添加RGD短肽的新型培养基对细胞生长、融合及外源基因表达的影响

The Influence of New Medium with RGD on Cell Growth, Cell Fusion and Expression of Exogenous Gene

中文关键词:  新型培养基RGD细胞融合外源基因

英文关键词:New culture mediumRGDCell fusion techniqueExogenous gene

中文摘要:

      目的在普通培养基中添加RGD短肽制备新型培养基，观察新型培养基对细胞生长状态、杂交瘤细胞融合和外源基因表达的影响。方法以普通培养基为对照，在普通培养基中添加不同质量浓度的RGD制备新型培养基，通过观察人胰腺上皮细胞株HPDE6-C7的生长增殖情况确定RGD的最佳质量浓度，然后，在培养基中接种不同浓度（5×10⁴、10⁵、5×105 mL^－1）的HPDE6-C7细胞，通过倒置显微镜观察细胞的形态、贴壁、长势和密度情况；再分别用普通培养基和添加RGD短肽的新型培养基进行杂交瘤细胞的融合，比较两种培养基融合后克隆形成率和克隆阳性率的差异；通过转染含绿色荧光蛋白(GFP)的质粒观察其荧光强度的方法和转染过表达KRAS质粒观察其蛋白表达的方法，观察添加RGD短肽的新型培养基相比普通培养基在转染外源基因表达水平方面的优势。结果RGD短肽使用的最佳质量浓度为10 ng/mL；添加RGD短肽的新型培养基所培养的细胞相比普通培养基形态更佳、贴壁更好、增殖更快。同时，添加RGD短肽的新型培养基应用于细胞融合所形成克隆的百分率和克隆阳性率均高于普通培养基（P<0.05）；与普通培养基比较，此新型培养基在转染外源基因GFP后荧光强度更高（P<0.05），表达外源基因KRAS的蛋白水平也更高（P<0.05）。结论添加RGD短肽的新型培养基在细胞生长状态、杂交瘤细胞融合及外源基因表达方面有突出优势，RGD短肽应用于细胞培养可能具有广泛前景和潜在的价值。

英文摘要:

      Objective To investigate the influence of a new culture medium added with RGD on cell growth, cell fusion and expression of exogenous gene. Methods A new medium was prepared by adding different concentrations of RGD to ordinary culture medium. The optimum concentration of RGD was determined by observation of the growth of human pancreatic epithelial cell line HPDE6-C7. After determining the optimum concentration of RGD, different concentrations of cells HPDE6-C7 (5×10⁴, 10⁵, 5×10⁵ mL^－1) were inoculated in the two mediums. The morphology, adherence, growth and density of the cells were observed by inverted microscope; The ratio of clone formation and the positive rate of cloning were compared between the two cultures after fusion; The fluorescence intensity after the transfection of plasmid with green fluorescent protein (GFP) and the protein expression after transfection of plasmid with KRAS were observed to campare the expression of exogenous genes between the new medium with ordinary medium. Results Firstly, the optimal concentration of RGD was 10 ng/mL. Compared with the normal medium, the cultured cells with RGD had better morphology, adhesion and faster proliferation. In addition, both of the number and positive rate of clones formed in the new medium were significantly higher than that in the ordinary medium (P<0.05);The fluorescence intensity after transfection of exogenous gene GFP in the new medium was significantly higher than that in normal medium (P<0.05); Expression level of exogenous gene KRAS of the new medium was also significantly higher than that in normal medium.Conclusion The new culture medium has highlighted advantages in cell growth, cell fusion and expression of exogenous genes. RGD peptide has widely prospect and potential value in the cell culture.