高体积分数氧对骨髓间充质干细胞含半胱氨酸的天冬氨酸蛋白水解酶-3和增殖细胞核抗原表达的影响

Effect of hyperoxia on cysteinyl aspartate specific proteinase -3 and proliferating cell nuclear antigen expres-sions in bone mesenchymal stem cells

目的探讨高体积分数氧（高氧）对骨髓间充质干细胞（BMSCs）增殖细胞核抗原（PCNA）和含半胱氨酸的天冬氨酸蛋白水解酶一3（Caspase一3）表达的影响。方法原代培养SD大鼠BMSCs，待生长至融合度70％～80％时，将细胞分为2组：高氧暴露组和空气组，每组分为5个亚组，分别培养0、3、6、12和24h。采用倒置相差显微镜观察高氧暴露对BMSCs形态学的影响，Western blot法检测2组细胞Caspase-3和PCNA蛋白的表达。结果1．与空气组相比，高氧暴露组随着暴露时间的延长（〉12h），细胞之间间隙逐渐增大，细胞体积缩小，部分呈圆形，并逐渐出现细胞脱落、漂浮。2．与空气组相比，高氧暴露组细胞随着高氧时间的延长，Caspase-3表达水平逐渐增高，高氧处理6h细胞内Caspase-3表达即与空气组的差异有统计学意义（0．27±0．04比0．14±0．02，t=5．03，P=0．007）。3．与空气组相比，高氧暴露组细胞随着高氧时间的延长，PCNA表达水平逐渐降低，高氧处理6h细胞内PCNA表达即与空气组的差异有统计学意义（0．27±0．04比0．38±0．04，t=3．37，P=0．028）。结论高氧暴露促进Caspase-3的表达，PCNA蛋白的表达，BMSCs在高氧条件下增殖和凋亡受影响。

Objective To investigate the effect of hyperoxia on the expressions of cysteinyl aspartate specific proteinase- 3（ Caspase -3 ） and proliferating cell nuclear antigen （PCNA） in bone marrow mesenchymal stem cells （BMSCs）. Methods Primary BMSCs from SD rats were cultured in vitro, BMSCs grew to 70% - 80% degrees of fu- sion and then were randomly divided into room air group and hyperoxia exposure group. Each group was divided into 5 sub - groups, and cultured for O, 3,6,12, and 24 h respectively. Morphology investigation with inverted phase contrast microscope was adopted. The expressions of Caspase - 3 and PCNA protein levels were detected by Western blot. Results （1）As time wenty by, compared with room air group, the gap between cells increased, some of the cells be- came circular, and cell detachment and floating appeared in the hyperoxia -exposure group （ 〉 12 h） compared with room air group. （2）As time went on, compared with the room air group, the expression levels of Caspase -3 protein in the hyperoxia - exposure group were increased, and the difference was significant after 6 hours of culture （0.27 ± 0.04 vs 0. 14± 0. 02, t = 5.03, P = 0. 007 ）. （ 3 ） Compared with room air group, the PCNA levels of the hyperoxiaexpo- sure group（6 h） decreased, and the difference in PCNA protein expression levels was significant after 6 hours of culture （0.27 ±0.04 vs 0.38 ±0.04,t =3.37,P=0.028）. Conclusions Hyperoxia exposure increases Caspase -3 expres- sion levels and decreases PCNA expression,which may affect the proliferation and apoptosis of BMSCs.