β-catenin基因敲减与Sox9过表达联合对人间充质干细胞早期成软骨分化作用的实验研究  

Experimental study on early chondrogenic differentiation of human mesenchymal stemcells byβ-catenin gene knockdown combined with Sox9 overexpression

作　　者：张卫飞 孙军营 王方曦 李国庆 康斌[1] 王德利 曾晖[1] ZHANG Weifei;SUN Junying;WANG Fangxi;LI Guoqing;KANG Bin;WANG Deli;ZENG Hui(National&Local Joint Engineering Research Center of Orthopaedic Biomaterials,Department of Bone and Joint Surgery,Peking University Shenzhen Hospital,Shenzhen 518036,Guangdong,China)

机构地区：[1]骨科生物材料国家地方联合工程研究中心,北京大学深圳医院骨关节科,广东深圳518036

出　　处：《中华骨与关节外科杂志》2020年第1期56-61,共6页Chinese Journal of Bone and Joint Surgery

基　　金：深圳市科创委资助项目(JCYJ20170307111755218);深圳“医疗卫生三名工程”项目(SZSM201612092)。

摘　　要：背景:β-catenin基因抑制人间充质干细胞(hMSCs)成软骨分化,而Sox9基因促进hMSCs成软骨分化,但两者联合在hMSCs早期成软骨分化中的作用及其可能机制目前尚不明确。目的:探究β-catenin基因敲减与Sox9过表达联合对hMSCs早期成软骨分化的作用。方法:将骨髓来源hMSCs分为4组:阴性对照组,β-catenin基因敲减组,Sox9过表达组,β-catenin基因敲减与Sox9过表达联合组,加入成软骨分化培养基培养7 d。转染24 h,RT-PCR检测β-catenin基因敲减效率与Sox9过表达效率,RT-PCR检测COL2A1和ACAN的m RNA表达量,番红固绿染色、甲苯胺蓝染色、免疫细胞化学检测COL2A1和Aggrecan蛋白。结果:RT-PCR结果显示转染成功,β-catenin基因敲减效率与Sox9过表达效率较高(P<0.05)。β-catenin基因敲减与Sox9过表达联合组中COL2A1和ACAN的mRNA表达量显著高于其他3组(P<0.05),软骨成分染色更深(P<0.05)。免疫组化结果显示β-catenin基因敲减与Sox9过表达联合组的COL2A1和Aggrecan蛋白表达量显著高于其他3组(P<0.05)。结论:β-catenin基因敲减与Sox9过表达联合对hMSCs早期成软骨分化有促进作用。Background:β-catenin inhibits human mesenchymal stem cells(hMSCs)into chondrogenic differentiation,while Sox9 promotes chondrogenic differentiation of hMSCs.However,the role of the combination ofβ-catenin and Sox9 in hMSCs in early chondrogenic differentiation and its possible mechanism are still unclear.Objective:To investigate the effect ofβ-catenin gene knockdown combined with Sox9 overexpression on early chondrogenic differentiation of hMSCs.Methods:Bone marrow-derived hMSCs were divided into four groups:negative control group(empty vector),β-catenin gene knockdown group(rAAV-β-catenin-RNAi),Sox9 overexpression group(rAAV-FLAG-Sox9),andβ-catenin gene knockdown and Sox9 overexpression group(combined group,β-catenin-RNAi+rAAV-FLAG-Sox9).The chondrogenic differentiation medium was cultured for 7 d.After transfection for 24 h,the efficiency ofβ-catenin gene knockdown and Sox9 overexpression were detected by RT-PCR.The mRNA expression of COL2A1 and ACAN was detected by RT-PCR.Blue staining,immunocytochemistry detection of COL2A1 and Aggrecan proteins.Results:RT-PCR results showed that transfection was successful,β-catenin gene knockdown efficiency and Sox9 overexpression efficiency were higher(P<0.05).RT-PCR results showed that the mRNA expression level of COL2A1 and ACAN of combined group was higher than that of the first three groups(P<0.05).The staining results showed that the combined group stained more deeply than the cartilage components of the previous three groups(P<0.05).Immunohistochemistry showed that the protein expression levels of COL2A1 and Aggrecan in the combined group were higher than those in the first three groups(P<0.05).Conclusions:The combination ofβ-catenin gene knockdown and Sox9 overexpression can promote early chondrogenic differentiation of hMSCs.

关 键 词：Β-CATENIN基因 SOX9 HMSCS 成软骨分化 

分 类 号：R68[医药卫生—骨科学]