肝细胞生长因子对兔骨髓基质细胞生物学特性的影响  

The effect of hepatocyte growth factor on the biological characteristics of marrow-derived stromal cells in rabbits

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

机构地区：[1]解放军总医院骨科研究所,100853 [2]解放军军事医学科学院二所三室

出　　处：《中国药物与临床》2003年第5期392-395,共4页Chinese Remedies & Clinics

基　　金：国家自然科学基金资助项目 (3 0 170 949)

摘　　要：目的　观察腺病毒携带肝细胞生长因子cDNA(adHGF)转染兔骨髓基质细胞 (MSC)后的主要生物学特性。方法　抽取成年雄性新西兰白兔骨髓 ,密度梯度离心获得MSC。取兔膝关节软骨 ,体外培养软骨细胞至第 3代。adHGF转染第 5代MSC ,噻唑蓝 (MTT)法检测细胞增生活力 ,阿尔新蓝法检测细胞培养上清液中氨基己糖多糖 (GAG)含量。酶联免疫吸附实验 (ELISA)检测adHGF转染MSC后HGF的表达。免疫组织化学染色及反转录 聚合酶链反应 (RT PCR)检测Ⅰ、Ⅱ型胶原表达。结果　原代MSC为短梭形、簇状生长 ,传代细胞呈长梭形、旋涡样生长。adHGF转染前后细胞增生状态差异无显著性。MSC转染后GAG含量增加。免疫组织化学染色MSC转染前后I型胶原阳性 ,转染后Ⅱ型胶原弱阳性。转染后HGF表达量在转染 1周内最高 ,达 12 0 5ng/ml,并可持续至 6周。RT PCR表明MSC在adHGF转染前后均表达I型胶原 ,转染后微量表达Ⅱ型胶原。结论　MSC在体外培养过程中的自然转归是趋向于成骨。MSC不仅是HGF的源细胞 。Objective To observe the main biological characteristics of bone marrow derived stromal cells(MSC) after gene modified with hepatocyte growth factor (adHGF). Methods The bone marrow derived stromal cells from an adult New Zealand white rabbit were isolated with density gradient centrifugation and cultivated till the 5th passage.Chondrocytes were obtained with Ⅱ collagenase from knee joints of the rabbit and culture expanded till the 3th passage.The 5th MSC were transduced by adHGF.The capacity of cell proliferation was tested by MTT;quantitative secretion of glycosaminoglycan (GAG) was tested by alcian blue;expression of hepatocyte growth factor (HGF) of transduced MSC was tested by ELISA;collagen Ⅰ,Ⅱ were tested by immunohistochemical and RT PCR.Results Primary MSC proliferated as visible symmetric colonies and short spindle shape while the 5th MSC showed long spindle shape.No significant difference showed among 5th MSC,adHGF transduced MSC and chondrocytes on proliferation by MTT ( P =0 269 8).The content of GAG in adenoviral hepatocyte growth factor (adHGF) modified was higher than that of 5th MSC, but no significant difference was seen between them.Type Ⅰ collagen was identified in MSC with or without adHGF transfected.Not more than slight positive stain of type Ⅱ collagen showed and a low level of mRNA type Ⅱ collagen expressed after adHGF modified MSC.Conclusion MSC had a natural tendency of osteogenic differentiation in vitro culture.Besides being a source of HGF,MSC could be transfected efficiently as seed cells to accept target genes by adHGF,and had a higher level of expression in vitro ,which persisted 6 weeks at least.

关 键 词：肝细胞生长因子 兔 骨髓基质细胞 生物学特性 免疫组织化学 检测 

分 类 号：R96[医药卫生—药理学]