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三七总皂苷对大鼠骨髓间充质干细胞凋亡的抑制作用

区泳芳, 伏学坤, 梅兴莎, 郑惠珍^*

1广东医科大学基础医学院生理学教研室，东莞 523808；2广东医科大学附属医院病理诊断与研究中心，湛江 524001

摘要

本文旨在研究三七总皂苷(total saponins of Panax notoginseng, tPNS)对二氯化钴(CoCl2)诱导的大鼠骨髓间充质干细胞
(rat bone marrow mesenchymal stem cells, rBMSCs)凋亡的抑制作用及机制。采用密度梯度离心法分离SD大鼠BMSCs。不同浓
度tPNS (1、10和100 μg/mL)处理rBMSCs 48 h后，行流式细胞术检测细胞增殖(EdU法)和细胞周期。300 μmol CoCl2孵育细胞
24 h诱导凋亡，在CoCl2处理的同时加入不同浓度tPNS (1、10和100 μg/mL)。Annexin V-FITC/PI染色后，用流式细胞仪检测
细胞凋亡率；罗丹明123染色后，在荧光显微镜下观察线粒体膜电位改变；qRT-PCR检测细胞Bcl-2家族的基因表达。结果显
示，tPNS各浓度组细胞增殖率均较对照组升高，100 μg/mL tPNS组G2+S期细胞百分比较对照组升高。与对照组相比，CoCl2
组细胞凋亡率增加14.2%，而tPNS三个浓度组细胞凋亡率分别较CoCl2组减少14.4%、12.8%和13.9% (均P < 0.01)。与对照组
相比，CoCl2组线粒体膜电位明显降低，而tPNS各浓度组膜电位降低程度明显低于CoCl2组。tPNS各浓度组的Bcl-2和Bcl-xl
mRNA表达均高于CoCl2组(均P < 0.05)；10和100 μg/mL tPNS组的Bax/Bcl-2比值较CoCl2组显著降低。以上结果提示，tPNS对
CoCl2诱导的rBMSCs凋亡有抑制作用，其机制是通过提高细胞线粒体膜电位，上调抗凋亡基因Bcl-2和Bcl-xl的表达，降低
Bax/Bcl-2比值而实现的。

关键词： 三七总皂苷; 骨髓间充质干细胞; 细胞增殖; 细胞周期; 细胞凋亡; 线粒体膜电位; 大鼠

Inhibitory effect of total saponins of Panax notoginseng on rat bone marrow mesenchymal stem cell apoptosis

OU Yong-Fang, FU Xue-Kun, MEI Xing-Sha, ZHENG Hui-Zhen^*

1Department of Physiology, College of Basic Medicine, Guangdong Medical University, Dongguan 523808, China； 2Pathological Diagnosis and Research Center, Affiliated Hospital of Guangdong Medical University, Zhanjiang 524001, China

Abstract

The study was aimed to investigate the effects of total saponins of Panax notoginseng (tPNS) on cobalt chloride (CoCl2)-induced apoptosis of rat bone marrow mesenchymal stem cells (rBMSCs) and the underlying mechanism. rBMSCs were isolated by density gradient centrifugation from Sprague Dawley (SD) rats. After being incubated with different concentrations of tPNS (1, 10, 100 μg/mL) for 48 h, the rBMSCs were stained with EdU and PI for proliferation and cell cycle assay, respectively. CoCl2 group was
treated with 300 μmol CoCl2 for 24 h, and different concentrations tPNS groups were treated with 300 μmol CoCl2 plus 1, 10 or 100 μg/mL tPNS. After Annexin V-FITC/PI staining, flow cytometry was applied to measure the cell apoptosis. For mitochondrial
membrane potential assay, rhodamine123 and Hoechst33342 staining were used. qRT-PCR was applied to analyze gene expression of Bcl-2 family. The results showed that the proliferation rates of the three concentrations tPNS groups were all higher than that of the control group (all P < 0.05). Compared with control group, only 100 μg/mL tPNS group exhibited increased cell percentage of S and G2 phase. Compared with that in control group (without CoCl2), the apoptotic rate was increased by 14.2% in CoCl2 group. And the apoptotic rates were reduced by 14.4%, 12.8% and 13.9% in three concentrations tPNS groups, compared with that in CoCl2 group (all P < 0.01). CoCl2 could decrease the mitochondrial membrane potential, while different concentrations of tPNS reversed the inhibitory effect of CoCl2. Bcl-2 and Bcl-xl mRNA expressions in all tPNS groups were higher than those in CoCl2 group (all P < 0.05). Moreover, 10 and 100 μg/mL tPNS groups showed lower ratios of Bax/Bcl-2, compared with CoCl2 group. The results suggest that tPNS protects the rBMSCs against CoCl2-induced apoptosis through improving the cell mitochondrial membrane potential, up-regulating the expressions of anti-apoptosis genes Bcl-2 and Bcl-xl, and reducing the Bax/Bcl-2 gene expression ratio.

Key words: total saponins of Panax notoginseng (tPNS); bone marrow mesenchymal stem cells; cell proliferation; cell cycle; apoptosis; mitochondrial membrane potential; rat

收稿日期：　　录用日期：

通讯作者：郑惠珍　　E-mail: zhenghz6@gdmc.edu.cn

引用本文：

区泳芳, 伏学坤, 梅兴莎, 郑惠珍. 三七总皂苷对大鼠骨髓间充质干细胞凋亡的抑制作用[J]. 生理学报 2016; 68 (3): 285-292.

OU Yong-Fang, FU Xue-Kun, MEI Xing-Sha, ZHENG Hui-Zhen. Inhibitory effect of total saponins of Panax notoginseng on rat bone marrow mesenchymal stem cell apoptosis. Acta Physiol Sin 2016; 68 (3): 285-292 (in Chinese with English abstract).