人髓核细胞体外培养及冻存的实验研究  

Culture of human nucleus pulposus cells in vitro and cryopreservation

作　　者：薛鹏举[1] 敖俊[2] 赵利涛[1] 郭明柯[1] 吕海文[1] 姬文慧[3] 黄欢欢[4] 

机构地区：[1]河南科技大学第二附属医院骨一科,河南洛阳471000 [2]遵义医学院脊柱外科,贵州遵义563003 [3]河南科技大学第二附属医院麻醉科,河南洛阳471000 [4]河南科技大学第二附属医院病理科,河南洛阳471000

出　　处：《中国现代医学杂志》2016年第23期53-58,共6页China Journal of Modern Medicine

基　　金：贵州省科技厅科学技术基金(No:黔科合J字20102179号)

摘　　要：目的研究颈椎手术所取髓核组织培养髓核细胞的可行性,适合作为种子的代次,冻存复苏是否影响其传代。方法测量传1-5代次髓核细胞免疫组织化学法照片光密度（OD）值,观察胞质内Ⅱ型胶原蛋白含量的差异。通过噻唑蓝（MTT）试验观察冻存（实验组）与未冻存（对照组）髓核细胞增殖活力的差异。通过免疫组织化学法观察冻存对髓核细胞胞质内Ⅱ型胶原蛋白含量的影响。结果 1采用酶消化法可成功分离并培养出人髓核细胞,传1、2代细胞形态呈短梭型、三角形或多边形,传3代以后,细胞形态逐渐变为长梭型。2Ⅱ型胶原蛋白免疫组织化学法测OD值,传1与2代比较差异无统计学意义（P〉0.05）。3实验组细胞胞质内Ⅱ型胶原蛋白免疫组织化学法测得OD值与对照组细胞比较,差异无统计学意义（P〉0.05）,实验组细胞与对照组细胞MTT比色法测OD值比较,差异无统计学意义（P〉0.05）。结论 1经颈椎手术取得的髓核组织,可体外培养出髓核细胞,并可获得传代细胞。2传1、2代髓核细胞胞质内Ⅱ型胶原蛋白含量差异较小,细胞形态分化不明显,适宜做髓核组织工程学种子细胞。3冻存复苏处理未能使髓核细胞胞质内Ⅱ型胶原蛋白含量减少,细胞增殖活力亦未降低。Objective To search for the best generation of the nucleus pulposus cells suitable as seed cells in tissue engineering, which come from the nucleus pulposus tissues of anterior cervical operation, and study the effects of cryopreservation and recovery process on type Ⅱ collagen synthesis and proliferation of nucleus pulposus cells. Methods The mean optical density of the first, second, third, fourth and fifth generations of nucleus pulposus cells in immunohistochemical staining photographs was measured, so as to observe the difference between type Ⅱ collagen content in cytoplasm. The primary nucleus pulposus cells were divided into groups A and B. The cells in the group A were frozen and the cells of the group B were not cryopreserved. The proliferation activity of the nucleus pulposus cells in the group A and the group B was measured by MTT method and compared. The mean optical density values of immunohistochemical staining photos of the nucleus pulposus cells in the groups A and B were measured. Results Using enzyme digestion method,human nucleus pulposus cells were successfully extracted from the human nucleus pulposus tissues obtained from anterior cervical operation and cultured. The first and second generation cells were short fusiform, triangle or polygon. After the cells passaged to the third generation, they became spindle. The mean optical density of the first generation and the second generation of nucleus pulposus cells had no significant difference in the type Ⅱ collagen immunohistochemical staining（P〉0.05）. There was no obvious statistical difference in the mean optical density of type Ⅱ collagen between the two groups（P〉0.05）. There was no significant difference in optical density of MTT assay between the groups A and B（P〉0.05）. Conclusions The cells of the human nucleus pulposus tissues obtained from anterior cervical operation could be cultured in vitro and passaged cells could be obtained. The first and the second generations of nucleus pulposus cells have little differ

关 键 词：髓核细胞 组织工程学 种子细胞 Ⅱ型胶原蛋白 

分 类 号：R681.5[医药卫生—骨科学]