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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 mouse hepatoma cell supernatant on the proliferation and expression of α-smooth muscle actin (α-SMA), cyclooxygenase-2 (COX-2) Effect of hepatocyte growth factor (HGF). Methods Mouse fibroblasts were prepared and cultured in the medium of normal medium, the conditioned medium of mouse liver cancer cell supernatant and the culture medium containing transforming growth factor-β1 (TGF-β1) cell. The proliferation of fibroblasts cultured in three culture media was detected by MTT assay. The transcriptional level of mRNA of α-SMA, COX-2 and HGF in fibroblasts cultured in three culture media were detected by RT-PCR and immunohistochemistry And protein expression. Results Conditioned culture medium and medium containing TGF-β1 promoted the proliferation of fibroblasts, but the effect of conditioned medium was stronger. The average transcription level of α-SMA mRNA in cultured fibroblasts cultured on day 5 was the highest, and the mRNA level of α-SMA mRNA in TGF-β1-conditioned medium and fibroblasts cultured in conditioned medium was higher than that on day 3 Which was significantly higher than that of normal medium at day 5, and remained stable after day 3; no transcription of COX-2 and HGF was found in fibroblasts cultured in normal medium; and the conditioned medium and medium containing TGF-β1 The mRNA level of COX-2 and HGF mRNA in cultured fibroblasts cultured on day 5 was the highest, and there was no significant decrease after 5 days. Α-SMA protein expressed in cytoplasm and membrane of fibroblasts cultured in different culture medium to varying degrees; COX-2 and HGF proteins in fibroblasts cultured in normal medium were not expressed, while TGF-β1-containing fibroblasts COX-2 and HGF protein in culture medium and conditioned medium were positive for COX-2 and HGF protein, all expressed in cytoplasm. Conclusion The mouse hepatoma cell supernatant can effectively and stably activate fibroblasts into myofibroblasts stably expressing COX-2 and HGF, which is expected to become a cell that promotes the survival and revascularization of transplanted cells in cell transplantation.