基质金属蛋白酶基因在兔关节软骨细胞体外损伤后不同时间点的表达变化
|
摘要
目的:检测基质金属蛋白酶(MMPs)在兔膝关节软骨细胞损伤后不同时间点的表达变化。方法:无菌条件下获取兔膝关节软骨细胞,原代体外培养软骨细胞,6孔板内制备细胞损伤模型。显微镜观察正常细胞和划伤后1、3和7 d的软骨细胞的增殖情况。实时荧光定量PCR检测正常软骨细胞和损伤后1、3和7 d MMPs的mRNA表达水平。结果:成功分离关节软骨细胞,原代培养后成功建立细胞损伤模型。MMP-2 mRNA表达水平在损伤后1、3、7 d比正常组均升高,其中损伤后7 d最高(P<0.05～P<0.01)。MMP-3 mRNA表达水平在细胞损伤后1、3、7 d与正常组均明显下降,损伤后7 d最低(P<0.01)。MMP-9 mRNA表达水平在损伤后1、3、7 d均比正常组升高,划伤后3 d最高,随后开始下降(P<0.01)。结论:MMPs在细胞损伤后的不同时间点表达不同,可为调节细胞外基质基因治疗关节软骨损伤提供实验依据。
Objective: To explore the mRNA expressions of matrix metalloproteinases( MMPs) in rabbit articular chondrocytes at different time-points after injury. Methods: The rabbit knee joint cartilage cells were obtained under sterile condition,primarily cultured in vitro,and established the cell scratch model in six-well plates. The proliferation of chondrocytes after 1,3 and 7 days of scratching and normal cells were observed under the microscope. The MMPs mRNA expression levels in normal cells and different time-points after injury were detected using real-time PCR. Results: The articular chordrocyte was successfully isolated,the scratch model of chondrocytes was established after primary culture. Compared with normal cells,the mRNA expression levels of MMP-2 gene in chondrocytes increased at 1,3 and 7 days aftere injury,and which was the highest on the seventh day( P < 0. 05 to P < 0. 01). Compared with normal cells,the mRNA expression levels of MMP-3 gene in chondrocytes decreased significantly at 1,3 and 7 days after injury,and which was the lowest at on the seventh day( P < 0. 01). Compared with normal cells,the mRNA expression levels of MMP-9 gene in chondrocytes increased at 1,3 and 7 days after injury,and which was the highest on the third day and then began to decrease( P < 0. 01).Conclusions: The mRNA expression levels of MMPs are different after injury,which can provide the experimental evidence in gene therapy of articular cartilage injury.
Objective: To explore the mRNA expressions of matrix metalloproteinases( MMPs) in rabbit articular chondrocytes at different time-points after injury. Methods: The rabbit knee joint cartilage cells were obtained under sterile condition,primarily cultured in vitro,and established the cell scratch model in six-well plates. The proliferation of chondrocytes after 1,3 and 7 days of scratching and normal cells were observed under the microscope. The MMPs mRNA expression levels in normal cells and different time-points after injury were detected using real-time PCR. Results: The articular chordrocyte was successfully isolated,the scratch model of chondrocytes was established after primary culture. Compared with normal cells,the mRNA expression levels of MMP-2 gene in chondrocytes increased at 1,3 and 7 days aftere injury,and which was the highest on the seventh day( P < 0. 05 to P < 0. 01). Compared with normal cells,the mRNA expression levels of MMP-3 gene in chondrocytes decreased significantly at 1,3 and 7 days after injury,and which was the lowest at on the seventh day( P < 0. 01). Compared with normal cells,the mRNA expression levels of MMP-9 gene in chondrocytes increased at 1,3 and 7 days after injury,and which was the highest on the third day and then began to decrease( P < 0. 01).Conclusions: The mRNA expression levels of MMPs are different after injury,which can provide the experimental evidence in gene therapy of articular cartilage injury.
引文
[1]MACRINI TE,COAN HB,LEVINE SM,et al.Reproductive status and sex show strong effects on knee OA in a baboon model[J].Osteoarthritis Cartilage,2013,21(6):839.
[2]马子君,赵海洋,任俊丽.凋亡与增殖相关基因在损伤关节软骨细胞中的表达[J].解剖学杂志,2013,36(5):884.
[3]KUCUKGUVEN A,KHALIL RA.Matrix metalloproteinases as potential targets in the venous dilation associated with varicose veins[J].Curr Drug Targets,2013,14(3):287.
[4]任俊丽,马子君,白笙君.细胞外基质因子在兔软骨细胞损伤模型中的表达变化[J].解剖学杂志,2015,38(3):1001.
[5]李叔强,扬帆,张新,等.先天性胫骨假关节骨膜组织中基质金属蛋白酶对骨生成的影响[J].中国组织工程研究与临床康复,2008,12(2):213.
[6]赵海洋,马子君,任俊丽.生长因子在兔软骨细胞损伤模型中表达[J].解剖学杂志,2014,34(2):154.
[7]JIN L,ZHAO J,JING W,et al.Role of miR-146a in human chondrocyte apoptosis in response to mechanical pressure injury in vitro[J].Int J Mol Med,2014,34(2):451.
[8]LEYH M,SEITZ A,DURSELEN L,et al.Osteoarthritic cartilage explants affect extracellular matrix production and composition in cocultured bone marrow-derived mesenchymal stem cells and articular chondrocytes[J].Stem Cell Res Ther,2014,5(3):77.
[9]BENJAMIN MM,KHALIL RA.Matrix metalloproteinase inhibitors as investigative tools in the pathogenesis and management of vascular disease[J].EXS,2012,103:209.
[10]XU X,XIAO L,XIAO P,et al.A glimpse of matrix metalloproteinases in diabetic nephropathy[J].Curr Med Chem,2014,21(28):3244.
[11]LOVETT DH,MAHIMKAR R,RAFFAI RL,et al.A novel intracellular isoform of matrix metalloproteinase-2 induced by oxidative stress activates innate immunity[J].PLo S One,2012,7(4):e34177.
[12]吴振彪,卢宁,王彦宏.基质金属蛋白酶MMP-2、MMP-9与类风湿性关节炎关节破坏的关系[J].中国免疫学杂志,2006,22(3):260.
[2]马子君,赵海洋,任俊丽.凋亡与增殖相关基因在损伤关节软骨细胞中的表达[J].解剖学杂志,2013,36(5):884.
[3]KUCUKGUVEN A,KHALIL RA.Matrix metalloproteinases as potential targets in the venous dilation associated with varicose veins[J].Curr Drug Targets,2013,14(3):287.
[4]任俊丽,马子君,白笙君.细胞外基质因子在兔软骨细胞损伤模型中的表达变化[J].解剖学杂志,2015,38(3):1001.
[5]李叔强,扬帆,张新,等.先天性胫骨假关节骨膜组织中基质金属蛋白酶对骨生成的影响[J].中国组织工程研究与临床康复,2008,12(2):213.
[6]赵海洋,马子君,任俊丽.生长因子在兔软骨细胞损伤模型中表达[J].解剖学杂志,2014,34(2):154.
[7]JIN L,ZHAO J,JING W,et al.Role of miR-146a in human chondrocyte apoptosis in response to mechanical pressure injury in vitro[J].Int J Mol Med,2014,34(2):451.
[8]LEYH M,SEITZ A,DURSELEN L,et al.Osteoarthritic cartilage explants affect extracellular matrix production and composition in cocultured bone marrow-derived mesenchymal stem cells and articular chondrocytes[J].Stem Cell Res Ther,2014,5(3):77.
[9]BENJAMIN MM,KHALIL RA.Matrix metalloproteinase inhibitors as investigative tools in the pathogenesis and management of vascular disease[J].EXS,2012,103:209.
[10]XU X,XIAO L,XIAO P,et al.A glimpse of matrix metalloproteinases in diabetic nephropathy[J].Curr Med Chem,2014,21(28):3244.
[11]LOVETT DH,MAHIMKAR R,RAFFAI RL,et al.A novel intracellular isoform of matrix metalloproteinase-2 induced by oxidative stress activates innate immunity[J].PLo S One,2012,7(4):e34177.
[12]吴振彪,卢宁,王彦宏.基质金属蛋白酶MMP-2、MMP-9与类风湿性关节炎关节破坏的关系[J].中国免疫学杂志,2006,22(3):260.