SD大鼠肋软骨细胞的原代培养和去分化现象的观察.docVIP

SD大鼠肋软骨细胞的原代培养和去分化现象的观察 　　作者：袁亚江 ， 梅晰凡，高蔚然，张赫 作者单位：(1.辽宁医学院附属第一医院骨科;2.辽宁医学院附属第一医院肿瘤科，辽宁 锦州 121001) 　　【摘要】 目的 建立大鼠肋软骨细胞(RCC)分离、培养的方法, 探讨其传代过程中细胞外基质及其形态的变化。方法采用胰酶和II型胶原酶两步消化法结合机械吹打获得分散的单个RCC，观察单层培养的不同代数RCC的细胞形态变化;采用免疫细胞化学方法检测不同代数RCC蛋白多糖和Ⅱ型胶原的表达。结果 大鼠肋软骨经两步消化后，可获得高纯度的软骨细胞，经免疫组化及甲苯胺蓝染色呈阳性结果，同时观察到RCC在传代过程中细胞发生了去分化现象。结论 软骨细胞可以通过传代培养获得扩增，但仅限于四代以内细胞。 　　【关键词】 大鼠;软骨细胞;单层培养;去分化 　　The Culturing of SD Rat Costochondral Chondrocyte and Observation of DedifferentiationYUAN Yajiang1, MEI Xifan1, GAO Weiran2 , ZHANG He 　　(1.Department of Orthopaedics, the First Affiliated Hospital of Liaoning Medical University 　　2.Cancer Tumour, the First Affiliated Hospital of Liaoning Medical University, Jinzhou 121001 China)Abstract: Objective To establish the separating and culturing methods for rat costochondral chondrocyte (RCC), and to investigate their morphous changes in the course of cell passage.Methods Under the help of collagenase II and trypsins digestion, the scattered single RCC was obtained. The observation of morphological changes of RCC cells cultured by monolayer culture in different generations was carried out. And the detection of the expression of collagen II and proteoglycan of RCC cells in different generations by immunocytochemical method was also implemented. Results High purity cartilage cells were obtained from rat costal cartilages after digested by collagenase II and trypsin. It was confirmed by immunohistochemistry and toluidine blue staining. The dedifferentiation of RCC cells was observed when the RCC cells were cultured by monolayer culture.Conclusions RCC cells can be amplified by passage Culture,but only within the cells of four generations. 　　Key words:rat; cartilage cell; monolayer culture; dedifferentiation 　　关节软骨损伤后，因局部血供缺乏，所以多年来一直被认为软骨细胞本身不具有修复与再生能力。1989年Webber[1]的研究结果却显示:软骨细胞能进行自我修复，只是它们的修复能力在体内环境中不能得到激发。 　　自20世纪80年代以来，人们对关节软骨的再造和修复研究的焦点已转向细胞疗法(cell-based therapy )，即移植足够数量的软骨细胞来修复关节软骨的缺损。Yanchun Liu等[2]偿试应用自体关节软骨细胞为种子细胞，以聚