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
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摘要
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 micro-gravity group(all P < 0.01). TEM indicated that the dosing group cells were genera lly 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.
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 micro-gravity group(all P < 0.01). TEM indicated that the dosing group cells were genera lly 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.
引文
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[5]牟欢,刘洋,李俊杰,等.骨髓间充质干细胞在重度烧伤大鼠心肌损伤中的保护作用[J].中国医药导报,2017,14(12):12-16.
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[8]孟芮,续惠云,王哲,等.模拟失重骨细胞条件培养基对成骨细胞活性的影响[J].航天医学与医学工程,2009,22(4):247-251.
[9]李静,陈军,李秀玉,等.模拟微重力对骨髓间充质干细胞全细胞瘤苗干预A549移植瘤增殖的研究[J].中国医药导报,2016,13(3):8-12.
[10]毛新建.模拟微重力对骨髓间充质干细胞增殖和迁移的影响及相关分子机理研究[D].重庆:重庆大学,2014.
[11]黄懿文.模拟微重力状态下过氧化物酶体增殖物激活受体γ基因表达对成骨分化的影响[D].广州:南方医科大学,2013.
[12]李钢.淫羊藿苷及富自体浓缩生长因子促进兔颅骨缺损修复的分子机制探讨[D].石家庄:河北医科大学2015.
[13]黄国平,郑强,杨金凤,等.模拟微重力对人骨髓间充质干细胞向成骨细胞分化中细胞信号通路影响的分析[J].空间科学学报,2008(1):87-96.
[14]石东燕.模拟微重力对人骨髓间充质干细胞向成骨细胞分化的影响[D].杭州:浙江大学,2010.
[15]刘天麟,张学斌,张忻,等.淫羊藿苷对犬骨髓间充质干细胞增殖、成骨和成脂分化的影响[J].口腔颌面外科杂志,2014,24(4):276-280.
[16]Balaburski GM,Hontz RD,Murphy ME.p53 and ARF:unexpected players in autophagy[J].Trends Cell Biol,2010,20(6):363-369.
[17]胡汪来,吴缅.p53磷酸化修饰及其功能研究进展[J].中国科学:生命科学,2015,45(11):1093-1100.
[18]杜联芳,刘望彭.诊断超声辐照后大鼠睾丸生精细胞P53m RNA、Bcl-2 m RNA表达改变[J].中华超声影像学杂志,2001,(3):53-55.
[19]李晓宁,迟蕾,吴磊,等.头针对急性脑梗死大鼠不同时间窗Bax与Bcl-2影响的研究[J].针灸临床杂志,2016,32(4):73-76,97.
[20]Salakou S,Kardamakis D,Tsamandas AC,et al.Increased Bax/Bcl-2 ratio up-regulates caspase-3 and increases apoptosis in the thymus of patients with myasthenia gravis[J].In Vivo,2007,21(1):123-132.
[2]赵文静,王历,王芝兰,等.淫羊藿的药理作用及临床应用研究进展[J].中医药信息,2016,33(2):105-108.
[3]Chen KM,Ge BF,Liu XY,et al.Icariin inhibits the osteoclast formation induced by RANKL and macrophage-colony stimulating factor in mouse bone marrow culture[J].Pharmazie,2007,62(5):388-391.
[4]朱鸿飞,郑军,徐小燕,等.不同浓度淫羊藿苷修复骨损伤:争议与探索[J].中国组织工程研究,2014,18(2):301-306.
[5]牟欢,刘洋,李俊杰,等.骨髓间充质干细胞在重度烧伤大鼠心肌损伤中的保护作用[J].中国医药导报,2017,14(12):12-16.
[6]Korn C,Méndez-Ferrer S.Myeloid malignancies and the microenvironment[J].Blood,2017,129(7):811-822.
[7]李军,张春秋,宋光明,等.高重力下成骨细胞的力学生物学响应[J].医用生物力学,2016,31(3):278-283,290.
[8]孟芮,续惠云,王哲,等.模拟失重骨细胞条件培养基对成骨细胞活性的影响[J].航天医学与医学工程,2009,22(4):247-251.
[9]李静,陈军,李秀玉,等.模拟微重力对骨髓间充质干细胞全细胞瘤苗干预A549移植瘤增殖的研究[J].中国医药导报,2016,13(3):8-12.
[10]毛新建.模拟微重力对骨髓间充质干细胞增殖和迁移的影响及相关分子机理研究[D].重庆:重庆大学,2014.
[11]黄懿文.模拟微重力状态下过氧化物酶体增殖物激活受体γ基因表达对成骨分化的影响[D].广州:南方医科大学,2013.
[12]李钢.淫羊藿苷及富自体浓缩生长因子促进兔颅骨缺损修复的分子机制探讨[D].石家庄:河北医科大学2015.
[13]黄国平,郑强,杨金凤,等.模拟微重力对人骨髓间充质干细胞向成骨细胞分化中细胞信号通路影响的分析[J].空间科学学报,2008(1):87-96.
[14]石东燕.模拟微重力对人骨髓间充质干细胞向成骨细胞分化的影响[D].杭州:浙江大学,2010.
[15]刘天麟,张学斌,张忻,等.淫羊藿苷对犬骨髓间充质干细胞增殖、成骨和成脂分化的影响[J].口腔颌面外科杂志,2014,24(4):276-280.
[16]Balaburski GM,Hontz RD,Murphy ME.p53 and ARF:unexpected players in autophagy[J].Trends Cell Biol,2010,20(6):363-369.
[17]胡汪来,吴缅.p53磷酸化修饰及其功能研究进展[J].中国科学:生命科学,2015,45(11):1093-1100.
[18]杜联芳,刘望彭.诊断超声辐照后大鼠睾丸生精细胞P53m RNA、Bcl-2 m RNA表达改变[J].中华超声影像学杂志,2001,(3):53-55.
[19]李晓宁,迟蕾,吴磊,等.头针对急性脑梗死大鼠不同时间窗Bax与Bcl-2影响的研究[J].针灸临床杂志,2016,32(4):73-76,97.
[20]Salakou S,Kardamakis D,Tsamandas AC,et al.Increased Bax/Bcl-2 ratio up-regulates caspase-3 and increases apoptosis in the thymus of patients with myasthenia gravis[J].In Vivo,2007,21(1):123-132.