共培养条件下肌卫星细胞C2C12对软骨细胞ATDC5活性的影响  

Effect of skeletal muscle satellite cells C2C12 on the activity of chondrocytes ATDC5 in the co-cultured condition

作　　者：陈泽华 王毅 申震 李俊毅 欧梁 CHEN Zehua;WANG Yi;SHEN Zhen;LI Junyi;OU Liang(The Orthopedics Hospital of Traditional Chinese Medicine Zhuzhou city,Zhuzhou 412007,Hunan,China;The Fifth Clinical Medical College of Guangzhou University of Chinese Medicine,Guangzhou 510095,Guangdong,China;Kunming Municipal Hospital of Traditional Chinese Medicine,Kunming 650599,Yunnan,China;Hunan Academy of Chinese Medicine,Changsha 410006,Hunan,China)

机构地区：[1]株洲市中医伤科医院,湖南株洲412007 [2]广州中医药大学第五临床医学院,广东广州510095 [3]昆明市中医医院,云南昆明650599 [4]湖南省中医药研究院,湖南长沙410006

出　　处：《中医正骨》2024年第2期17-22,31,共7页The Journal of Traditional Chinese Orthopedics and Traumatology

基　　金：湖南省中医药科研课题(B2023153);贵州省中医药管理局中医药、民族医药科学技术研究课题(QZYY-2019-031)。

摘　　要：目的:观察共培养条件下肌卫星细胞C2C12对软骨细胞ATDC5活性的影响。方法:①将肌卫星细胞C2C12分为对照组和地塞米松组,对照组常规培养,地塞米松组采用地塞米松干预,通过CCK8法和BCA法测定地塞米松对C2C12细胞增殖和蛋白合成的影响,以划痕实验和Transwell小室观察地塞米松对C2C12细胞修复和迁移能力的影响。②采用Transwell小室将正常肌卫星细胞C2C12(共培养组)和经地塞米松预处理的肌卫星细胞C2C12(预处理组)分别与软骨细胞ATDC5共培养,对照组Transwell下层小室内接种ATDC5细胞、上层小室内为无细胞的常规培养基,采用CCK8法和二氯二氢荧光素-乙酰乙酸酯荧光探针检测ATDC5细胞增殖率和细胞内活性氧含量。结果:①C2C12细胞增殖率和蛋白含量测定结果。地塞米松组C2C12细胞增殖率和蛋白含量均低于对照组[(78.402±5.401)%,(100.000±3.096)%,t=8.498,P=0.000;(5080.367±296.657)μg·mL-1,(5775.577±150.476)μg·mL-1,t=3.620,P=0.022]。②C2C12细胞伤口修复率测定结果。地塞米松组C2C12细胞伤口修复率低于对照组[(53.173±1.800)%,(79.979±10.176)%,t=4.493,P=0.011]。③C2C12细胞迁移数量测定结果。培养24 h和48 h时,地塞米松组C2C12细胞迁移数量均少于对照组[24 h:(24.200±5.630)个,(57.000±2.449)个,t=11.945,P=0.000;48 h:(57.600±8.820)个,(91.000±4.743)个,t=7.457,P=0.000]。④ATDC5细胞增殖率测定结果。3组ATDC5细胞增殖率比较,差异有统计学意义[(100.000±1.663)%,(116.894±7.917)%,(89.130±2.980)%,F=23.700,P=0.001]。对照组和共培养组ATDC5细胞增殖率均高于预处理组(P=0.037,P=0.006),共培养组ATDC5细胞增殖率高于对照组(P=0.001)。⑤ATDC5细胞内活性氧含量测定结果。3组ATDC5细胞内活性氧含量比较,差异有统计学意义(20.148±5.636,13.959±4.110,40.691±3.146,F=30.096,P=0.001)。预处理组ATDC5细胞内活性氧含量高于对照组和共培养组(P=0.001,P=0.000),对照组和共培养组ATDC5细胞Objective:To observe the effect of skeletal muscle satellite cells(MuSCs)C2C12 on the activity of chondrocytes ATDC5 in the case of co-cultured condition.Methods:①The C2C12 cells were cultured,and the third-generation cells were collected and divided into control group and dexamethasone(DEX)group.The C2C12 cells in the control group were cultured in the conventional Dulbecco’s Modified Eagle’s Medium(DMEM),while the ones in DEX group were intervened with DEX.After that,the effects of DEX on proliferation and protein synthesis in C2C12 cells were determined by using the cell counting kit-8(CCK8)assay and bicinchoninic acid(BCA)assay,respectively,meanwhile,the wound scratch assay and Transwell chamber were employed for observing the effects of DEX on repairing and migration ability of C2C12 cells.②The normal C2C12 cells(co-cultured group)and DEX-pretreated C2C12 cells(pre-treatment group)were co-cultured with ATDC5 chondrocytes,respectively,in the Transwell chambers;In the Transwell chamber of control group,the upper chamber was the cells-free conventional culture medium,and the lower was inoculated with ATDC5 cells.After that,the proliferation rate and intracellular reactive oxygen species(ROS)level of ATDC5 cells were detected by using CCK8 assay and 2′,7′-dichlorofluorescin diacetate(DCFH-DA)fluorescent probe,respectively.Results:①The proliferation rate and protein level of C2C12 cells were lower in DEX group compared to control group(78.402±5.401 vs 100.000±3.096%,t=8.498,P=0.000;5080.367±296.657 vs 5775.577±150.476μg/mL,t=3.620,P=0.022).②The wound repairing rate of C2C12 cells was lower in DEX group in contrast to control group(53.173±1.800 vs 79.979±10.176%,t=4.493,P=0.011).③On hour 24 and 48,the number of migrated C2C12 cells was less in DEX group compared with that of control group(on hour 24:24.200±5.630 vs 57.000±2.449 cells,t=11.945,P=0.000;on hour 48:57.600±8.820 vs 91.000±4.743 cells,t=7.457,P=0.000).④There was statistical difference in proliferation rate of ATDC5 cells a

关 键 词：卫星细胞 骨骼肌 软骨细胞 骨关节炎 肌萎缩 共同培养技术 

分 类 号：R68[医药卫生—骨科学]