稳定表达人CNTF诱导PC12细胞分化作用的研究

Study on differentiation of PC12 cells induced Stable expression of human Ciliary neurotophic factor

目的:探讨CNTF对PC12细胞的诱导分化作用。方法:应用转基因技术将pcDNA-S-hCNTF质粒转入COS7细胞进行表达,并对CNTF表达进行检测,然后将hCNTF修饰过的COS7细胞与PC12细胞进行联合培养,4天后对CNTF作用后的PC12进行形态学观察,β-tubulin免疫细胞化学染色及不同培养时间4天,5天后阳性细胞的指数进行分析,同时通过MTF实验对诱导培养后的PC12细胞内增殖进行检测。结果:PC12细胞与hCHTF修饰过的COS7细胞经过4天的联合培养,细胞大部分形态呈现出类似于神经元的形态,胞体立体感较强,并且细胞长出较长的突起。另外细胞呈现β-tubulin反应阳性,并且随培养时间延长,β-tubulin阳性细胞的数目有增加的趋势(从76.6％增加到88.3％),最后,反映细胞增殖程度的OD值为0.328±0.019,明显低于对照组0.586±0.028,0.597±0.032。结论:hCNTF对PC12细胞向神经元分化有明显的促进作用。

Objective: To explore differentiation of PC12 cells induced by stable expression of Ciliary neurotrophic factor. Methods: Biologically active human Ciliary neurotrophic factor was expressed and produced by transfection pc DNA-S-hCNTF into COS7 cells, CNTF production by the COS7 cell was determined with immtinocytorhemiral; stain ing method. PC12 cells were then co-cultured with CNTF genetically modified COST cells. After A days in vitro. The morphological changes of the co-cultured PC12 cells were observed under a phase constrast microsocope, Those PC 12 cells β-tubilin imnlunocytochemical staining were undertaken to evaluate PC',12 cells differentiation. And the number of the β-tubulin-positive cells were counted to analyse differentiation into neural cells at different culture time. Furthermore, The proliferation of cells by CNTF-induced were assessed with MTT assays. Result; After 4 days of co-culture with hCNTF-modified COS7, The majority of PC12 cells show neuron-like morphology, and they possess strong ste teognoisis in soma with thin, long processes. In addition, these cells show β-tubulin-immunocytochemical staining positive, and the percentage of β-tubulin positive cell increase from 76. 6% to 88. 3% with the culture time prolongation. As for these cells proliferation, The OD value were 0. 328 ± 0. 019, whereas OD value of control groups represented 0. 586 ±0. 028. 0. 597 ± 0. 032, respectively. Conclusion: The human CNTF can be expressed in COST cells, and human CNTF is able to significantly induce the differentiation of PC12 cells to neuronal cells.