胰岛素促进结肠平滑肌细胞合成干细胞因子的信号转导途径

The signal transduction pathway of insulin promoting stem cell factor expression in colonic smooth muscle cells

目的探讨胰岛素（Ins）促进结肠平滑肌细胞（SMC）表达干细胞因子（SCF）的细胞内信号传导途径。方法分离培养SD大鼠结肠SMC，采用a-actin免疫荧光鉴定。分3大组进行实验。①Ins浓度梯度组：SMC添加不同浓度Ins（0、2．5、5、20、40和80mg／L），培养16h。②Ins时间梯度组：SMC＋Ins（40mg／L），分别培养0、8、16和24h。③信号通路阻断组：SMC加LY-294002或PD-98059预处理后，再予以40mg／L的Ins诱导16h。四甲基偶氮唑盐（MTT）法检测结肠SMC增殖情况，Western印迹和反转录PCR检测SCF的变化。结果①Ins在5mg／L浓度即能显著促进SMC增殖，其效应与20、40和80mg／L时差异无统计学意义（P值均d0．05）。②与未添加Ins组（即空白对照组）比较，低中剂量Ins（2．5、5和20mg／L）即有促进结肠SMC表达SCFmRNA和SCF蛋白的作用（P〈0．05），这种作用在Ins40mg／L时达到最大（P〈0．05），80mg／L时与40mg／L时差异无统计学意义（P〉0．05）。③与0h时相比，40mg／L的Ins作用于SMC8h后SCF表达增加（P〈0．05），16h后达峰值（P〈0．05），24h与16h差异无统计学意义（P〉0．05）。④与空白对照组和溶剂对照组相比，LY-294002、PD-98059处理16h，均能减少Ins诱导的SCF的表达（P值均〈0．05）。结论Ins能促进SMC增殖；在一定范围内呈剂量与时间依赖性诱导结肠SMC合成SCF，该效应可能与PI3K与MAPK两条信号通路有关。

Objective To investigate the intracellular signal transduction pathway of insulin promoting stem cell factor （SCF） expression in colonic smooth muscle cells （SMCs）. Methods Colonic SMCs from SD rats were isolated and cultured, which were identified by a-actin immunofluorescence cytochemistry staining. SMCs were divided into 3 groups for experiment. （1） Insulin concentration gradient group： Adding different insulin concentrations （0, 2.5, 5, 20, 40 and 80 mg/L） into SMCs culture and cultured for 16 hours; （2） Insulin time point group.. SMCs were cultured with insulin （40 mg/L） for different time durations （0, 8, 16, 24 hours） ; （3） Signal pathway blocking group： SMCs were pretreated with LY-294002 or PD-98059, and then induced with insulin （40 mg/L） for 16 h. The proliferation of SMCs was examined by MTT method. The change of SCF expression was determined by Western blot and RT-PCR. Results （1） Insulin at concentration of 5 mg/L remarkably promoted SMCs proliferation, and there was no significant difference in its effect with insulin concentrations at 20, 40, 80 mg/L （all P〈0.05）. （2） Compared with no insulin adding group, low and medium dose of insulin （2. 5, 5, 20 mg/L） already had a role in promoting SCF mRNA and SCF protein expression （all P 〈 0.05）. It reached maxium effect when insulin concentration was at 40 mg/L （P〈 0. 05）, and there was no significant difference between insulin concentration at 40 mg/L and 80 mg/L （P〉0.05）. （3） Compared with time 0 hour, the expression of SCF increased after insulin worked on SMC （40 mg/L） for 8 hours （P〈0.05）, and reached the peak after 16 hours （P〈0. 05）, and there was no significant difference between 24 hours and 16 hours （P〉0.05）. （4） Compared with blank control group and solvent control group, both insulin-induced SCF expression reduced after pretreated with LY-294002 or PD-98059 （all P〈0.05）. Conclusions Insulin can promote SMCs proliferation, and in certain range the induction of SCF synthesis in colon SMCs is in dose and time dependent manners. The effect may be related to PI3K and MAPK signal pathway.