比较单独消化法及分步消化法培养新生大鼠原代软骨细胞的生物学特性  

Biological properties of primary chondrocytes isolated by one-step digestion versus stepwise digestion in newborn rats

作　　者：邓林峡 余慕雪[1] 潘思年[2] 郭楚怡[1] 丘小汕[1] 曹志毓 Deng Lin-xia;Yu Mu-xue;Pan Si-nian;Guo Chu-yi;Qiu Xiao-shan;Cao Zhi-yu(Department of Pediatrics,the First Affiliated Hospital of Sun Yat-Sen University,Guangzhou 510080,Guangdong Province,China;Department of Pediatrics,the Third Affiliated Hospital of Sun Yat-Sen University,Guangzhou 510630,Guangdong Province,China;Zhongshan School of Medicine,Sun Yat-Sen University,Guangzhou 510080,Guangdong Province,China)

机构地区：[1]中山大学附属第一医院儿科,广东省广州市510080 [2]中山大学附属第三医院儿科,广东省广州市510630 [3]中山大学中山医学院,广东省广州市510080

出　　处：《中国组织工程研究》2018年第25期4047-4052,共6页Chinese Journal of Tissue Engineering Research

基　　金：广东省自然科学基金(2015A030313148);广东省科技计划项目(2012B031800077;2011B031800143);国家级大学生创新训练计划项目(201701101)~~

摘　　要：背景:原代软骨细胞体外培养是研究骨骼生长的重要方法之一。目前原代软骨细胞的分离方法主要有单独消化法和分步消化法,但两种方法对软骨细胞生物学性能的影响尚不清楚。目的:比较两种方法对新生大鼠软骨细胞生物学性能的影响,为软骨发育的程序化研究提供实验基础。方法:无菌条件下取出新生SD大鼠关节软骨,分别采用Ⅱ型胶原蛋白酶单独消化法、胰蛋白酶和Ⅱ型胶原蛋白酶分步消化法分离软骨细胞,并进行体外培养。光学显微镜下观察软骨细的形态,CCK-8法检测软骨细胞的增殖活性,半定量免疫荧光分析软骨细胞的Ⅱ型胶原蛋白表达。结果与结论:(1)单独消化法和分步消化法都可以得到大量软骨细胞,但分步消化法较单独消化法所得软骨细胞的纯度更高;(2)光学显微镜下观察,两种方法消化所得细胞在形态学上无明显差异;(3)单独消化法所得细胞生长速度更快;(4)免疫荧光半定量分析显示两种方法所得软骨细胞Ⅱ型胶原蛋白表达一致;(5)以上结果表明,分步消化法更适于新生大鼠原代软骨细胞的分离培养。BACKGROUND:In vitro culture of primary chondrocytes is one of the most important methods to study the bone growth.Currently,the main methods of primary chondrocyte separation are one-step digestion and stepwise digestion.However,the biological properties of chondrocytes isolated by these two methods have not been well documented.OBJECTIVE:To compare the biological properties of chondrocytes in newborn rats isolated using these two methods and to provide experimental basis for the programmed study of cartilage development.METHODS:Articular cartilage of newborn SD rats was removed under aseptic conditions.Chondrocytes were isolated by one-step digestion(type II collagen)and stepwise digestion(type II collagen and trypsin).Primary culture and subculture were performed.Cell morphology was observed under light microscope,cell proliferation was detected by cell counting kit-8 assay,and the expression of type II collagen of chondrocytes was analyzed by semi-quantitative immunofluorescence.RESULTS AND CONCLUSION:A large number of chondrocytes were yielded by two digestion methods,but the purity of chondrocytes obtained by stepwise digestion method was higher than that by one-step digestion method.Under the light microscope,there was no significant difference in the cell morphology between the two groups.The growth of chondrocytes obtained by one-step digestion was faster than that by stepwise digestion method.Results from the semi-quantitative immunofluorescence analysis showed that the chondrocytes obtained by these two digestion methods showed the same ability to express type II collagen.These findings indicate that compared with one-step digestion,stepwise digestion is more suitable for primary chondrocytes isolation in newborn rats.

关 键 词：原代软骨细胞 细胞培养 Ⅱ型胶原蛋白酶 胰蛋白酶 半定量免疫荧光 细胞增殖 生长曲线 Ⅱ型胶原蛋白 干细胞 广东省自然科学基金 软骨细胞 细胞 培养的 胰蛋白酶 胶原酶类 胶原Ⅱ型 组织工程 

分 类 号：R394.2[医药卫生—医学遗传学]