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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 ,而且可作为靶细胞接受外源目的基因的转染并有效表达
Objective To observe the main biological characteristics of adenovirus carrying hepatocyte growth factor cDNA (adHGF) transfected rabbit bone marrow stromal cells (MSC). Methods The bone marrow of adult male New Zealand white rabbits was drawn and the MSCs were obtained by density gradient centrifugation. Rabbit knee articular cartilage, in vitro cultured chondrocytes to the third generation. AdHGF was transfected into the fifth generation of MSCs. Cell viability was measured by MTT assay. The content of hexosaminoglycan (GAG) in cell culture supernatant was assayed by Alcian blue method. The expression of HGF in adHGF transfected MSCs was detected by enzyme-linked immunosorbent assay (ELISA). Immunohistochemical staining and reverse transcriptase polymerase chain reaction (RT PCR) were used to detect the expression of type I and type II collagen. The results of the original MSC short shuttle-shaped, cluster-like growth, passage cells were fusiform, vortex-like growth. AdHGF transfection before and after the proliferation of cells was no significant difference. GAG content increased after MSC transfection. Immunohistochemical staining before and after transfection MSC type I collagen positive, type Ⅱ collagen positive after transfection. After transfection, the expression of HGF was the highest within 1 week after transfection, reaching 125 ng / ml, and could last up to 6 weeks. RT-PCR showed that MSC expressed both type I collagen before and after adHGF transfection, and expressed microform type II collagen after transfection. Conclusion The natural outcome of MSC in vitro culture tends to be osteogenic. MSC is not only the source of HGF cells, but also as a target cell transfected with gene of exogenous gene and effective expression