淫羊藿苷对微重力下骨髓间充质干细胞增殖与凋亡的影响

Effects of icariin on proliferation and apoptosis of bone mesenchymal stem cell under microgravity

目的探讨淫羊藿苷(ICA)对微重力下骨髓间充质干细胞(BMSCs)增殖与凋亡的影响。方法将BMSCs随机分为3组:微重力组、加药组和对照组,其中微重力组固定于平行平板旋转微重力效应模拟器于培养箱中培养,加药组将微重力和1×10-6mol/L ICA同时应用于细胞,对照组正常培养,不进行任何附加操作。采用MTT法检测BMSCs增殖,每组设置3个复孔;流式细胞术(FCM)检测BMSCs凋亡,每组细胞分3次测量;实时定量PCR(q PCR)检测BMSCs凋亡相关基因,每个样本的每种基因设置3个复孔;透射电镜(TEM)观察BMSCs亚细胞结构的变化。结果 MTT法检测显示,微重力组吸光度值显著低于对照组(P<0.01),加药组细胞吸光度值明显高于对照组(P<0.01)。流式细胞术检测显示,微重力组细胞凋亡率明显高于对照组(P<0.01);加药组细胞凋亡率明显低于对照组(P<0.01)。q PCR检测提示,微重力组Bcl-2、Bax/Bcl-2、P53和Bax的表达水平均较对照组明显升高(均P<0.05);加药组P53、Bcl-2、Bax和Bax/Bcl-2的表达均较微重力组显著降低(均P<0.01)。透射电镜下可见,加药组细胞形态大致正常,与对照组比较,未发生明显变化,微重力组部分细胞可见细胞膜皱缩、细胞膜表面微绒毛显著减少甚至消失,胞浆浓缩,核糖体、线粒体等聚集在一起,细胞核均一固缩,染色质浓缩致密、呈向核膜下聚集。结论微重力可抑制BMSCs增殖、促进细胞凋亡,但适当浓度ICA可逆转微重力所引起的这种有害影响,对BMSCs起相应的保护作用。

Objective To investigate the effect of icariin （ICA） on the proliferation and apoptosis of bone mesenchymal stem cell （BMSCs） under microgravity. Methods The BMSCs were randomly divided into three groups： microgravity group, dosing group and control group. The microgravity group and dosing group was treated with microgravity, besides, 1×10-6 mol/L ICA was applied to the cells in dosing group; the control group was cultured without any additional operation. Cell proliferation was detected by MTT assay, each group was set up 3 reduplicateholes; the apoptosis was detected by FCM, each group was detected 3 times; the expression of apoptosis related genes were detected by QPCR with 3 reduplicate holes, and the subcellular structure was observed by TEM. Results The result of MTT examination showed that the absorbance value of microgravity group was significantly lower than that of the control group （P 〈 0.01）, and the dosing group was significantly higher than that of the control group （P 〈 0.01）. FCM method showed that the apoptosis rate of microgravity group was significantly higher than control group （P 〈 0.01）, and the apoptosis rate of the dosing group was significantly lower than control group （P 〈 0.01）. Compared with the control group, the expression of Bcl-2, Bax/bcl-2, Bax, and P53 gene was up-regulated in microgravity group （all P 〈 0.05）; in dosing group, the expression of Bax, Bcl-2, P53 and the Bax/bcl-2 gene was significantly down-regulated when compared with microgravity group （all P 〈 0.01）. TEM indicated that the dosing group cells were generally normal and with no obvious changes when compared with the control group; in microgravity group, the cells existed membrane shrinking, microvilli decreasing, cytoplasmic concentration, ribosome and mitochondria gathering, nucleus pyknosis, and dense chromatin gathering to the margin of nuclear membrane. Conclusion Microgravity can inhibit osteoblast′s proliferation and promote apoptosis of BMSCs. But the appropriate concentration of ICA can protect BMSCs from the harmful effect caused by microgravity.