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目的:研究补肾益气方对人早孕细胞滋养层细胞增殖和早期凋亡的影响及其可能的信号转导通路。方法:体外分离培养人早孕细胞滋养层细胞,并添加不同浓度的喂服3d补肾益气方中药的大鼠血清,培养48h,采用噻唑蓝(MTT)比色法测定细胞的增殖活力,流式细胞术检测增殖细胞核抗原(PCNA)表达、Annexin Ⅴ FITC/PI双染色法检测细胞早期凋亡。结果:与体积分数20%对照血清组(B2组)比较,体积分数10%(A1组)及20%(A2组)补肾益气方含药血清作用48h,上调细胞滋养层细胞增殖活力、促进PCNA表达、降低凋亡细胞百分率(P<0.05),上述指标均呈剂量-效应关系。哺乳动物雷帕霉素靶分子(mTOR)通路的特异性抑制剂雷帕霉素抑制20%含药血清诱导的细胞增殖活力及PCNA蛋白表达,增加凋亡细胞百分率。结论:补肾益气方对人早孕细胞滋养层细胞的生长具有促进作用,mTOR信号通路可能是中药促进细胞滋养层细胞增殖、减少细胞凋亡的主要信号转导通路之一。
Objective: To study the effects of Bushengyiqi recipe on the proliferation and early apoptosis of human trophoblast cells and its possible signal transduction pathway. METHODS: Human gestational trophoblast cells were isolated and cultured in vitro. Different concentrations of serum from rats fed Chinese herbs of Nourishing Kidney and Invigorating Qi were added and cultured for 48 hours. Cell viability was measured by MTT colorimetric assay. Flow cytometry The expression of proliferating cell nuclear antigen (PCNA) was detected by cytology. The early apoptosis was detected by Annexin Ⅴ FITC / PI double staining. Results: Compared with 20% (B2) control group, the volume fraction of 10% (A1) and 20% (A2) Bushen Yiqi prescription serum for 48h, increased the proliferation of trophoblast cells and promoted PCNA expression and decrease the percentage of apoptotic cells (P <0.05). The above indexes all showed dose-effect relationship. Rapamycin, a specific inhibitor of mammalian target of rapamycin (mTOR) pathway, inhibited cell proliferation and PCNA protein expression induced by 20% drug-containing serum and increased the percentage of apoptotic cells. Conclusion: Bushen Yiqi prescription can promote the growth of human trophoblastic cells in human early pregnancy. The mTOR signaling pathway may be one of the major signal transduction pathways by which traditional Chinese medicine can promote cell proliferation of trophoblast cells and reduce apoptosis.