摘要
目的探讨缝隙连接蛋白43(connexin 43,Cx43)对骨髓间充质干细胞(bone marrow mesen chymal stem cells,BMSCs)成骨分化的影响。方法将Cx43目的基因克隆后与慢病毒载体pHBLV CMVIE Puro进行连接,重组载体与包装载体共同转染293T细胞后包装生产病毒。获取小鼠BMSCs,流式细胞术检测细胞表面标志。将慢病毒载体pHBLV Cx43 Puro(Cx43组)、pHBLV CMVIE Puro(空载体组)转染BMSCs,同时设立空白对照组;Western blot和qRT PCR检测三组细胞中Cx43的表达。成骨诱导培养基诱导三组细胞成骨分化,qRT PCR检测成骨相关基因Runt相关转录因子2(runt related transcrip tion factor 2,Runx2)、锌指结构转录因子(Osterix/Sp7)、转录活化因子4(activating transcription factor 4,ATF4)、骨唾蛋白(bone sialoprotein,BSP)的表达;碱性磷酸酶(alkaline phosphatase,ALP)活性检测试剂盒定量分析三组BMSCs的成骨分化能力。结果流式细胞术检查示BMSCs中CD29、CD44、CD90呈阳性表达,而CD14和CD34呈阴性表达,pHBLV Cx43 Puro转染BMSCs后Cx43蛋白和mRNA表达显著增加。成骨诱导后Cx43组成骨相关基因Runx2、Osterix/Sp7、ATF4和BSP的mRNA表达显著高于其他两组(P均<0.05),ALP活性明显高于其他两组(P<0.05)。结论Cx43高表达能够显著促进BMSCs向成骨细胞分化,其可能成为骨损伤修复的有效靶点。
Objective To investigate the effect of Connexin 43(Cx43)on the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).Methods The Cx43 gene was cloned and connected to the lentiviral expression vector pHBLV CMVIE Puro.The recombinant lentiviral vectors and packaging plasmids were mixed and co transfected into 293T cells for packaging and producing virus.BMSCs were isolated from cultured mice.Flow cytometry was used to identify the surface markers.BMSCs were transfected with the lentiviral vector pHBLV Cx43 Puro(Cx43 group)and pHBLV CMVIE Puro(empty body group),and a blank control group was established.Cx43 mRNA and protein were detected by qRT PCR and Western blotting in these groups.The expression of osteoblast specific genes in BMSCs,such as runt related transcription factor 2(Runx2),Osterix/Sp7,activating transcription factor(ATF4)and bone sialoprotein(BSP)was detected by qRT PCR after the osteoinductive culture.The expression of ALP enzyme activity was determined by ALP activity assay.Results Flow cytometry showed that BMSCs were positive for CD29,CD44,CD90,and negative for CD14,CD34.The Cx43 mRNA and protein expression of BMSCs transfected with pHBLV Cx43 Puro was significantly higher than other two groups.The expression levels of osteogenic related genes(Runx2,Osterix/Sp7,ATF4,BSP)and ALP activity in Cx43 lentiviral transfected BMSCs after induction were significantly higher than other two groups(P<0.05).Conclusion The osteoblastic differentiation of BMSCs can be remarkably promoted by Cx43 high expression.Cx43 may be an effective intervention target for the repair of bone injury.
作者
谢锡洪
张振伟
林泽金
林利忠
XIE Xi hong;ZHANG Zhen wei;LIN Ze jin;LIN Li zhong(Guangzhou Medical University,Guangzhou 511400,China;Department of Hand Surgery,Shenzhen Shajing People s Hospital Affiliated to Guangzhou Medical University,Shenzhen 518104,China;Department of Traumatic Orthopaedics,Shenzhen Longhua District Center Hospital,Shenzhen 518110,China)
出处
《骨科》
CAS
2020年第2期149-154,共6页
ORTHOPAEDICS
基金
龙华区科技计划医疗卫生项目(2030228)。
关键词
骨髓间充质干细胞
缝隙连接蛋白43
成骨分化
慢病毒
Bone marrow mesenchymal stem cells
Connexin 43
Osteogenic differentiation
Lentiviral
