骨髓基质细胞与关节软骨细胞生物学特性的比较研究  

COMPARATIVE STUDY ON THE MAIN BIOLOGICAL CHARACTERISTICS OF MARROW-DERIVED STROMAL CELLS AND CHONDROCYTES IN VITRO CULTURE IN RABBITS

作　　者：张东[1] 黄靖香[1] 哈小琴[2] 赵斌[1] 余征[1] 卢世璧[1] 

机构地区：[1]解放军总医院骨科研究所,北京100853 [2]解放军军事医学科学院

出　　处：《中国修复重建外科杂志》2004年第1期53-57,共5页Chinese Journal of Reparative and Reconstructive Surgery

摘　　要：目的　观察兔骨髓基质细胞 (MSCs)诱导和基因修饰后的主要生物学特性 ,并与关节软骨细胞进行比较。　方法　抽取成年雄性新西兰大白兔髂骨骨髓 ,密度梯度离心获得骨髓基质细胞 ,培养传至第 5代 ,按处理方法分为常规培养液组 (A组 )、条件培养液组 (B组 )及重组缺陷型腺病毒携带肝细胞生长因子 c DNA转染组 (C组 )。条件培养液为常规培养液中含转化生长因子 - β1 (10 ng/ml)、碱性成纤维细胞生长因子 (2 5 ng/ml)和地塞米松 (10 - 7mol/L)。切取兔膝关节软骨 ,3mg/ml 型胶原酶消化传代培养至第 3代 (D组 )。观察原代 MSCs及第 5代 MSCs(体外培养 8～ 10周后 )细胞形态 ,对第 5代 MSCs及第 3代软骨细胞进行 、 型胶原免疫组织化学染色 ,MTT法检测细胞增殖情况。阿利新蓝法检测细胞培养上清液中糖胺多糖 (GAG)含量。提取各组培养细胞总 RNA,RT- PCR检测 、 型胶原表达。　结果　原代 MSCs为短梭形、簇状生长 ,传代细胞呈长梭形、旋涡样生长。A组细胞爬片 型胶原免疫组织化学染色阳性 , 型胶原免疫组织化学染色阴性 ,GAG含量低 ,与 D组比较 ,差异有统计学意义 (P<0 .0 5 )。B组细胞爬片 、 型胶原免疫组织化学染色阳性 ,GAG含量升高 ,与 D组比较差异无统计学意义 (P>0 .0 5 ) ;C组转染后第Objective To observe the main biological characteristics and chondrogenesis potency of bone marrow -derived stromal cells(MSCs) after cytokines induction or gene modification in vitro. Methods MSCs from an adult New Zealand white rabbit were isolated and cultivated, and then MSCs were divided into the common medium group(Group A, 15%FBS in DMEM), the induced group by cytokines (Group B), the transfected group(Group C)with adenovirus-hepatocyte growth factor transgene (adHGF). The medium of group B consisted of transforming growth factor-β 1(TGF-β 1,10 ngml), basic fibroblast growth factor(bFGF,25 ngml) and dexamethasone (DEX,10 -7 molL) with 15%FBS in DMEM. Cartilage slices were obtained from femoral condyles and patellar grove in the same rabbit. The minced cartilage was digested in Ⅱ collagenase (3 mgml) to obtain chondrocytes(Group D). The change of cell appearance, proliferation capacity, glycosaminoglycans(GAG), immunohistochemical staining for type Ⅰ, Ⅱ collagen were observed during the 5th passage MSCs and MSCs after induction or gene modification. Expression of mRNA for type Ⅰ and Ⅱ collagen was detected by RT-PCR. Results Primary MSCs proliferated as short-spindle shape, while the 5th MSCs showed long-spindle shape. Positive stain of type Ⅰ collagen could be found in groups A, B and C, while positive stain of type Ⅱ collagen was shown in groups B and D. The content of GAG in group B was higher than that in group A, but there was no significant difference between them(P>0.05), and there was significant difference between groups A and D(P<0.05). No significant difference was noted in groups A,B and C on proliferation by MTT(P>0.05), except that of at the fourth day after transfection between groups A and C(P<0.05). RT-PCR demonstrated that MSCs always had higher levels of mRNA type Ⅰ collagen in groups A, B and C. The expression of mRNA type Ⅱ collagen was identified in groups B and D, and only low levels of mRNA type Ⅱ collagen in group C. Conclusion The above results i

关 键 词：骨髓基质细胞 关节软骨 细胞生物学特性 基因修饰 兔 组织工程 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]