肿瘤细胞上清液对成纤维细胞表型转变及血管内皮生长因子-A表达的影响

目的检测肿瘤细胞上清液对成纤维细胞的激活情况及激活后血管内皮生长因子-A(VEGF-A)表达的变化规律。方法 MTT法检测普通培养液、含转化生长因子-β1(TGF-β1)的普通培养液以及肿瘤细胞上清液组成的条件培养液对成纤维细胞生长情况的影响,用RT-PCR、Western blot及免疫组织化学法检测不同培养条件下成纤维细胞的α-平滑肌肌动蛋白(α-SMA)与VEGF-A的表达。结果肿瘤细胞上清液对成纤维细胞的生长有一定的促进作用。含TGF-β1的普通培养液比普通培养液更有利于成纤维细胞转化为肌成纤维细胞,并且能维持肌成纤维细胞的表型,但是二者均不表达VEGF-A;条件培养液能促进成纤维细胞稳定表达α-SMA和VEGF-A,二者在培养后第1天均开始表达,第3天表达量达到峰值,第3天以后表达稳定。结论肿瘤细胞上清液能够有效、稳定地激活成纤维细胞为肌成纤维细胞,成纤维细胞的激活程度影响VEGF-A的表达,二者均具有最佳的激活时间点,并且最佳时间点相一致,最佳激活点的成纤维细胞有望成为一种可用于移植的促进血管重建的细胞。

Influences of Tumor Cells Supernatant on Phenotypic Transition of Fibroblasts and Expression of Vascular Endothelial Growth Factor-A

Objective To investigate the expression of vascular endothelial growth factor-A(VEGF-A) and the phenotypic transition after the activation of fibroblasts by the supernatant of cultured tumor cells.Methods The growth tendency of fibroblasts was tested by the MTT assay.The expressions of alpha-smooth muscle actin(α-SMA) and VEGF-A mRNA were tested by RT-PCR.The expressions of α-SMA and VEGF-A protein were tested by immunohistochemistry and Western blot.Results The MTT assay indicated that the conditional medium which contained tumor cells supernatant could obviously promote the growth of the fibroblasts.RT-PCR and Western blot manifested that α-SMA expressed by the fibroblasts which cultured by normal medium reached its peak on day 5,then decreased to a low level on day 7.When the medium contained 2 ng/ml transforming growth factor-β1(TGF-β1),the fibroblasts could steadily express more α-SMA.But the above two mediums could not make the fibroblasts express the VEGF-A.When using the conditional medium,the α-SMA peak advanced on the third day and maintained at a high level,so as the expression of the VEGF-A.Conclusions The results suggested that fibroblasts can be activated to be myofibroblasts when using the conditional medium.The best activation time of the fibroblasts is consistent with the time of the VEGF-A expression at the highest level by the activated fibroblasts.The fibroblasts which activated at the best time are expected to become a kind of cells which can be used for promoting revascularization.