模拟微重力环境下威灵仙维持兔膝关节软骨细胞表型的效应与机制

Effects of Clematis Chinensis Osbeck Mediated by Simulated Microgravity Enviroment on Chondrocyte Phenotype Maintenance

目的研究模拟微重力培养环境下威灵仙提取物对兔软骨细胞表型维持的作用及机制。方法利用液动力聚焦细胞培养系统体外模拟微重力培养环境,同时使用威灵仙提取物进行干预,将兔膝关节软骨细胞分为空白组(平面培养)、威灵仙组(平面培养+威灵仙提取物)、微重力组(微重力培养)、微重力-威灵仙组(微重力培养+威灵仙提取物)4组,培养7 d;观察各组软骨细胞形态学变化,CCK-8检测各组细胞增殖活性,流式细胞技术检测各组软骨细胞凋亡率;RT-qPCR检测成软骨相关mRNAⅡ型胶原蛋白、蛋白多糖、TGF-β、SOX9,以及与软骨去分化相关mRNA MMP13、Ⅰ型胶原蛋白;Western blotting检测Ⅱ型胶原蛋白、蛋白聚糖、TGF-β及MMP13蛋白表达;同时对细胞内葡萄糖、ATP、乳酸含量进行检测,评估各组软骨细胞能量代谢状态。结果形态学观察显示微重力组、微重力-威灵仙组软骨细胞保持良好形态,空白组、威灵仙组梭形细胞增多。与空白组相比,模拟微重力培养条件与威灵仙提取物均能提高软骨细胞48,72 h的增殖活性,降低细胞凋亡率,上调蛋白聚糖、Ⅱ型胶原蛋白、TGF-β的蛋白及mRNA表达,降低MMP13的蛋白及mRNA表达,上调SOX9 mRNA表达,降低Ⅰ型胶原蛋白mRNA表达,上调软骨细胞内葡萄糖、ATP、乳酸含量,两者结合效果最为显著(P<0.01)。结论模拟微重力培养环境与威灵仙提取物均能够促进软骨细胞能量代谢,保持增殖活性以及软骨细胞表型维持,而二者联合应用可以发挥协同作用。

OBJECTIVE To study the effect and mechanism of the extract of Clematis chinensis Osbeck(CCO) on the phenotypic maintenance of rabbit chondrocytes in simulated microgravity culture environment. METHODS Rotary Cell Culture system(RCC) was used to simulate microgravity environment in vitro, and the extract of CCO was used to interfere with chondrocyte. The rabbit knee joint cartilage cells were extracted and divided into 4 groups: blank(two dimensions on plate),CCO(two dimensions on plate, with CCO), microgravity group(cultured in RCC), RCC-CCO(cultured in RCC, with CCO). All were cultured for 7 d. Morphological changes of chondrocytes were observed. Chondrocyte proliferation was detected by CCK8 and apoptotic rate were detected using flow cytometry. The chondrogenic differentiation related mRNA expression of Collagen type Ⅱ, aggrecan, TGF-β, SOX9 and chondrogenic dedifferentiation related mRNA MMP13, collagen type Ⅰ were detected. The protein expression of type Ⅱ collagen, aggrecan, TGF-β and MMP13 were detected by Western blotting. And energy metabolism was assessed by detecting concentration of intracellular glucose, ATP and lactate. RESULTS Chondrocytes in RCC group and RCC-CCO group maintained good morphology, while the spindle cells in control group and CCO group increased. Compared with the Control group, both the microgravity conditions and the CCO could increase the proliferation activity of chondrocytes at 48 h and 72 h, reduce the apoptotic rate of chondrocytes. Then, they could up-regulate the protein and mRNA expression of proteoglycan, type II collagen and TGF-β, decrease MMP13 protein and mRNA expression, up-regulate SOX9 mRNA expression, decrease type I collagen mRNA expression, and up-regulate glucose, ATP, and lactic acid content in chondrocytes. Combining of the two was more significant(P<0.01). CONCLUSION The simulated microgravity culture environment and the extract of CCO can promote the energy metabolism of chondrocytes, maintain the proliferative activity and the phenotype of chondrocytes, and the combined application of the two can play a synergistic role.