属第一医院眼科,辽宁沈阳110001 [3]中国医科大学附属第一医院病理生理学教研室,辽宁沈阳110001 [4]中国医科大学附属第一医院组织与胚胎学教研室,辽宁沈阳110001

出　　处：《中国组织化学与细胞化学杂志》2007年第5期538-543,共6页Chinese Journal of Histochemistry and Cytochemistry

摘　　要：目的探讨大鼠骨髓间充质干细胞(MSCs)在缺氧环境下是否存在凋亡及Fas和Fas-L蛋白在MSCs凋亡时是否表达及与细胞凋亡的相关性。方法将接种的P3细胞置于94%N2、1%O2和5%CO2缺氧箱中37℃孵育,分别于0.5h、1h、2h、4h、6h、8h和12h取出进行缺口末端标记(TUNEL)计算细胞凋亡指数(ApoptoticIndex,AI)和透射电镜观察细胞超微结构的改变,用Fas和Fas-L免疫组化试剂盒检测Fas-L和Fas蛋白的表达。结果1.培养的骨髓单个核细胞CD29、CD71、CD44免疫细胞化学染色均阳性,CD34染色阴性,5-氮胞苷诱导后表达TnT,提示其为MSCs。2.MSCs在缺氧前、缺氧0.5、1、2、4、6、8和12h时AI分别为0.11±2.03%、15.4±3.19%、16.7±2.51%、16.9±0.25%、17.7±2.50%、18.3±3.15%、18.4±4.22%、19.7±4.58%,缺氧不同时间点AI较缺氧前均显著性增高(P<0.01),并随着缺氧时间延伸,AI显著增加(P<0.05),不过,缺氧6h、8h和12h时AI没有统计学意义(P>0.05)。3.MSCs在不同缺氧时间点Fas、Fas-L蛋白表达较缺氧前均显著性增高(P<0.05),且随着缺氧时间延伸,表达显著增加,而在缺氧6h-12h时间点表达没有统计学意义(P>0.05)。3.缺氧0.5、1、2、4、6、8和12h,AI与Fas和Fas-L均显著正相关。结论缺氧是促进MSCs凋亡的重要因素,Fas和Fas-L蛋白可能在MSCs缺氧凋亡的调控中起着重要作用。Objeetiw To elucidate whether bone mesenchymal stem cells （MSCs） of rats undergo apoptosis under hypoxia and whether there exists the expression of Fas and Fas-L proteins in apoptotic MSCs. Methods The passage 3 MSCs were cultured in a chamber with 94% N2, 1% O2, 5% CO2 at 37℃. At different hypoxia time points （0, 0.5, 1, 2, 4, 6, 8 and 12 h）, MSCs were labeled by TdT-mediated d-UTP nick end labeling （TUNEL） to calculate the apoptotic index （AI） of MSCs. Immunocytochemistry was used to observe the expression of Fas and Fas-L proteins. Transmission electron microscopy was used to observe the ultrastructural changes of MSCs. Results 1. Immunocytochemical staining for CD29^＋ , CD71^＋ , CIM4^＋ , CD34^- and Tn T^＋ after 5-azacytidine induction suggested they were MSCs. 2. At different hypoxia time points （0, 0. 5, 1,2, 4, 6, 8 and 12 h）, the apoptotic index （AI） of MSCs was 0. 11 ±2. 03%, 15.4 ±3.19%, 16. 7 ±2. 51%, 16. 9 ±0. 25%, 17.7 ± 2. 50%, 18.3 ±3.15%, 18.4 ±4. 22%, 19. 7 ±4.58%, respectively. The AI of MSCs at different hypoxia time points were significantly increased than before hypoxyia （P 〈 0.01 ）, and was significantly increasing （P 〈 0. 05）, though with out statistic significane at 6 h, 8 h and 12 h （P〉0. 05） . 3. At the above different hypoxia time points, the expression of Fas and Fas-L proteins was significantly increased than before hypoxyia （ P 〈 0. 05 ）, and was increasing with time （P 〈0. 05 ）, though with out statistic significane （P 〉0. 05 ） at 6 h, 8 h and 12 h. 3. At different hypoxia time points （0, 0. 5, 1, 2, 4, 6, 8 and 12 h）, correlation analysis suggested that the AI of MSCs was positive correlated with the values of Fas and Fas-L proteins, respecive. Conclusion Hypoxia is one of the important factors provoking apoptosis of MSCs, and the proteins Fas and Fas-L may play an indispensible role in the process of MSC apoptosis.

关 键 词：骨髓间充质干细胞 细胞凋亡指数 FAS蛋白 FAS-L蛋白 

分 类 号：Q255[生物学—细胞生物学]