瘦素对HTR8-SVneo细胞增殖的影响及调控机制

目的研究瘦素(Leptin)对HTR8-SVneo滋养细胞系增殖的影响及对DNA结合和分化抑制剂(ID)分子表达水平的影响,探讨Leptin对HTR8-SVneo细胞增殖作用的调节机制。方法体外培养HTR8-SVneo滋养细胞,MTT法检测Leptin不同质量浓度(0、10、50、100、250、500 ng/mL)刺激后细胞增殖情况。HTR8-SVneo细胞培养体系中加入Leptin(0、100、500 ng/mL),RT-PCR检测瘦素受体(Leptin R)各亚型表达,实时定量PCR检测ID分子表达,Western Blot检测ID2表达。SiRNA干扰ID2分子后,分为Leptin(0 ng/mL)、Leptin(100 ng/mL)、Leptin(500 ng/mL)、Leptin(0 ng/mL)+ID2-siRNA、Leptin(100 ng/mL)+ID2-siRNA、Leptin(500 ng/mL)+ID2-siRNA,MTT检测Leptin对HTR8-SVneo滋养细胞增殖的影响。结果与Leptin(0 ng/mL)比较,高质量浓度Leptin(100、250、500 ng/mL)促进HTR8-SVneo细胞增殖(P<0.05);HTR8-SVneo细胞表达除HuB219.2外的全部受体亚型;Leptin以剂量依赖方式促进HTR8-SVneo细胞ID2分子及OB-RL表达(P<0.05);ID2-siRNA抑制ID2表达并逆转Leptin对滋养细胞增殖的促进作用。结论 Leptin可能通过Leptin R-ID2途径促进HTR8-SVneo细胞增殖,为阐明滋养细胞增殖调控机制提供了新的基础数据。

Effect of leptin on HTR8-SVneo cell proliferation in vitro and its mechanism

Objective To investigate the effects of leptin on proliferation of HTR8-SVneo cells in vitro and the level of ID(inhibitor of DNA binding and differentiation),and to explore the regulatory mechanism of leptin underlying HTR8-SVneo cell proliferation.Methods The proliferation of HTR8-SVneo cells cultured in vitro under diverse concentrations(0,10,50,100,250 and 500 ng/mL) of leptin was tested by means of MTT.Then,HTR8-SVneo cells were treated with leptin(0,100 and 500 ng/mL),and leptin receptor(leptin R) profiling,ID mRNA profiling and the level of the ID2 protein were detected by RT-PCR,real-time PCR and Western blot,respectively.To observe the effect of leptin on HTR8-SVneo cell proliferation after ID2-siRNA treatment,HTR8-SVneo cells were divided into 6 groups: leptin(0 ng/mL),leptin(100 ng/mL),leptin(500 ng/mL),leptin(0 ng/mL)+ID2-siRNA,leptin(100 ng/mL)+ ID2-siRNA and leptin(500 ng/mL)+ID2-siRNA.Results Proliferation of HTR8-SVneo cells was enhanced by high concentrations of leptin(100,250 and 500 ng/mL) compared with leptin(0 ng/mL)(P<0.05).Leptin receptor subtypes except HuB219.2 were detected on HTR8-SVneo cells.The up-regulation of ID2 or OB-RL was induced by leptin in a dose-dependent manner.ID2-siRNA inhibited expression of the ID2 protein and reversed the enhancement of HTR8-SVneo cell proliferation induced by leptin.Conclusion Leptin enhances HTR8-SVneo cell proliferation by the pathway of leptin R-ID2,which offers new basal data for elucidating the regulatory mechanism of proliferation of trophocytes.