急性消化成年兔膝关节软骨单位与软骨细胞基因表达分析  

Study on gene expression level of chondrocytes and cartilage in adult rabbits

作　　者：段王平[1] 郝永壮[1] 宋文杰 赵瑞鹏[1] 任晓春[1] 赵昱[1] 李琦[1] 孙振伟[1] 李鹏翠[1] 卫小春[1] Duan Wangping;Hao Yongzhuang;Song Wenjie;Zhao Ruipeng;Ren Xiaochun;Zhao Yu;Li Qi;Sun Zhenwei;Li Pengcui;Wei Xiaochun(Department of Orthopaedics,the Second Clinical Medical College of Shanxi Medical University,Shanxi Key Lab of Bone and Soft Tissue Injury Repair,Taiyuan 030001,China)

机构地区：[1]山西医科大学第二医院骨科、骨与软组织损伤修复山西省重点实验室,太原030001

出　　处：《中华风湿病学杂志》2023年第7期459-463,共5页Chinese Journal of Rheumatology

基　　金：国家自然科学基金项目(U21A20353,82172503);山西省重点研发计划项目(201903D421019);中央引导地方科技发展资金(YDZJSX2022B011)。

摘　　要：目的探讨急性消化成年兔膝关节软骨单位和软骨细胞基因表达的差异。方法8月龄新西兰兔5只,无菌条件剖取双膝关节股骨髁全层软骨组织,左侧膝关节软骨组织采用0.4%Pronase酶和0.025%Ⅱ型胶原酶依次酶解消化获取软骨细胞,右侧膝关节软骨组织采用0.3%dispase酶和0.2%Ⅱ型胶原酶联合搅拌酶解消化3 h获取兔膝关节骨单位。通过实时定量PCR对急性消化软骨细胞和软骨单位基因表达进行分析,包括基质蛋白[聚集蛋白聚糖(Agg)、胶原蛋白(Col)-2、Col-6A6、Col-10、Col-11]、MMPs及MMPs抑制因子(TIMPs)(MMP-1、MMP-3、MMP-9、MMP-13、TIMP-1、TIMP-2、TIMP-3)、性别决定区Y-框蛋白9(Sox)-9及细胞骨架蛋白(黏着斑蛋白、微管蛋白、肌动蛋白)、炎症因子(IL-1β、TNF-α)。采用SPSS 16.0统计软件进行分析,采用t检验,以P<0.05表示差异具有统计学意义。结果与软骨细胞相比,软骨单位中Agg[(5.78±0.90)与(1.89±0.27),t=9.26,P<0.001]、Col-2[(6.29±0.76)与(3.06±0.60),t=7.46,P<0.001]、Col-6A6[(0.89±0.18)与(0.22±0.06),t=7.90,P<0.001]、Col-10[(3.83±0.76)与(1.00±0.26),t=7.88,P<0.001]及TIMP-1[(1.98±0.85)与(1.03±0.34),t=2.32,P=0.049]、TIMP-2[(3.46±1.50)与(1.52±1.06,t=2.36,P=0.046]、TIMP-3[(3.96±0.50)与(1.36±0.18),t=10.94,P<0.001]、Sox-9[(7.09±2.93)与(3.24±0.77),t=2.84,P=0.022]、黏着斑蛋白[(3.42±1.69)与(1.46±0.68),t=2.41,P=0.043]、微管蛋白(9.34±0.71)与(2.35±0.80),t=14.61,P<0.001]表达较高,差异具有统计学意义。与软骨细胞相比,在软骨单位中MMP-1[(1.02±0.30)与(2.67±0.45),t=6.91,P<0.001]、MMP-3[(1.21±0.32)与(2.52±0.79),t=3.44,P=0.009]、MMP-13[(1.23±0.34)与(3.42±0.86),t=5.30,P=0.007]、IL-1β[(1.02±0.14)与(2.70±0.49),t=7.37,P<0.001]、TNF-α[(0.99±0.08)与(3.15±0.54),t=8.85,P<0.001]表达低,差异具有统计学意义。Col-11、MMP-9、肌动蛋白在软骨单位及软骨细胞中表达差异无统计学意义(P>0.05)。结论与单纯软骨细胞相比,急性消化软骨�Objective To investigate the differences in gene expression levels in knee chondrocytes and chondrons in vitro.Methods The chondrocytes and chondrons were isolated from full thickness of the 8-months(n=5)rabbit knees cartilage.Chondrons from right knee were enzymatically isolated using 0.3%dispase and 0.2%collagenase-2 with shaking for 3 hours.Chondrocytes were isolated by 0.4%Pronase and 0.025%collagenase-2 from left knee.The mRNA levels in chondrocytes and chondrons were analyzed by quantitative real-time PCR,including matrix proteins[aggrecan(Agg),collagen(Col-2),Col-6A6,Col-10,Col-11],MMPs and inhibitors(MMP-1,MMP-3,MMP-9,MMP-13,TIMP-1,TIMP-2,TIMP-3),cytoskeletal proteins(Sox-9,vinculin,tubulin,actin),cytokines(IL-β,TNF-α).Analysis was performed using SPSS 16.0 statistical software,and the two-group comparisons were considered as significant by t-test at P<0.05.Results Compared to the chondrocytes,the Agg[(5.78±0.90)vs(1.89±0.27),t=9.26,P<0.001],Col-2[(6.29±0.76)vs(3.06±0.60),t=7.46,P<0.001],Col-6A6[(0.89±0.18)vs(0.22±0.06),t=7.90,P<0.001],Col-10[(3.83±0.76)vs(1.00±0.26),t=7.88,P<0.001]and TIMP-1[(1.98±0.85)vs(1.03±0.34),t=2.32,P=0.049],TIMP-2[(3.46±1.50)vs(1.52±1.06),t=2.36,P=0.046],TIMP-3[(3.96±0.50)vs(1.36±0.18),t=10.94,P<0.001],Sox-9[(7.09±2.93)vs(3.24±0.77),t=2.84,P=0.022],vinculin[(3.42±1.69)vs(1.46±0.68),t=2.41,P=0.043],tubulin[(9.34±0.71)vs(2.35±0.80),t=14.61,P<0.001]showed higher expression in the chondrons.Compared to the chondrocytes,the MMP-1[(1.02±0.30)vs(2.67±0.45),t=6.91,P<0.001],MMP-3[(1.21±0.32)vs(2.52±0.79),t=3.44,P=0.009],MMP-13[(1.23±0.34)vs(3.42±0.86),t=5.30,P=0.007],IL-1β[(1.02±0.14)vs(2.70±0.49),t=7.37,P<0.001],TNF-α[(0.99±0.08)vs(3.15±0.54),t=8.85,P<0.001]showed lower expression in the chondrons.There were no difference between chondrons and chondrocytes for Col-11,MMP-9,actin(P>0.05).Conclusion The gene expression of extracellular matrix components are higher and the gene expression levels of inflammatory factors and MMPs are decreased in chondrons co

关 键 词：软骨细胞 细胞外基质 基质金属蛋白酶类 软骨单位 

分 类 号：R684[医药卫生—骨科学]