hBMSCs诱导分化类髓核细胞  

DIFFERENTIATION OF INTERVERTEBRAL NUCLEUS PULPOSUS-LIKE CELLS FROM hBMSCs

作　　者：丰干钧[1] 刘浩[1] 陈晓禾[2] 李秀群[2] 赵献峰[1] 梁涛[1] 

机构地区：[1]四川大学华西医院骨科,成都610041 [2]生物治疗国家重点实验室·干细胞与组织工程研究室

出　　处：《中国修复重建外科杂志》2008年第12期1470-1475,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(30572163)~~

摘　　要：目的探讨人椎间盘细胞和关节软骨细胞的分子表型差异,分析hBMSCs经TGF-β3和BMP-7联合诱导后能否分化为软骨细胞和髓核细胞。方法取9例自愿捐赠者髂嵴骨髓20～40mL分离培养hBMSCs。取第4代hBMSCs行三维微球培养。根据基础培养基中加入的生长因子不同,实验分成4组,分别为加入10ng/mLTGF-β3组(A组)、200ng/mLBMP-7组(B组)、同时加入两种生长因子组(C组)以及空白对照组(D组)。于培养后21d,行组织学及免疫组织化学观察;于培养后4、21d进行PCR检测Ⅰ、Ⅱ、Ⅹ型胶原、蛋白多糖和SOX9基因的表达。结果培养后21d,HE染色示A组和C组细胞呈软骨细胞样形态特征,甲苯胺蓝染色及免疫组织化学染色Ⅱ型胶原表达呈阳性。B组和D组细胞形态无明显改变,PCR检测示培养后21d,A、C组SOX9、蛋白多糖、Ⅰ型胶原及Ⅱ型胶原表达较4d时明显增加(P<0.05)。B、D组Ⅰ型胶原表达较4d时明显增加(P<0.05),SOX9、蛋白多糖及Ⅱ型胶原较4d时无明显变化(P>0.05)。其中仅A组Ⅹ型胶原表达呈阳性。结论TGF-β3和BMP-7联合应用能促进hBMSCs分化更接近于椎间盘细胞,可能为椎间盘组织工程提供种子细胞。Objective To compare the molecular phenotype of human intervertebral disc cells and articular chondrocytes and to analyze whether hBMSCs can differentiate into both chondrocytes and nucleus pulposus cells after combined induction of TGF-β3 and BMP-7 in vitro. Methods The cells with the characteristics of hBMSCs were isolated from marrow aspirates of the volunteer donors＇ iliac crest. Human bone marrow was removed and fractionated, and adherent cell cultures were established. The 4th passage cells were then translated into an aggregate culture system in a serum-free medium. The pellet cultures ofhBMSCs were divided into four groups： 10 ng/mL TGF-β3 group （group A）, 200 ng/mL BMP-7 group （group B）, combination group of TGF-β3 and BMP-7 （group C） and blank group as the control （group D）. Histological observation, RT-PCR and RQ-PCR were applied to measure the expressions of collagen type I, II, X, aggrecan and SOX9 on the 4th and 21st day after cell induction, respectively. Results As was shown by histological observation, the induced cells expressed the feature of chondrocytes in morphology and ECM in groups A and C on the 21st day after the culture. And the collagen type II was positive after staining in groups A and C. The cell morphology of the induced cells in groups B and C had no obviouly changed. PCR detection showed that the expressions of SOX9, aggrecan, collagen type I, II in groups A and C at 21st day were more increased than those at 4th day （P 〈 0.05）. The only expressions of collagen type I in groups B and D at 21st day were more increased than those at 4th day （P 〈 0.05）. The expressions of collagen type X only was positive in group A. Conclusion Combination of TGF-β3 and BMP-7 can make the differentiated cells from hBMSCs much closer to intervertebral disc cells, so it perhaps could provide seed cells for intervertebral disc tissue engineering.

关 键 词：椎间盘组织工程hBMSCs 诱导分化 BMP-7 TGF-Β3 软骨形成 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]