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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。
Objective To detect the activation of tumor cell supernatant on fibroblasts and the changes of vascular endothelial growth factor-A (VEGF-A) expression after activation. Methods MTT assay was used to detect the effect of culture medium containing common culture medium containing TGF-β1 (TGF-β1) and tumor-cell supernatant on the growth of fibroblasts. RT-PCR and Western blot Immunohistochemistry was used to detect the expression of α-smooth muscle actin (α-SMA) and VEGF-A in fibroblasts under different culture conditions. The results of tumor cell supernatant on the growth of fibroblasts have a certain role in promoting. The common culture medium containing TGF-β1 is more conducive to the transformation of fibroblasts into myofibroblasts than ordinary culture medium, and can maintain the phenotype of myofibroblasts, but neither expression VEGF-A; conditioned medium can The results showed that α-SMA and VEGF-A could be stably expressed in fibroblasts. Both of them expressed on the first day after culture, reaching the peak on the third day and stable expression on the third day. Conclusion The tumor cell supernatant can effectively and stably activate fibroblasts into myofibroblasts. The activation of fibroblasts affects the expression of VEGF-A, both of which have the best activation time, and the optimal time point Consistent with the best activation point of fibroblasts is expected to become a transplant can promote vascular reconstruction of cells.