鹿茸Ⅰ型胶原对骨髓间充质干细胞增殖的影响及其与Ⅱ型胶原和聚集蛋白聚糖表达的关系  

Effects of velvet antler collagen typeⅠon proliferation of bone marrow mesenchymal stem cells and its relationships with typeⅡcollagen and aggrecan expressions

作　　者：隋欣 徐岩[2] 周佳 王伟楠[3] 张明天 韩冬[2] 李娜 杨擎 曲晓波[2] 黄晓巍[2] SUI Xin;XU Yan;ZHOU Jia;WANG Weinan;ZHANG Mingtian;HAN Dong;LI Na;YANG Qing;QU Xiaobo;HUANG Xiaowei(Experimental Center,Affiliated Hospital,Changchun University of Chinese Medicine,Changchun 130117,China;Department of Clinical Pharmacy and Pharmacology of Chinese Medicine,School of Pharmacy,Changchun University of Chinese Medicine,Changchun 130117,China;Department of Medicinal Chemistry and Chinese Medicine Chemistry,School of Pharmacy,Changchun University of Chinese Medicine,Changchun 130117,China;Department of Pharmacology,Jilin Ginseng Academy,Changchun University of Chinese Medicine,Changchun 130117,China)

机构地区：[1]长春中医药大学附属医院实验中心,吉林长春130117 [2]长春中医药大学药学院临床药学与中药药理教研室,吉林长春130117 [3]长春中医药大学药学院药物化学与中药化学教研室,吉林长春130117 [4]长春中医药大学吉林省人参科学研究院药理组,吉林长春130117

出　　处：《吉林大学学报（医学版）》2020年第5期992-997,I0004,共7页Journal of Jilin University：Medicine Edition

基　　金：吉林省卫计委青年科技骨干培养计划项目资助课题(2017Q046)。

摘　　要：目的:观察鹿茸Ⅰ型胶原(VACTⅠ)对骨髓间充质干细胞(BMSCs)增殖及细胞周期的影响,探讨其与Ⅱ型胶原和聚集蛋白聚糖表达的关系。方法:选取健康雄性4周龄SD大鼠,差时贴壁法提取BMSCs并传代培养。实验分为空白对照组、转化生长因子β3(TGF-β3)(10μg·L^-1)组和不同浓度(1.25、2.50、5.00、10.00和20.00 g·L^-1)VACTⅠ组,CCK-8法检测各组BMSCs增殖率,ELISA法检测各组细胞上清液中骨形态生成蛋白4(BMP-4)和转录因子Sox9水平,流式细胞术检测各组不同细胞周期BMSCs百分率,RT-PCR法和Western blotting法分别检测各组细胞中Ⅱ型胶原和聚集蛋白聚糖mRNA和蛋白表达水平。结果:与空白对照组比较,2.50~20.00 g·L^-1 VACTⅠ组大鼠BMSCs增殖率明显降低(P<0.05或P<0.01);ELISA法,与空白对照组比较,TGF-β3组和不同浓度VACTⅠ组细胞上清液中BMP-4和Sox9水平明显升高(P<0.05或P<0.01);流式细胞术,与空白对照组比较,TGF-β3组和不同浓度VACTⅠ组不同细胞周期BMSCs百分率差异无统计学意义(P>0.05);RT-PCR法,与空白对照组比较,TGF-β3组和不同浓度VACTⅠ组细胞中Ⅱ型胶原和聚集蛋白聚糖mRNA表达水平明显升高(P<0.05);Western blotting法,与空白对照组比较,TGF-β3组和不同浓度VACTⅠ组细胞Ⅱ型胶原和聚集蛋白聚糖蛋白表达水平明显升高(P<0.05或P<0.01)。结论:VACTⅠ可促进BMSCs释放BMP-4和Sox9,提高Ⅱ型胶原和聚集蛋白聚糖表达水平,抑制BMSCs增殖,是一种潜在的成软骨分化诱导剂。Objective:To observe the effects of velvet antler collagen typeⅠ(VACTⅠ)on the proliferation and cell cycle of bone marrow mesenchymal stem cells(BMSCs),and to explore its relationships with the expressions of typeⅡcollagen and aggrecan.Methods:The healthy male SD rats aged 4 weeks were chosen and the BMSCs were extracted by differential adhesion method and cultivated.The experiment was divided into blank control group,transforming growth factorβ3(TGF-β3)(10μg·L^-1)group,and different concentrations(1.25,2.50,5.00,10.00,and 20.00 g·L^-1)of VACT I groups.CCK-8 assay was used to detect the proliferation rates of BMSCs in various groups.The levels of bone morphogenetic protein-4(BMP-4)and transcription factor Sox9 in the cell supernatant in various groups were detected by ELISA method.The percentages of BMSCs in different cell cycles in various groups were detected by flow cytometry.The expression levels of typeⅡcollagen and aggrecan mRNA and proteins in BMSCs in various groups were detected by RT-PCR and Western blotting methods,respectively.Results:Compared with blank control group,the proliferation rates of BMSCs of the rats in 2.50-20.00 g·L^-1 VACTⅠgroups were significantly decreased(P<0.05 or P<0.01).The ELISA results showed that compared with blank control group,the levels of BMP-4 and Sox9 in the cell supernatant of the rats in TGF-β3 group and different concentrations of VACTⅠgroups were significantly increased(P<0.05 or P<0.01).The flow cytometry results showed that compared with blank control group,the percentages of BMSCs in different cell cycles of the rats in TGF-β3 group and different concentration of VACTⅠgroups had no significant differences(P>0.05).The RT-PCR results showed that compared with blank control group,the mRNA expression levels of typeⅡcollagen and aggrecan in TGF-β3 group and different concentrations of VACTⅠgroups were significantly increased(P<0.05).The Western blotting results showed that compared with blank control group,the expression levels of type Ⅱ co

关 键 词：鹿茸Ⅰ型胶原 骨髓间充质干细胞 成软骨 Ⅱ型胶原 聚集蛋白聚糖 

分 类 号：R329.[医药卫生—人体解剖和组织胚胎学]