Sox9基因过表达对人脂肪干细胞增殖、凋亡及成软骨分化的影响及机制研究
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李春亮等 Sox9 基因过表达对人脂肪干细胞增殖、凋亡及成软骨分化的影响及机制研究 第 17 期 doi：10. 3969/j. issn. 1000-484X. 2021. 17. 0因过表达对人脂肪干细胞增殖、凋亡及成软骨分化的影响及机制 研究①
李春亮 秦 凤 齐普良 孙凯华 韩雪来 张金柱 刘延蓉 李钊伟 王 凯 李泽清 （青海省人民医院骨科，西宁 810001）
中图分类号 R687. 4 文献标志码 A 文章编号 1000-484X（2021）17-2059-06 ［摘 要］ 目的：研究 Sox9 基因过表达对人脂肪干细胞（hADSCs）增殖、凋亡及成软骨分化的影响及机制。方法：酶消化 法分离 hADSCs 体外培养扩增，Lenti-Sox9-EGFP、Lenti-EGFP 慢病毒液分别感染 hADSCs，设为过表达组和空载组，另以无菌生 理盐水代替设为对照组。比较各组 Sox9 蛋白表达、细胞增殖、转化生长因子 β1（TGF-β1）、信号转导蛋白 3（Smad 3）蛋白表达、 细胞凋亡情况；hADSCs 成软骨诱导分化 2 周检测成软骨分化情况；比较各组 TGF-β1、Smad 3 蛋白表达情况。结果：显微镜下 观察 hADSCs 细胞形态正常，流式细胞计数鉴定 hADSCs 表面抗原符合生物学特征。与空载组和对照组比较，过表达组 Sox9 蛋 白相对表达量更高，MTT 试验 24 h、48 h、72 h 的 A 值与培养 24 h、48 h、72 h 后 TGF-β1、Smad 3 蛋白相对表达量较高，凋亡率降 低（P<0.001）。成软骨诱导分化 2 周后甲苯胺蓝染色显示过表达组 hADSCs 胞浆及细胞核为蓝紫色，空载组和对照组为微弱淡 蓝色；Ⅱ型胶原免疫组化染色过表达组为强阳性，空载组和对照组为弱阳性。与对照组和空载组比较，过表达组 TGF-β1、 Smad 3 蛋白相对表达量更高（P<0.001）。结论：Sox9 基因过表达可促进 hADSCs 增殖及成软骨分化，抑制 hADSCs 凋亡，其机制 可能与上调 TGF-β1、Smad 3 蛋白的表达有关。 ［关键词］ 性别决定区 Y 框蛋白 9；人脂肪干细胞；成软骨分化；增殖；凋亡
Effect of Sox9 gene overexpression on proliferation，apoptosis and chondrogenic differentiation of human adipose derived stem cells and its mechanism
LI Chun-Liang，QIN Feng，QI Pu-Liang，SUN Kai-Hua，HAN Xue-Lai，ZHANG Jin-Zhu，LIU Yan-Rong，LI Zhao-Wei，WANG Kai，LI Ze-Qing. Department of Orthopaedics，Qinghai Provincial People's Hospital，Xining 810001，China
［Abstract］ Objective：To study the effect and mechanism of Sox9 gene overexpression on proliferation，apoptosis and chondro⁃ genic differentiation of human adipose derived stem cells（hADSCs）. Methods：hADSCs were isolated by enzyme digestion and cul⁃ tured in vitro. Lenti-Sox9-EGFP and Lenti-EGFP lentivirus solutions infected hADSCs respectively，and set them as overexpression group and no-load group. In addition，sterile saline was used as a control group. The Sox9 protein expression，cell proliferation，trans⁃ forming growth factor β1（TGF-β1）and signal transduction protein 3（Smad 3）protein expression and apoptosis were compared. The chondrogenic differentiation of hADSCs was detected 2 weeks after induction. The expression of TGF- β1，Smad 3 protein in each group were compared. Results：The morphology of hADSCs was normal under microscope，and the surface antigen of hADSCs was con⁃ sistent with biological characteristics by flow cytometry. Compared with no-load group and control group，the relative expression of Sox9 protein，the A value of MTT test at 24 h，48 h and 72 h，the expression of TGF-β1 and Smad 3 protein after 24 h，48 h and 72 h of culture in the overexpression group were higher，the apoptosis rate was lower（P<0.001）. After 2 weeks of chondrogenic differentia⁃ tion，toluidine blue staining showed that the cytoplasm and nucleus of hADSCs in the overexpression group were blue purple，while those in the no-load group and the control group were weak light blue. Type Ⅱ collagen was strongly positive in the over expression group and weakly positive in the no-load and the control group. Compared with the control group and the no-load group，the relative ex⁃ pression of TGF-β1，Smad 3 protein in the over expression group were higher（P<0.001）. Conclusion：Overexpression of Sox9 gene can promote the proliferation and chondrogenic differentiation of hADSCs，and inhibit the apoptosis of hADSCs，which the mechanism may be related to the up regulation of TGF-β1，Smad 3 protein expression.
李春亮 秦 凤 齐普良 孙凯华 韩雪来 张金柱 刘延蓉 李钊伟 王 凯 李泽清 （青海省人民医院骨科，西宁 810001）
中图分类号 R687. 4 文献标志码 A 文章编号 1000-484X（2021）17-2059-06 ［摘 要］ 目的：研究 Sox9 基因过表达对人脂肪干细胞（hADSCs）增殖、凋亡及成软骨分化的影响及机制。方法：酶消化 法分离 hADSCs 体外培养扩增，Lenti-Sox9-EGFP、Lenti-EGFP 慢病毒液分别感染 hADSCs，设为过表达组和空载组，另以无菌生 理盐水代替设为对照组。比较各组 Sox9 蛋白表达、细胞增殖、转化生长因子 β1（TGF-β1）、信号转导蛋白 3（Smad 3）蛋白表达、 细胞凋亡情况；hADSCs 成软骨诱导分化 2 周检测成软骨分化情况；比较各组 TGF-β1、Smad 3 蛋白表达情况。结果：显微镜下 观察 hADSCs 细胞形态正常，流式细胞计数鉴定 hADSCs 表面抗原符合生物学特征。与空载组和对照组比较，过表达组 Sox9 蛋 白相对表达量更高，MTT 试验 24 h、48 h、72 h 的 A 值与培养 24 h、48 h、72 h 后 TGF-β1、Smad 3 蛋白相对表达量较高，凋亡率降 低（P<0.001）。成软骨诱导分化 2 周后甲苯胺蓝染色显示过表达组 hADSCs 胞浆及细胞核为蓝紫色，空载组和对照组为微弱淡 蓝色；Ⅱ型胶原免疫组化染色过表达组为强阳性，空载组和对照组为弱阳性。与对照组和空载组比较，过表达组 TGF-β1、 Smad 3 蛋白相对表达量更高（P<0.001）。结论：Sox9 基因过表达可促进 hADSCs 增殖及成软骨分化，抑制 hADSCs 凋亡，其机制 可能与上调 TGF-β1、Smad 3 蛋白的表达有关。 ［关键词］ 性别决定区 Y 框蛋白 9；人脂肪干细胞；成软骨分化；增殖；凋亡
Effect of Sox9 gene overexpression on proliferation，apoptosis and chondrogenic differentiation of human adipose derived stem cells and its mechanism
LI Chun-Liang，QIN Feng，QI Pu-Liang，SUN Kai-Hua，HAN Xue-Lai，ZHANG Jin-Zhu，LIU Yan-Rong，LI Zhao-Wei，WANG Kai，LI Ze-Qing. Department of Orthopaedics，Qinghai Provincial People's Hospital，Xining 810001，China
［Abstract］ Objective：To study the effect and mechanism of Sox9 gene overexpression on proliferation，apoptosis and chondro⁃ genic differentiation of human adipose derived stem cells（hADSCs）. Methods：hADSCs were isolated by enzyme digestion and cul⁃ tured in vitro. Lenti-Sox9-EGFP and Lenti-EGFP lentivirus solutions infected hADSCs respectively，and set them as overexpression group and no-load group. In addition，sterile saline was used as a control group. The Sox9 protein expression，cell proliferation，trans⁃ forming growth factor β1（TGF-β1）and signal transduction protein 3（Smad 3）protein expression and apoptosis were compared. The chondrogenic differentiation of hADSCs was detected 2 weeks after induction. The expression of TGF- β1，Smad 3 protein in each group were compared. Results：The morphology of hADSCs was normal under microscope，and the surface antigen of hADSCs was con⁃ sistent with biological characteristics by flow cytometry. Compared with no-load group and control group，the relative expression of Sox9 protein，the A value of MTT test at 24 h，48 h and 72 h，the expression of TGF-β1 and Smad 3 protein after 24 h，48 h and 72 h of culture in the overexpression group were higher，the apoptosis rate was lower（P<0.001）. After 2 weeks of chondrogenic differentia⁃ tion，toluidine blue staining showed that the cytoplasm and nucleus of hADSCs in the overexpression group were blue purple，while those in the no-load group and the control group were weak light blue. Type Ⅱ collagen was strongly positive in the over expression group and weakly positive in the no-load and the control group. Compared with the control group and the no-load group，the relative ex⁃ pression of TGF-β1，Smad 3 protein in the over expression group were higher（P<0.001）. Conclusion：Overexpression of Sox9 gene can promote the proliferation and chondrogenic differentiation of hADSCs，and inhibit the apoptosis of hADSCs，which the mechanism may be related to the up regulation of TGF-β1，Smad 3 protein expression.