体外器官培养建立软骨退变模型  

Establishment of a cartilage degeneration model in vitro

作　　者：丁道芳[1,2] 庞坚[1,2] 杜国庆[1,2] 郭海玲[1,2] 秦梦[1,2] 曹月龙[1,2] 詹红生[1,2] 郑昱新[1,2] 

机构地区：[1]上海中医药大学附属曙光医院石氏伤科医学中心,上海201203 [2]上海市中医药研究院骨伤科研究所,上海201203

出　　处：《中国矫形外科杂志》2015年第15期1405-1410,共6页Orthopedic Journal of China

基　　金：国家自然科学基金项目(编号:81073114;81072830;81373665);上海市教委创新项目(编号:11YZ64);上海市高校青年教师培养资助计划项目(编号:ZZszy12017)

摘　　要：[目的]体外培养大鼠的股骨头并诱导其快速退变,为药物筛选和治疗提供可靠的软骨退变模型。[方法]取2周龄SD幼鼠的股骨头,每只左右股骨头对应为对照组和实验组,分别培养于正常培养基和诱导培养基中(含50ng/ml的TNFa),每隔48 h换一次培养液,至第5 d,取出股骨头,固定于4%多聚甲醛,经过脱钙和脱水,石蜡包埋,切片后经甲苯胺蓝和臧红-固绿染色及细胞免疫荧光检测Ⅱ型胶原、MMP13、Sox9、ADAMTS5等基因的表达。[结果]在诱导培养基中培养5 d的股骨头外缘,相对于正常培养基中培养的股骨头外缘,甲苯胺蓝和臧红染色明显淡染,固绿深染,提示软骨退变。进一步免疫荧光检测软骨标志基因Ⅱ型胶原和Sox9表达减弱,促软骨退变基因MMP13和ADAMTS5表达增强。且经过诱导培养的股骨头的整体大小明显小于正常培养的股骨头。[结论]体外培养大鼠股骨头快速诱导其退变,建立软骨退变的体外模型。[Objective] To establish a reliable model of drug screening and therapy by culturing rat femoral heads and quickly inducing cartilage degeneration in vitro. [Methods] Femoral heads from the same 2- month- old Sprague- Dawley rats were divided into a control group and an experimental group. The femoral heads were cultured with Dulbecco＇s modified Eagle＇s medium（ DMEM） plus 10% fetal bovine serum or DMEM plus 10% fetal bovine serum plus 50 ng / m L tumor necrosis factor（ TNF-） for 5 days. The femoral heads were fixed in 4%paraformaldehyde,decalcified,dehydrated,embedded in paraffin,and cut into slices. Specimens were stained with toluidine blue and safranin O- fast green FCF. The protein expression levels of type 2 collagen, MMP13, Sox9, and ADAMTS5 were analyzed by using immunofluorescence. [Results] Both the toluidine blue and safranin O stainings were pale in the margin of the femoral heads that were stimulated with TNF- for 5 days,compared with the control group. The fast green FCF staining was positive at the edge of the femoral head in the experimental group,which indicated cartilage degeneration. The expression levels of type 2 collagen and Sox9 were decreased significantly,whereas the expression levels of MMP13 and ADAMTS5 were increased in the experimental group. We also found that the size of the femoral heads in the experiment group was smaller than that in the control group. [Conclusion] A model of cartilage degeneration can be made by culturing and quickly inducing femoral head degeneration in vitro.

关 键 词：股骨头 染色 蛋白表达 诱导 软骨退变 

分 类 号：R-332[医药卫生] R681.3