威灵仙提取物可促进体外牵张应力环境下软骨细胞表型的维持  

Phenotypic maintenance of chondrocytes in vitro under tensile stress enhanced by the extract of Clematis chinensis

作　　者：涂鹏程 郭杨[1,2] 马勇 潘娅岚[1] 郑苏阳 王礼宁 吴承杰 过俊杰 Tu Pengcheng;Guo Yang;Ma Yong;Pan Yalan;Zheng Suyang;Wang Lining;Wu Chengjie;Guo Junjie(Institute of Traumatology&Orthopedics and Laboratory of New Techniques of Restoration&Reconstruction of Orthopedics and Traumatology,Nanjing University of Chinese Medicine,Nanjing 210023,Jiangsu Province,China;Department of Traumatology&Orthopedics,Affiliated Hospital of Nanjing University of Chinese Medicine,Nanjing 210029,Jiangsu Province,China)

机构地区：[1]南京中医药大学骨伤研究所骨伤修复与重建新技术实验室,江苏省南京市210023 [2]南京中医药大学附属医院骨伤科,江苏省南京市210029

出　　处：《中国组织工程研究》2020年第8期1182-1187,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金面上项目（81673995），项目负责人：马勇;江苏省自然科学基金青年基金项目（BK20151007），项目负责人：郭杨;江苏省研究生科研创新计划（KYCX17_1308），项目负责人：潘娅岚;2018年地方高校国家级大学生创新训练计划（201810315008），项目负责人：过俊杰~~

摘　　要：背景:机械负荷是软骨细胞退行性改变及骨关节炎发展过程中的重要因素,威灵仙能够改善骨关节炎炎性微环境,但其对机械负荷诱导的软骨细胞退行性改变的作用尚未阐明。目的:探讨威灵仙提取物对体外间歇性循环牵张拉伸诱导的软骨细胞退行性改变的影响及机制。方法:Ⅱ型胶原酶消化法分离兔膝关节软骨细胞并通过阿利新蓝染色鉴定;实验将软骨细胞分为5组:空白组、间歇性循环牵张拉伸组、威灵仙低、中、高质量浓度组。空白组无任何干预,其他4组利用FLEXCELL-5000T牵张拉伸系统对软骨细胞施加间歇性循环牵张拉伸(10%拉伸强度、8 h/d、0.5 Hz、2 d)刺激,诱导软骨细胞退行性改变,威灵仙各组在施加相同的刺激同时添加0.5,1,2 g/L威灵仙提取物,干预48 h;CCK-8法检测各组软骨细胞增殖活性;FITC-鬼笔环肽染色观察各组细胞骨架形态;实时定量PCR和蛋白免疫印迹法检测各组细胞Ⅱ型胶原、基质金属蛋白酶13、转化生长因子β的表达。结果与结论:①与空白组相比,间歇性循环牵张拉伸刺激后软骨细胞骨架呈长条状拉伸状态,细胞增殖活性降低,转化生长因子β、Ⅱ型胶原表达均下调,而基质金属蛋白酶13表达上调;②与间歇性循环牵张拉伸组相比,威灵仙提取物能够促进软骨细胞增殖,上调转化生长因子β、Ⅱ型胶原表达,下调基质金属蛋白酶13表达,且效果与质量浓度相关;③结果表明,威灵仙提取物能够通过调控转化生长因子β表达抑制间歇性循环牵张拉伸诱导的软骨细胞分解代谢,促进软骨细胞外基质的合成,维持软骨细胞表型稳定。BACKGROUND: Mechanical load is crucial for the degeneration of chondrocytes and the development of osteoarthritis. Clematis chinensis can improve the inflammatory microenvironment of osteoarthritis, but its effect on mechanical load-induced degeneration of chondrocytes has not been elucidated. OBJECTIVE: To study the effect and mechanism of the extracts of Clematis chinensis on the degenerative changes of chondrocytes induced by intermittent cyclic mechanical tension (ICMT) in vitro. METHODS: Chondrocytes of the rabbit knee joint were isolated by type II collagenase digestion method and identified by Alcian blue staining. There were five groups in the experiment: blank group, ICMT group, high-, medium- and low-dose Clematis chinensis groups. There was no intervention in the blank group, and the other groups were subjected to ICMT (10% tensile strength, 0.5 Hz, 8 hours per day, for a total of 2 days) for inducing chondrocyte degeneration. Three Clematis chinensis groups were concurrently given 0.5, 1, 2 g/L extracts of Clematis chinensis, respectively. The intervention time was 48 hours. Cell counting kit-8 assay was used for detection of chondrocyte proliferation. FITC-phalloidin staining was used for observation of cytoskeleton morphology. Real-time quantitative PCR and western blot assay were used for determination of collagen type II, matrix metalloproteinase 13, and transforming growth factor β at protein and gene levels, respectively. RESULTS AND CONCLUSION: (1) Compared with the blank group, the cytoskeleton of chondrocytes stimulated by ICMT was long- stretched, the proliferation activity of chondrocytes decreased, and the expressions of collagen type II and transforming growth factor β were down-regulated, while the expression of matrix metalloproteinase 13 was up-regulated. (2) Compared with the ICMT group, the extract of Clematis chinensis could promote the proliferation of chondrocytes, up-regulate the expressions of transforming growth factor β and collagen II, and down-regulate the expression of m

关 键 词：威灵仙 软骨细胞 牵张拉伸 机械力 基质金属蛋白酶 Ⅱ型胶原 转化生长因子Β 细胞表型 国家自然科学基金 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学]