M-CSF对卵泡颗粒细胞功能调节及其分子机制
发布时间：2019-06-04 22:43
【摘要】：背景:巨噬细胞集落刺激因子(M-CSF,又称集落刺激因子-1,CSF-1)是细胞因子调控网络中的的重要成员,其对单核-巨噬细胞的作用已广为人知,近年来的研究表明,M-CSF除了能刺激巨噬细胞的增殖外,还具有调节女性生殖细胞生长、增殖和分化的作用。Witt BR在1997年首次发现在人卵泡细胞中有M-CSF及其受体mRNA,并且有动物模型证实M-CSF可促进卵泡的生长和排卵。缺乏M-CSF基因的OP/OP小鼠动情周期延长,窦前卵泡及成熟卵泡减少,排卵率降低,注射M-CSF可使其动情周期恢复正常,发育的卵泡数增加,排卵率升高,并增加颗粒细胞的增殖能力。随后又有研究证实,颗粒细胞存在M-CSF受体,其合成分泌受垂体促性腺激素及雌激素(E2)的影响。由此推测,M-CSF可能在维持卵子的发育及促进颗粒细胞的增殖分化、雌激素的合成分泌过程中起了重要作用本研究通过测定颗粒细胞表面FSH受体mRNA及M-CSF受体mRNA的表达,进一步探讨FSH、M-CSF和E2之间错综复杂的交互关系,同时研究M-CSF调节颗粒细胞功能的可能信号通路机制,从而更深刻地揭示M-CSF与颗粒细胞相互调控的全貌。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:2014年7月至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,平均年龄(30.8±2.1)岁,经肌内注射人绒毛膜促性腺激素(hCG)后34-36h在阴道超声探头引导下经阴道取卵,从剩余卵泡液中提取颗粒细胞培养24小时后,分为空白对照组、M-CSF组(10、25、50、100ng/mlrhM-CSF)、M-CSF+来曲唑组(0、10、25、50、100ng/mlrhM-CSF+10-65mol/l 来曲唑)、FSH 组(10、25、50、100IU/mlrhFSH)、FSH+来曲唑组(0、10、25、50、100IU/ml rhFSH+10-6mol/l来曲唑),继续培养24小时,留取上清液,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24小时,MTS比色实验测定细胞增殖率。用Western Blot法检测rhM-CSF(50ng/ml)处理后不同时间点(0,15,30分钟1,2,6小时)COV434细胞丝裂原活化蛋白激酶(MAPK)通路相关蛋白:细胞外调节的蛋白激酶(ERKs)、c-JunN末端蛋白激酶(JNKs)、p38蛋白激酶(p38kinase)的表达。结果:颗粒细胞培养液中E2浓度与M-CSF或FSH浓度呈正相关(P0.05)。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(p0.05)。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显著升高(p0.05)。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(p0.05)。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P-38信号通路,使其磷酸化水平增高,但总蛋白表达水平未受影响,对ERK1/2通路则无明显激活作用。分别使用JNK抑制剂SB203580,P38抑制剂SP600125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了重要作用。
[Abstract]:BACKGROUND: The macrophage colony-stimulating factor (M-CSF, also called the colony-stimulating factor-1, CSF-1) is an important member of the cytokine regulatory network, and its role in the mononuclear-macrophage is well known. In recent years, studies have shown that M-CSF, in addition to the ability to stimulate the proliferation of macrophages, It also has the effects of regulating the growth, proliferation and differentiation of female germ cells. Witt BR, for the first time in 1997, found M-CSF and its receptor mRNA in human follicular cells, and an animal model confirmed that M-CSF could promote the growth and ovulation of the follicle. In the absence of the M-CSF gene, the activity of the OP/ OP mouse is prolonged, the premenstrual follicle and the mature follicle are reduced, the ovulation rate is reduced, the injection of M-CSF can restore the estrus cycle of the mouse, the number of the developed follicles is increased, the ovulation rate is increased, and the proliferation ability of the granulosa cells is increased. It was then confirmed that the M-CSF receptor was present in the granulosa cells, and its synthesis was affected by the pituitary gonadotropins and the estrogen (E2). It is suggested that M-CSF may play an important role in maintaining the development of the ovum and promoting the proliferation and differentiation of the granulosa cells and the synthesis and secretion of the estrogen. The complex interaction between M-CSF and E2, and the possible signaling pathway mechanism of M-CSF in the regulation of the granular cell function, are also studied, so that the whole picture of the regulation of M-CSF and granulosa cells is more deeply revealed. Objective: To study the function regulation and molecular mechanism of macrophage colony-stimulating factor (M-CSF) on the granulosa cells of the follicle. Methods: In the period from July 2014 to October 2014,30 cases of in-vitro fertilization and embryo transfer were received in the Affiliated Hospital of Obstetrics and Gynecology of Zhejiang University Medical College. The average age (30.8-2.1) years. 34-36h after intramuscularly injection of human chorionic gonadotropin (hCG) under the guidance of the vaginal ultrasound probe under the guidance of the vaginal ultrasound probe for 24 hours, the cells were divided into the blank control group, the M-CSF group (10,25,50,100 ng/ ml of rhM-CSF), the M-CSF + group (0,10,25,50,100 ng/ ml rhM-CSF + 10-65 mol/ l), The expression of FSH receptor mRNA and M-CSF receptor mRNA in granulosa cells was determined by RT-PCR. The human ovarian tumor granulosa cell line COV434 in the exponential growth phase was taken, and the human recombinant M-CSF with different concentrations (0,10,25,50,100 ng/ ml) was given. The cells were cultured for 24 hours at 37.degree. C. and 5% CO2 incubator, and the rate of cell proliferation was determined by MTS colorimetric assay. The expression of protein kinase (ERKs), c-JunN terminal protein kinase (JNKs), p38 protein kinase (p38kinase) in the extracellular regulated protein kinase (ERKs), c-JunN terminal protein kinase (JNKs), p38 protein kinase (p38kinase) was detected by Western Blot method at different time points (0,15,30 min.1,2,6 h) after treatment with rhM-CSF (50 ng/ ml). Results: The concentration of E2 in granulosa cell culture medium was positively correlated with the concentration of M-CSF or FSH (P0.05). rhFSH could promote the expression of M-CSF receptor mRNA in granulosa cells (p0.05) in a certain concentration range (25 IU/ ml). The expression of M-CSF receptor mRNA in the granulosa cells of different concentration groups was significantly higher than that of the control group (p0.05). As the concentration of rhM-CSF increased, the proliferation ability of COV434 cells was also on the rise. rhM-CSF at 50 ng/ ml concentration could activate the signal pathway of JNK and P-38 in a time-dependent manner to increase the level of phosphorylation, but the level of total protein expression was not affected, and the ERK1/2 pathway did not significantly activate. After pre-treatment with JNK inhibitor SB203580 and P38 inhibitor SP600125, the levels of estrogen in each group were not affected. Conclusion: M-CSF may play an important role in promoting the proliferation and differentiation of granulosa cells and the synthesis and secretion of estrogen.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R714.8
本文编号：2493052
[Abstract]:BACKGROUND: The macrophage colony-stimulating factor (M-CSF, also called the colony-stimulating factor-1, CSF-1) is an important member of the cytokine regulatory network, and its role in the mononuclear-macrophage is well known. In recent years, studies have shown that M-CSF, in addition to the ability to stimulate the proliferation of macrophages, It also has the effects of regulating the growth, proliferation and differentiation of female germ cells. Witt BR, for the first time in 1997, found M-CSF and its receptor mRNA in human follicular cells, and an animal model confirmed that M-CSF could promote the growth and ovulation of the follicle. In the absence of the M-CSF gene, the activity of the OP/ OP mouse is prolonged, the premenstrual follicle and the mature follicle are reduced, the ovulation rate is reduced, the injection of M-CSF can restore the estrus cycle of the mouse, the number of the developed follicles is increased, the ovulation rate is increased, and the proliferation ability of the granulosa cells is increased. It was then confirmed that the M-CSF receptor was present in the granulosa cells, and its synthesis was affected by the pituitary gonadotropins and the estrogen (E2). It is suggested that M-CSF may play an important role in maintaining the development of the ovum and promoting the proliferation and differentiation of the granulosa cells and the synthesis and secretion of the estrogen. The complex interaction between M-CSF and E2, and the possible signaling pathway mechanism of M-CSF in the regulation of the granular cell function, are also studied, so that the whole picture of the regulation of M-CSF and granulosa cells is more deeply revealed. Objective: To study the function regulation and molecular mechanism of macrophage colony-stimulating factor (M-CSF) on the granulosa cells of the follicle. Methods: In the period from July 2014 to October 2014,30 cases of in-vitro fertilization and embryo transfer were received in the Affiliated Hospital of Obstetrics and Gynecology of Zhejiang University Medical College. The average age (30.8-2.1) years. 34-36h after intramuscularly injection of human chorionic gonadotropin (hCG) under the guidance of the vaginal ultrasound probe under the guidance of the vaginal ultrasound probe for 24 hours, the cells were divided into the blank control group, the M-CSF group (10,25,50,100 ng/ ml of rhM-CSF), the M-CSF + group (0,10,25,50,100 ng/ ml rhM-CSF + 10-65 mol/ l), The expression of FSH receptor mRNA and M-CSF receptor mRNA in granulosa cells was determined by RT-PCR. The human ovarian tumor granulosa cell line COV434 in the exponential growth phase was taken, and the human recombinant M-CSF with different concentrations (0,10,25,50,100 ng/ ml) was given. The cells were cultured for 24 hours at 37.degree. C. and 5% CO2 incubator, and the rate of cell proliferation was determined by MTS colorimetric assay. The expression of protein kinase (ERKs), c-JunN terminal protein kinase (JNKs), p38 protein kinase (p38kinase) in the extracellular regulated protein kinase (ERKs), c-JunN terminal protein kinase (JNKs), p38 protein kinase (p38kinase) was detected by Western Blot method at different time points (0,15,30 min.1,2,6 h) after treatment with rhM-CSF (50 ng/ ml). Results: The concentration of E2 in granulosa cell culture medium was positively correlated with the concentration of M-CSF or FSH (P0.05). rhFSH could promote the expression of M-CSF receptor mRNA in granulosa cells (p0.05) in a certain concentration range (25 IU/ ml). The expression of M-CSF receptor mRNA in the granulosa cells of different concentration groups was significantly higher than that of the control group (p0.05). As the concentration of rhM-CSF increased, the proliferation ability of COV434 cells was also on the rise. rhM-CSF at 50 ng/ ml concentration could activate the signal pathway of JNK and P-38 in a time-dependent manner to increase the level of phosphorylation, but the level of total protein expression was not affected, and the ERK1/2 pathway did not significantly activate. After pre-treatment with JNK inhibitor SB203580 and P38 inhibitor SP600125, the levels of estrogen in each group were not affected. Conclusion: M-CSF may play an important role in promoting the proliferation and differentiation of granulosa cells and the synthesis and secretion of estrogen.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R714.8
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