肝癌细胞上清液对成纤维细胞表达α-平滑肌肌动蛋白、环氧合酶-2和肝细胞生长因子的影响

Effect of hepatic carcinoma cell supernatant on expressions of α-smooth muscle actin,cyclooxygenase-2 and hepatocyte growth factor in fibroblasts

目的观察小鼠肝癌细胞上清液对成纤维细胞增殖及表达α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、环氧合酶-2(cyclooxygenase-2,COX-2)、肝细胞生长因子(hepatocyte growth factor,HGF)的影响。方法制备小鼠成纤维细胞,并分别用普通培养液、小鼠肝癌细胞上清液的条件培养液及含转化生长因子-β1(transforming growth factor-β1,TGF-β1)的培养液培养成纤维细胞。采用MTT法检测3种培养液培养的成纤维细胞的增殖活力;RT-PCR和免疫组织化学法检测3种培养液培养的成纤维细胞中α-SMA、COX-2、HGF基因mRNA的转录水平及蛋白的表达情况。结果条件培养液及含TGF-β1的培养液对成纤维细胞的增殖均有促进作用,但条件培养液的促进作用更强。普通培养液培养的成纤维细胞在第5天时α-SMA基因mRNA的转录水平最高,含TGF-β1的培养液及条件培养液培养的成纤维细胞在第3天时α-SMA基因mRNA的转录水平最高,且明显高于普通培养液在第5天时的转录水平,第3天以后转录水平稳定;普通培养液培养的成纤维细胞中无COX-2和HGF转录;条件培养液及含TGF-β1的培养液培养的成纤维细胞在第5天时COX-2和HGF基因mRNA的转录水平最高,第5天后无明显下降。不同培养液培养的成纤维细胞中α-SMA蛋白在胞浆、胞膜均有不同程度的表达;普通培养液培养的成纤维细胞中COX-2和HGF蛋白不表达,而含TGF-β1的培养液及条件培养液培养的成纤维细胞中COX-2和HGF蛋白为阳性表达,表达部位均为胞浆。结论小鼠肝癌细胞上清液能有效、稳定地将成纤维细胞激活为稳定表达COX-2和HGF的肌成纤维细胞,其有望成为在细胞移植中促进移植细胞存活及血管重建的细胞。

Objective To observe the effect of hepatic carcinoma cell supematant on expressions of α-smooth muscle actin （α-SMA）, cyclooxygenase-2 （COX-2） and hepatocyte growth factor （HGF） in fibroblasts. Methods Mouse fibroblasts were prepared and cultured in common medium, conditional medium containing mouse hepatic carcinoma cell supematant and medium containing transforming growth factor-β1 （TGF-β1） respectively. The proliferative activities of fibroblasts in three media were determined by MTTassay, in which the mRNA transcription and protein expression levels of α-SMA, COX-2 and HGF by RT-PCR and immunohistochemical assay. Results Both conditional medium and the medium containing TGF-β1 promoted the proliferation of fibroblasts, while the former showed stronger effect. The transcription level of α-SMA mRNA in fibroblasts in common medium reached the maximum on day 5, while those in the medium containing TGF-β1 and conditional medium on day 3, after culture. The transcription levels of α-SMA mRNA in fibroblasts in the medium containing TGF-β1 and conditional medium on day 3 were significantly higher than that in common medium on day 5 after culture, and were stable afterwards. No transcription of COX-2 or HGF mRNA was observed in common medium. The transcription levels of COX-2 and HGF mRNAs in fibroblasts in conditional medium and the medium containing TGF-β1 reached the maximum on day 5 after culture and showed no significant decrease afterwards. The α-SMA was expressed at various degrees in cytoplasm and cell membrane of fibroblasts in various media. However, both COX-2 and HGF were negative in the fibroblasts in common medium, while were positive in the cytoplasm of those in medium containing TGF-β1 and conditional medium. Conclusion Mouse liver cancer cell supernatant effectively and stably activated fibroblasts into myofibroblasts in which COX-2 and HGF were expressed, which might be used for promoting the survival of transplanted cells and the revascularization in cell transplantation.