骨髓基质细胞与关节软骨细胞生物学特性的比较研究

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cDNA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(25 ng/ml)和地塞米松(10-7 mol/L).切取兔膝关节软骨,3 mg/ml Ⅱ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Ⅰ、Ⅱ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Ⅰ、Ⅱ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Ⅰ型胶原免疫组织化学染色阳性,Ⅱ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＜0.05).B组细胞爬片Ⅰ、Ⅱ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＞0.05);C组转染后第4天增殖率降低,与A组比较差异有统计学意义(P＜0.05),其余时间点各组间无统计学意义(P＞0.05).RT-PCR表明A、B、C组均表达Ⅰ型胶原,B、D组可表达Ⅱ型胶原,C组有较弱的Ⅱ型胶原表达. 结论 MSCs体外培养过程中自然转归趋向于成骨.传代后经向成软骨方向诱导,具有向软骨分化的能力;体外传代培养的MSCs具有干细胞自我增殖和定向分化的特性,可作为靶细胞接受外源目的基因转染并能有效表达.

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

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-beta 1 (TGF-beta 1, 10 ng/ml), basic fibroblast growth factor (bFGF, 25 ng/ml) and dexamethasone (DEX, 10(-7) mol/L) 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 mg/ml) 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 indicate MSCs have a natural tendency of osteogenic differentiation in vitro culture, and also demonstrate the chondrogenic potency with the technique of cytokines induction or gene modification after passage. MSCs can be transfected efficiently being seed cells in tissue engineered bone or cartilage to accept target genes such as adHGF, and have a higher levels of expression in vitro, which lasted 4 weeks at least.