不同浓度肿瘤细胞上清液对成纤维细胞生长及表型转化的影响

Effect of different concentrations of tumor cell supernatant on growth and phenotypic transition of fibroblasts

目的利用含不同浓度肿瘤细胞上清液的条件培养液培养成纤维细胞,观察其对成纤维细胞的生长及表型变化的影响。方法用MTT法和流式细胞凋亡术测定成纤维细胞的生长变化与凋亡率。用RT-PCR检测成纤维细胞转化为肌成纤维细胞后α-平滑肌肌动蛋白(α-SMA)与转化生长因子β1(TGF-β1)的表达。用Western blot检测α-SMA、TGF-β1蛋白的表达。结果 MTT法发现肿瘤细胞上清液与2×DMEM∶F12比例为1∶1时,对成纤维细胞的生长有明显的促进作用,其生长速度快于其他浓度(P<0.05)。流式细胞凋亡术检测表明随着肿瘤细胞上清液浓度的提高,成纤维细胞的凋亡率也上升,1∶1的凋亡率与1.5∶1时有差异(P<0.05)。RT-PCR、Western blot表明普通培养液培养的成纤维细胞α-SMA与TGF-β1的表达高峰在第5天,第7天下降明显(P<0.05)。当比例为1∶1时,α-SMA与TGF-β1的表达增加,高峰提前到第3天(P<0.05),第3天以后α-SMA的表达稳定(P=0.96,P=0.21)。结论当肿瘤细胞上清液与2×DMEM∶F12比例为1∶1时,能有效促进成纤维细胞的生长,并促使成纤维细胞向肌成纤维细胞稳定的转化。

Objective To observe the effect of different concentrations of tumor cell supernatant on the growth and phenotype transition of fibroblasts.Methods Fibroblasts were cultured in conditioned medium containing different concentrations of tumor cell supernatant.Fibroblasts growth tendency was tested by MTT assay,and apoptotic rate was analyzed by flow cytometry.The mRNA and protein expressions of α-SMA and TGF-β1 were tested by RT-PCR and Western blotting,respectively.Results MTT assay showed that when the ratio of tumor cell supernatant and 2×DMEM∶F12 was 1∶1,the growth of the fibroblasts was significantly promoted（P〈0.05）,and when the ratio was 2∶1 or the supernatant was completely used,the growth could be restrained.As the concentration of tumor cell supernatant increased,the cells apoptotic rate was rising.The apoptotic rate exhibited significant difference between the ratio of 1∶1 and 1.5∶1（P〈0.05）.RT-PCR and Western blotting demonstrated that α-SMA and TGF-β1 expressed by fibroblasts that were cultured in normal medium reached the peak level at the fifth day,and then decreased significantly at the seventh day（P〈0.05）.When the ratio of tumor cell supernatant and 2×DMEM∶F12 was 1∶1,the expressions of α-SMA and TGF-β1（P=0.96,P=0.21） increased and reached the peak at the third day（P〈0.05）,after that time the α-SMA expression was sustained.Conclusion When the ratio of tumor cell supernatant in 2×DMEM∶F12 is 1∶1,the growth of fibroblasts and fibroblasts transformation into myofibroblasts could be effectively promoted.