缺氧环境下大鼠骨髓间充质干细胞凋亡相关蛋白和mRNA的表达

目的探讨大鼠骨髓间充质干细胞（MSCs）在缺氧环境下凋亡相关蛋白和mRNA的表达。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取出分别应用Annexin V／PI双染法进行流式细胞仪（FCM）分析MSCs凋亡率（Apoptotic Rate，AR），并同步用免疫细胞化学、western blotting和Rt-PCR等方法检测Bax／Bcl-2，Fas／FasL和Caspase-3蛋白和mRNA的表达。结果1．缺氧前，免疫细胞化学法未检测到Bcl-2、Bax、Fas、FasL和Caspase-3蛋白表达，缺氧0．5h后均可较强表达；2．各缺氧时间点Bcl-2、Bax、Fas、FasL、Caspase-3蛋白和mRNA表达较缺氧前均显著性增高（P均〈0．05）；随缺氧时间延伸，Bcl-2蛋白和mRNA表达不显著增加（P〉0．05），而Bax、Fas、FasL、Caspase-3蛋白和mRNA表达均显著增加（P均〈0．05），但缺氧6-12h时间点之间表达均没有统计学意义（P均〉0．05）；3．AR和Bcl-2／Bax蛋白（r1=0．417，P=0．043）及mRNA（r2=-0．435，P=0．040）呈显著负相关，而和Fas（r1=0．639，P=0．025；r2=0．711，P=0．018）、Fas-L（r1=0．581，P=0．022；r2=0．605，P=0．037）、Caspase-3（r1=0．704，P=O．014；r2=0．657，P=0．026）蛋白及mRNA均呈显著正相关。结论在缺氧促进MSCs凋亡的过程中，Bcl-2蛋白和mRNA可能起着保护作用，而Bax、Fas-L、Fas、Caspase-3蛋白和mRNA可能在MsCs凋亡的进程中起着促进作用。

EXPRESSION OF APOPTOSIS RELATED PROTEIN AND mRNA IN BONE MESENCHYMAL STEM CELLS OF RATS UNDER HYPOXIA

Objective To elucidate the expression of apoptosis related protein and mRNA, such as Bcl-2, Bax, Fas, Fas L and caspase-3 in bone mesenchymal stem cells （MSCs） of rats under hypoxia. Methods MSCs of passage 3 were cultured in chamber with 94%N2, 1%O2, 5%CO at 37℃. At different hypoxia time points （0, 0. 5, 1, 2, 4, 6, 8 and 12 h） the apoptotic rate （AR） was analyzed by flow cytometry （FCM） after Annexin V/PI staining, and the expression of Bcl-2, Bax, Fas, Fas L and caspase-3 protein and mRNA was observed by immunocytochemistry, Western blot and RT-PCIL Results 1. At normoxia, the proteins of Bcl-2, Bax, Fas, Fas L and Caspase-3 were not found by immunocytochemistry, but all of them were found after undergoing hypoxia. 2. At different hypoxia time points, the expression of the above protein and mRNA was significantly increased than at normoxia （P〈0.05） There was no statistically significant difference （P〉0. 05） in the expression of Bcl-2 during hypoxia. However, the protein and mRNA of Bax, Fas, Fas L and caspase-3 increased with time （P〈0.05）, but the increase was not significant （P〉0. 05） betwcen 6-12 h （P〉0.05）. 3. There was a significant positive relation between the AR and the ratio of Bcl-2/Bax （protein： r1 =-0. 417, P=0.043; mRNA： r2=-0. 435, P=0. 040） and a negative relation between AR and Fas （r1 =0. 639, P=0. 025; rz =0. 711, P=0. 018）, Fas-L （rl = 0. 581, P=0. 022; r2=0. 605, P=0. 037）, caspase-3 （r1 =0. 704, P=0. 014; r2 =0. 657, P=0. 026）. Conclusion During hypoxia which promotes apoptosis of MSCs, the protein and mRNA of Bcl-2 may be a protective factor, while, the protein and mRNA of Bax, Fas-L, Fas and Caspase-3 may provoke apoptosis.