M-CSF对卵泡颗粒细胞功能调节及其分子机制

目的 探讨巨噬细胞集落刺激因子(M-CSF)对卵泡颗粒细胞的功能调节及其分子机制。方法 取2014年7月~2014年10月间,在浙江大学医学院附属妇产科医院接受体外受精-胚胎移植的30例因男性因素不孕妇女的卵泡液,患者平均年龄30.8±2.1岁,提取颗粒细胞培养24h后,分为空白对照组、M-CSF组(10、25、50、100ng/ml rhM-CSF)、M-CSF+来曲唑组(0、10、25、50、100ng/ml rhM-CSF+10^-6mol/L来曲唑)、FSH组(10、25、50、100IU/ml rhFSH)、FSH+来曲唑组(0、10、25、50、100IU/ml rhFSH+10^-6mol/L来曲唑),继续培养24h,用ELISA法测定培养液E₂浓度,RT-PCR法测定颗粒细胞FSH受体mRNA及M-CSF受体mRNA的表达。取指数生长期的人卵巢肿瘤颗粒细胞系COV434,给予不同浓度(0、10、25、50、100ng/ml)的人重组M-CSF,于37℃、5%CO₂温箱内培养24h,MTS比色实验测定细胞增殖率。用Western blot法检测rhM-CSF(50ng/ml)处理后COV434细胞MAPK通路相关蛋白的表达。结果 颗粒细胞培养液中雌二醇(E₂)浓度与rhM-CSF或rhFSH浓度呈正相关(P<0.05)。rhFSH在一定浓度范围(<25IU/ml)内可促进颗粒细胞M-CSF受体mRNA表达(P<0.05),与来曲唑联合作用后,不同浓度组颗粒细胞M-CSF受体mRNA的表达水平与对照组相比均明显升高(P<0.05)。rhM-CSF单独或与来曲唑联合作用,均可促进颗粒细胞FSH受体mRNA表达(P<0.05)。随着rhM-CSF浓度的升高,COV434细胞的增殖能力也呈上升趋势。50ng/ml浓度的rhM-CSF能以时间依赖的方式瞬间激活JNK及P38信号通路,分别使用JNK抑制剂SP600125、P38抑制剂SB203580预处理后,并不影响各组雌激素水平。结论 M-CSF可能在促进颗粒细胞的增殖分化以及雌激素的合成分泌过程中起重要作用。

Involvement of Macrophage Colony-stimulating Factor (M-CSF) in the Function of Follicular Granulosa Cells and Its Molecular Mechanism

Objective To investigate the effect of macrophage colony-stimulating factor(M-CSF) in follicular granulosa and its molecular mechanism. Mehtods Human luteinized granulosa cells(LCC) were isolated from follicular fluid of superovulated infertile patients(average age 30.8±2.1years old) undergoing in vitro fertilization and embryo transfer. LCC were cultured with DMEM/F12 medium plus various concentration of M-CSF(0,10,25,50,100ng/ml),M-CSF+Letrozole(10^-6mol/L),FSH(0,10,25,50,100IU/mL),FSH+Letrozole(10^-6mol/L).Media were collected at 24 hours after culture and estradiol(E₂) in media were measured by ELISA. Expression levels of FSH receptor and M-CSF receptor were deteced by quantiative RT-PCR. After cultured COV434 were treated with M-CSF, cell proliferation was quantified by MTs assay and protein expression was detected by Western blot. Results Either M-CSF or FSH stimulated the production of E₂. The production of FSH receptors was enhanced by M-CSF or M-CSF+Letrozole in vitro in a dose-dependent manner. Conversely, FSH was able to promote the expression of the receptor of M-CSF when present in an appropriate concentration. With the increasing of M-CSF concentration, the cell proliferation of COV343 showed an upward trend. M-CSF induced phosphorylation of JNK and P38. The level of estradiol(E₂) in media didn't change when cells were pretreated with the JNK inhibitor SP600125 or P38 inhibitor SB203580. Conclusion M-CSF may play an important role in promoting the proliferation and differentiation of granulosa cell,also the synthesis and secretion of estrogen.