兔膝关节软骨单位体外酶解法消化 分离的实验研究  

Enzymatically isolated chondrons from rabbit knee joint cartilage in vitro

作　　者：段王平[1] 孙振伟[1] 李琦[1] 任立新[1] 卫小春[1] 

机构地区：[1]山西医科大学第二医院骨科骨与软组织损伤修复山西省重点实验室,太原030001

出　　处：《中国药物与临床》2010年第8期874-877,共4页Chinese Remedies & Clinics

基　　金：国家重点基础研究发展计划资助项目(2009CB526514)

摘　　要：目的探讨兔膝关节软骨单位(Chondron)体外酶解法消化、分离的可行性及实验方法。方法 2月龄新西兰白兔8只,随机分为2组,各4只。无菌条件下剖取双膝关节全层软骨,一组采用常规0.4%Pronase酶和0.025%Ⅱ型胶原酶依次消化为软骨细胞;另一组采用0.3%dispase酶和0.2%Ⅱ型胶原酶联合搅拌消化3h为软骨单位。利用倒置显微镜观察、细胞爬片苏木素-伊红(HE)和Ⅵ型胶原免疫荧光染色以及微系统测试分析仪进行软骨细胞及单位形态学分析,Annexin-Ⅴ/PI流式细胞术检测软骨细胞及单位早期凋亡率。结果倒置显微镜下软骨单位主要由1或几个串珠状细胞形态构成,透亮程度较差;而软骨细胞均表现为单一透亮的圆形细胞形态。软骨细胞HE染色呈爬片形态,胞质膨松透亮;软骨单位细胞周围存在一层淡染基质成分,且Ⅵ型胶原免疫荧光染色强阳性表达,界限清晰,可见大量由其包裹1、2、3或4个软骨细胞的单位形态。微系统分析仪下软骨单位由高到低均分为细胞核、细胞质及PCM三层结构,每一层梯度高度约0.4μm。急性消化软骨细胞和单位在活细胞率、早期及晚期凋亡率方面差异无统计学意义。结论 0.3%dispase酶和0.2%Ⅱ型胶原酶联合搅拌消化3h可以成功获取兔膝关节软骨单位,为其体外进一步研究奠定基础。Objective To explore the feasibility of enzymatic procedure for isolating chondrons from rabbit knee joint cartilage in vitro. Methods Eight 2-month-old New Zealand rabbits were randomized into two groups, the chondroctye and chondron groups （n=4 in each group）. In chondrocyte groups, full articular cartilages from both knees were enzymatically isolated to chondrocytes firstly by 0.4% pronase and then by 0.025% collagenase type-Ⅱ. In the chondron group, chondrons were obtained from articular cartilage using 0.3% dispase （a neutral protease） and 0.2% collagenase type-Ⅱ at 37°C with shaking for 3 h. Chondroctyes and chondrons were cultured in plastic culture bottles and morphologic analysis performed by inverted microscopy, HE staining, fluorescence immunolabeling for collagen type-Ⅵ, and micro-measure system. Cell apoptosis was analyzed by Annexin-Ⅴ/PI flow cytometry. Results The chondrons consisted of one or several clustered chondrocytes under inverted microscopy with poor translucency. All chondrocytes appeared as single round-shaped cells. Chondrons were heterogeneous in composition and structure from chondrocytes in micro-measure system, and was classified into different zones （nucleolus, cytoplasm, and PCM region）, the calculated average height of the each region was about 0.4 μm. There was no significant differences in rate of viable cells, and early or late stage apoptotic cells after enzymatic isolation. Conclusion 0.3% dispase and 0.2% collagenase type-Ⅱ with shaking for 3 h was relatively simple for enzymatic isolation of chondrons, which can be a basis for further studies in vitro.

关 键 词：膝关节 兔 软骨细胞 软骨单位 酶解法 

分 类 号：R684[医药卫生—骨科学]