胰岛素样生长因子Ⅰ对结肠平滑肌细胞产生干细胞因子的调控

目的 探讨激活胰岛素样生长因子(IGF)-I受体后,大鼠结肠平滑肌细胞(SMC)合成干细胞因子(SCF)的变化及其信号途径.方法 酶解法分离培养SD大鼠结肠SMC,免疫组化鉴定.2.5×10~5/ml SMC接种培养,①加入100μg/L IGF-I分别培养0、8、16、24、48 h;②分别加入0、5、10、50、100、150μg/L IGF-I培养16 h.同时设空白对照组和1GF-I组.免疫荧光、Western印迹、逆转录(RT)-PCR法检测SMC经不同浓度和不同时间IGF-I刺激后SCF的表达.并用50μmol/L LY-294002、PD-98059分别阻断磷脂酰激醇3激酶(P13K)、丝裂原活化蛋白激酶(MAPK)信号途径,观察SCF的变化是否与P13K、MAPK信号途径有关.结果 在无血清培养条件下,SCF在大鼠结肠SMC中少量表达,低剂量IGF-I(5和10μg/L)对SCF表达无影响,中、高剂量IGF-I(50、100、150μg/L)诱导其表达增加,100 μg/L可能为体外最大有效浓度,其促SCF合成的最高峰在第16小时.分别用LY-294002、PD-98059预处理SMC、再用IGF-I诱导,LY-294002对SCF的表达无影响、PD-98059能部分阻断SCF的表达.结论 IGF-I可能通过MAPK信号通道诱导SCF在结肠SMC中的表达,提示其可能是IGF-I调控结肠SMC产生SCF的机制.

Regulation of insulin-like growth factor I on the expression of stem cell factor in colonic smooth muscle ceils

Objective To estimate the effect and the intracellular signal transduction pathway of insulin-like growth factor I (IGF-I) on the expression of stem cell factor(SCF) in colonic smooth-muscle cells(SMC). Methods The SMCs isolated from colon of the SD rats by enzymolysis were cultured and identified by α-actin immunocytochemical method. Colonic SMCs were cultured either with 100 μg/L of IGF-I at different time points (0, 8, 16, 24 and 48 hours) or with different concentrations (0,5,10,50,100 and 150 μg/L) of IGF-I for 16 hours. The expressions of SCF in colonic SMCs pretreated with or without speicific phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY-294002 or mitogen-activated protein (MAP) kinase (MEK1) inhibitor PD-98059 were examined by Western blotting and quantitative reverse transeription-polymerase chain reaction and immunofluorescence. Results Low expression of SCF was found in colonic SMCs cultured in the bovine serum free medium. There was no effect of 5 or 10 μg/L of IGF- I on the expression of SCF. However, the expressions of SCF mRNA and protein were increased when stimulated with high concentrations (50,100,150 μg/L) of IGF-I. The peak expression of SCF was showed at the 16th hour after stimulating with 100 μg/L of IGF- I that was considered as the most effective concentration in vitro. The expression of SCF was not influenced by LY-294002, but was partly blocked by PD-98059. Conclusions The expression of SCF in colon SMCs may be induced by IGF-Ⅰ through MAP kinase signaling pathway.