Toll样受体4激活对转化生长因子-β1致骨骼肌纤维化作用的影响  

The influence of Toll-like recepter 4 activation to the skeletal muscle fibrosis mediated by transforming growth factor-beta1

作　　者：聂铭博[1] 鲍远[1] 张滋洋[1] 施佳[1] 康皓[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院骨科,武汉430030

出　　处：《中华实验外科杂志》2017年第7期1177-1180,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金（81472106）

摘　　要：目的提取并培养小鼠骨骼肌内肌源性干细胞（MDSCs），观察其在Toll样受体4（TLR4）激活后，对转化生长因子-β1（TGF-β1）介导的骨骼肌纤维化作用的影响。方法细胞培养：采用差速贴壁法获得小鼠肌源性干细胞，传代培养。所得细胞分为3组，A组作为阴性对照，B组在培养液中加入重组人TGF-β1（rhTGF-β1），C组加入重组人TGF-β1和TLR-4激动剂热休克蛋白60（Hsp60）。上述各组细胞培养3 d后，分别行反转录-聚合酶链反应（RT-PCR）、Western blot、免疫荧光染色。检测各组细胞核因子（NF）-κB表达差异，明确B组细胞TLR-4是否被激活，再通过比较各组MDSCs在TLR-4激活后结缔组织生长因子（CTGF）和Ⅰ型胶原的表达水平。动物实验：取雄性小鼠24只（体重15～18 g），随机分为D、E、F 3组（每组8只）。每周一分别于左侧腓肠肌内侧头注射生理盐水（阴性对照）、rhTGF-β1、rhTGF-β1加Hsp60混合溶液，连续4周。第28天切取左侧腓肠肌内侧头测量肌湿重，Von Geison染色比较肌肉组织纤维化程度。结果细胞培养：RT-PCR测定A、B、C 3组所得核转录因子κB（NF-κB）相对表达量分别为：0.852±0.121、0.987±0.084、1.304±0.171（P=0.031）。CTGF相对表达量分别为：0.756±0.205、0.977±0.188、1.308±0.302（P=0.022）。Ⅰ型胶原相对表达量分别为：0.767±0.148、0.998±0.138、1.261±0.327（P=0.016）。Western blot测定A、B、C 3组NF-κB与内参的吸光度比值依次为：0.784±0.133、0.887±0.066、0.994±0.171（P=0.026）。CTGF与内参的吸光度比值依次为：0.644±0.125、0.863±0.109、0.998±0.512（P=0.018）。Ⅰ型胶原与内参的吸光度比值依次为：0.347±0.133、0.761±0.086、0.868±0.167（P=0.045）。与Western blot结果相对应，免疫荧光染色结果显示在A、B、C 3组CTGF和Ⅰ型胶原的表达依次增强，差异有统计学意义（P=0.025，P=0.012）。动物实验：D、E、F3�Objective Obtain the Muscle derived stem cells from the mouse skeletal muscle and observe the influence of Toll-like receptor 4 （TLR4） activation to the skeletal muscle fibrosis mediated by transforming growth factor-beta1.Methods Cell culture： muscle-derived stem cells （MDSCs） from primary cultures of mouse skeletal muscle were isolated and purified by the preplate technique. Then, the MDSCs were divided into 3 average groups （A, B, C） at random. Group A is the control, Group B cultured in vitro with the transforming growth factor-β1 （TGF-β1）, Group C cultured in vitro with the TGF-β1 and heat shock protein 60 （Hsp60）respectively for 3 days. At first, the level of Nuclear transcription factors κB （NF-κB）was tested by RT-PCR and Western blotting to make clear if the TLR4 was activated. Then the level of CTGF and collagen I in these three groups were tested by RT-PCR, Western blotting and immunofluorescence. Animal model： 24 adult male mouses （15-18 g） were divided into three groups （D, E, F） of 8 each at random. The saline, rhTGF-β1 solution and rhTGF-β1 with Hsp60 solution were injected into the left gastrocnemius medial head respectively per 7 days for four times. At the 28th day, the left gastrocnemius medial head was removed to test the wet weight of the muscles and Von Geison stain performed to observe the muscular fibrosis of each group.Results Cell culture： RT-PCR show that the relative expression of the NF-κB of group A, B and C were respectively 0.852±0.121, 0.987±0.084 and 1.304±0.171 （P=0.031）. The CTGF of group A, B and C were respectively 0.756±0.205, 0.977±0.188, 1.308±0.302 （P=0.022）. The collagen I of group A, B and C were respectively 0.767±0.148, 0.998±0.138, 1.261±0.327 （P=0.016）. Western blotting show that the optical density ratio of the NF-κB of group A, B and C were respectively 0.784±0.133, 0.887±0.066, 0.994±0.171 （P=0.026）. The CTGF of group A, B and C were respectively 0.644±0.125, 0.863±0.109, 0.998±0.512 （

关 键 词：肌源性干细胞 转化生长因子Β-1 TOLL样受体 结缔组织生长因子 纤维化 

分 类 号：R68[医药卫生—骨科学]