表没食子儿茶素没食子酸酯对大鼠干细胞成骨分化作用的研究  

Effect ofepigallocatechin gallate on osteogenic differentiation of rat stem cells

作　　者：程琳 姚洁 隋萍 李兵 陈习驰 靳攀 Cheng Lin;Yao Jie;Sui Ping;Li Bing;Chen Xichi;Jin Pan(Health Science Center,Yangtze University,Jingzhou 434023,Hubei,China)

机构地区：[1]长江大学医学部,湖北荆州434023

出　　处：《右江民族医学院学报》2022年第3期315-319,335,共6页Journal of Youjiang Medical University for Nationalities

基　　金：国家自然科学基金项目(81860386);长江大学“校级大学生创新创业训练计划项目”(Yz2021292)。

摘　　要：目的通过比较表没食子儿茶素没食子酸酯(EGCG)对大鼠干细胞的直接作用和与成骨诱导液的协同作用,探究EGCG在成骨分化中的作用。方法利用全骨髓贴壁法分离大鼠骨髓间充质干细胞(BMSCs)。培养至第三代,将细胞分为4组:A组(BMSCs):单纯用完全培养基培养BMSCs;B组(BMSCs+EGCG):将BMSCs在添加10μmol/L EGCG的完全培养基中培养;C组(BMSCs+Osteoinductive fluid):用成骨诱导液培养BMSCs;D组(BMSCs+Osteoinductive fluid+EGCG):将BMSCs在添加10μmol/L EGCG的成骨诱导液中培养。培养7 d和14 d,通过CCK-8检测细胞增殖情况,通过碱性磷酸酶(ALP)检测、ALP染色和成骨相关基因骨钙素(Ocn)、Ⅰ型胶原(Col1a1)、骨涎蛋白(Bsp)、Runt相关转录因子2(Runx2)等的表达水平评估EGCG在大鼠干细胞成骨分化中的作用。结果CCK-8检测发现C组和D组细胞增殖均明显低于A组和B组,但是C组与D组差异不显著,说明EGCG对大鼠干细胞的增殖影响不明显;ALP检测中A组与B组差异不显著,D组与C组比较差异有统计学意义,且ALP染色中D组染色明显高于C组,同时在成骨相关基因表达的检测中D组mRNA表达水平也明显高于C组,这些结果说明单纯EGCG对干细胞成骨作用比较微弱,但是在成骨诱导液存在时其成骨作用大大增强。结论EGCG发挥促进成骨作用需要依赖其它因素,其自身不能启动成骨过程。Objective To explore the influence of epigallocatechin gallate(EGCG)on osteogenic differentiation via comparing the direct effect of EGCG and synergistic effect with osteoinductive fluid on rat stem cells.Methods The bone marrow mesenchymal stem cells(BMSCs)of the rats were isolated by the method ofwhole bone marrow adherence.In the third generation,the cells were divided into four groups:Group A,Group B,Group C and Group D.In Group A(BMSCs),the BMSCs were cultured in the complete medium alone.In Group B(BMSCs+EGCG),the BMSCs were cultured in the complete medium supplemented with 10μmol/L EGCG.In Group C(BMSCs+osteoprosthesis fluid),the BMSCs were cultured with osteogenic induction fluid.In Group D(BMSCs+Osteoprosthesis fluid+EGCG),the BMSCs were cultured in osteogenic induction fluid supplemented with 10μmol/L EGCG.On the 7th and 14th day of culture,the cell proliferation was detected by CCK-8.The role of EGCG in osteogenic differentiation of rat stem cells was evaluated by alkaline phosphatase(ALP)assay,ALP staining and the expression levels of osteogenesis-relatedgenes such as osteocalcin(Ocn),type I collagen(Col1a1),bone sialoprotein(Bsp)and Runt-associated transcription factor 2(Runx2).Results CCK-8 detection showed that cell proliferations in group C and D were lower than those in group A and B.However,no significant difference was found between group C and group D,which demonstrated that EGCG had no obvious effect on the proliferation of rat stem cells.There was no significant difference between group A and group B in ALP detection.There was statistically significant difference between group D and group C,and ALP staining in group D was significantly higher than that in group C.Meanwhile,in the detection of osteogenesis-related gene expressions,the mRNA expression level of group D was also significantly higher than that of group C.These results indicated that EGCG alone had a weak osteogenic effect on stem cells,but its osteogenic effect was greatly enhanced in the presence of osteogenic induction fluid.

关 键 词：表没食子儿茶素没食子酸酯 干细胞 成骨分化 骨质疏松症 

分 类 号：R580[医药卫生—内分泌]