人类髓核细胞分离培养方法的优化选择  

Optimized method for isolating and culturing human nucleus pulposus cells

作　　者：张子言[1] 佟珅[1] 颜华东[1] 姜睿[1] 武汉[1] 

机构地区：[1]吉林大学中日联谊医院,吉林省长春市130033

出　　处：《中国组织工程研究》2013年第28期5151-5156,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30772209)~~

摘　　要：背景:关于人类髓核细胞的分离培养方法不一,所用消化酶组分及消化时间差异较大,如何提高人类髓核细胞获得效率的问题尚未解决。目的:优选人类髓核细胞获取的消化酶组分和消化方法。方法:髓核组织标本取自吉林大学中日联谊医院骨科成人椎间盘3例。无菌条件下取突出至椎管内的急性创伤后椎间盘组织,可见到外周白色的纤维环和中心胶冻样的髓核组织。按不同混合酶组分划分为2组,Ⅰ组成分为0.2%Ⅱ型胶原酶;Ⅱ组成分为先用0.25%胰酶消化30min,再用0.2%Ⅱ型胶原酶。每组再按消化时间分为2,4h,过夜3个亚组。最后重悬细胞终体积均定位2mL并计数,采用含胎牛血清的DMEM培养液体外培养细胞。观察不同组分酶对分离所得髓核细胞数量、活性及增殖能力的影响,采用锥虫蓝染色计数细胞总数及活细胞比值,MTT法测定髓核细胞生长曲线。结果与结论:采用2种不同酶组分进行消化,发现消化时间2,4h时,Ⅱ组消化细胞较Ⅰ组多,但差异无显著性意义(P>0.05)。孵育过夜时,Ⅰ组和Ⅱ组的存活细胞率较消化2,4h明显降低,差异有显著性意义(P<0.05)。在作用4h时,组织块消失,计数细胞数量最多。提示以Ⅱ型胶原酶为主要成分的Ⅰ组对分离髓核细胞有利,消化时间以4h为宜,该条件具有操作简单,效率较高,成本较低等优点,可以认为0.2%Ⅱ型胶原酶消化髓核组织块4h为获取髓核细胞的最佳条件。METHODS： Nucleus pulposus tissue specimens were selected from three adult discs in the Department of Orthopedics, China-Japan Union Hospital of Jilin University. The acute traumatic disc tissues that outstanding to the spinal canal were taken under aseptic conditions, and then the peripheral white annulus and jelly-like nucleus pulposus in the center could be seen. According to different mixed enzyme concentration ratio, the samples were divided into two groups. The enzyme I group was treated with 0.2% II collagenase; and the mixed enzyme II group was digested with 0.25% trypsin for 30 minutes, and then treated with 0.2% II collagenase. According to digestion time, each group was divided into three subgroups： 2 hours group, 4 hours group, and overnight group. Finally, suspended cell volume was decided as 2 mL to count cells. Dulbecco＇s modified Eagle＇s medium containing fetal bovine serum was used for cell culture in vitro. Trypan blue staining was performed to counttotal cell number and ratio of living cells. Methylthiazolyldiphenyl-tetrazolium bromide assay was used to detect the growth curve of nucleus pulposus cells. RESULTS AND CONCLUSION： Based on the two digestion enzyme concentration, the number of digested cells in the enzyme Ⅰ group was larger than that in the enzyme Ⅱ group after digested for 2 and 4 hours, but the difference was not significant （P 〉 0.05）. Overnight, cell survival rate was decreased in the enzyme I group after digested for 2 and 4 hours when compared with the enzyme H group, and the difference was significant （P 〈 0.05）. After digested for 4 hours, tissue blocks disappeared, and the number of cells reached maximum. The results indicate that enzyme 1 group composite with Ⅱ collagenase is benefit for the separation of nucleus pulposus cells, and the digestion time is appropriate to 4 hours. This condition has the advantages of simple operation, high efficiency and low cost, and it considered that digestion of nucleus pulposus tissues with 0.2% IX collagenase

关 键 词：组织构建 软骨组织构建 髓核细胞 人类 Ⅱ型胶原酶 胰酶 消化 分离培养 MTT 国家自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]