TDP43慢病毒载体转染人脐带间充质干细胞与软骨细胞共培养后的增殖与凋亡  

Proliferation and apoptosis of chondrocytes co-cultured with TDP43 lentivirus transfected-human umbilical cord mesenchymal stem cells

作　　者：黄永明[1] 黄启明 刘焱杰 王竣 曹振武 田振江 陈博鉴 麦秀钧[1] 冯恩辉 Huang Yongming;Huang Qiming;Liu Yanjie;Wang Jun;Cao Zhenwu;Tian Zhenjiang;Chen Bojian;Mai Xiujun;Feng Enhui(Department of Orthopedics,Guangdong Provincial Hospital of Chinese Medicine,Guangzhou 510120,Guangdong Province,China;Yinuobo(Beijing)Biomedical Technology Co.,Ltd.,Beijing 100088,China;the Second Clinical Medical College of Guangzhou University of Chinese Medicine,Guangzhou 510405,Guangdong Province,China)

机构地区：[1]广东省中医院骨科,广东省广州市510120 [2]怡诺博(北京)生物医学技术有限公司,北京市100088 [3]广州中医药大学附属第二临床医院,广东省广州市510405

出　　处：《中国组织工程研究》2020年第7期1016-1022,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(81273781)，项目负责人：黄永明;广东省自然科学基金(2018A030313643)，项目负责人：黄永明~~

摘　　要：背景:在缺血缺氧条件下TDP43可能为MAPK信号通路关键的负调控因子,但其在骨性关节炎中对JNK及p38 MAPK信号通路的作用并不清楚。目的:研究野生型TDP43介导骨性关节炎中软骨细胞病变的靶基因RACK1表达,分析其发挥应激作用的效应。方法:以TDP43慢病毒载体转染人脐带间充质干细胞,分析其体外分化为软骨细胞的能力。将TDP43慢病毒转染脐带间充质干细胞、空载体慢病毒转染脐带间充质干细胞、未转染脐带间充质干细胞分别与人软骨细胞共培养12 d,倒置显微镜下观察软骨细胞形态变化;共培养第0,3,6,9,12天,分析软骨细胞增殖水平;共培养第3天,流式细胞仪检测软骨细胞凋亡率;共培养第3天,qRT-PCR检测软骨细胞内TDP43、RACK1、p38、JNK、AP-1和cl-xl的基因表达。结果与结论:①TDP43慢病毒载体转染后,人脐带间充质干细胞可分化为软骨细胞;②与TDP43慢病毒转染脐带间充质干细胞共培养的软骨细胞形态发生显著改变,细胞变得粗大,并出现多个分枝情况;与空载体慢病毒转染脐带间充质干细胞、未转染脐带间充质干细胞共培养的软骨细胞形态未出现变化,呈梭形贴壁生长;③软骨细胞与TDP43慢病毒转染脐带间充质干细胞共培养后,促使软骨细胞凋亡而抑制了细胞增殖(P<0.05);④软骨细胞与TDP43慢病毒转染脐带间充质干细胞共培养后,TDP43、RACK1、JNK、AP-1和Bcl-xl基因表达高于与未转染脐带间充质干细胞、空载体慢病毒转染脐带间充质干细胞共培养的软骨细胞(P<0.05);⑤结果表明,软骨细胞高表达TDP43可激活RACK1的表达,进而调控软细胞增殖和凋亡。BACKGROUND:TDP43 may be a negative regulator of MAPK signaling pathway under hypoxic-ischemic conditions.However,its effect on JNK and p38 MAPK signaling pathways in osteoarthritis remains unclear.OBJECTIVE:To investigate the expression of chondrocyte lesion-related gene RACK1 in wild type TDP43 involved in osteoarthritis,and to analyze its stress effect.METHODS:Human umbilical cord mesenchymal stem cells were transfected by TDP43 lentivirus,and the ability to differentiate into chondrocytes in vitro was analyzed.Umbilical cord mesenchymal stem cells transfected by TDP43 lentivirus,empty vector and without transfection were co-cultured with chondrocytes for 12 days.The chondrocyte proliferation was detected at 0,3,6,9 and 12 days of co-culture.The chondrocyte apoptosis rate was detected by flow cytometry at 3 days of co-culture.The expression levels of TDP43,RACK1,p38,JNK,AP-1 and cl-xl in chondrocytes were detected by qRT-PCR at 3 days of co-culture.RESULTS AND CONCLUSION:(1)After TDP43 lentivirus transfection,human umbilical cord mesenchymal stem cells could differentiate into chondrocytes.(2)The morphology of chondrocytes co-cultured with TDP43 lentivirus transfected-umbilical cord mesenchymal stem cells showed significant change,and the cells became large with abundant branches.Chondrocytes co-cultured with empty vector transfected-or non-transfected umbilical cord mesenchymal stem cells were spindle-shaped in appearance and showed adherent growth with no morphological changes.(3)After co-cultured with TDP43 lentivirus transfected-umbilical cord mesenchymal stem cells,the apoptosis of chondrocytes was promoted,and the cell proliferation was inhibited(P<0.05).(4)The expression levels of TDP43,RACK1,p38,JNK,AP-1 and Bcl-xl in the chondrocytes co-cultured with TDP43 lentivirus transfected-umbilical cord mesenchymal stem cells were significantly higher than those in the chondrocytes co-cultured with non-transfected-and empty vector-transfected-umbilical cord mesenchymal stem cells.(5)To conclude,high expression o

关 键 词：骨性关节炎 软骨细胞 TDP43 RACK1 人脐带间充质干细胞 凋亡信号通路 细胞增殖 细胞凋亡 

分 类 号：R459.9[医药卫生—治疗学] R684.3[医药卫生—临床医学]