M-CSF對卵泡顆粒細胞功能調節(jié)及其分子機制
發(fā)布時間：2019-06-04 22:43
【摘要】：背景:巨噬細胞集落刺激因子(M-CSF,又稱集落刺激因子-1,CSF-1)是細胞因子調控網絡中的的重要成員,其對單核-巨噬細胞的作用已廣為人知,近年來的研究表明,M-CSF除了能刺激巨噬細胞的增殖外,還具有調節(jié)女性生殖細胞生長、增殖和分化的作用。Witt BR在1997年首次發(fā)現在人卵泡細胞中有M-CSF及其受體mRNA,并且有動物模型證實M-CSF可促進卵泡的生長和排卵。缺乏M-CSF基因的OP/OP小鼠動情周期延長,竇前卵泡及成熟卵泡減少,排卵率降低,注射M-CSF可使其動情周期恢復正常,發(fā)育的卵泡數增加,排卵率升高,并增加顆粒細胞的增殖能力。隨后又有研究證實,顆粒細胞存在M-CSF受體,其合成分泌受垂體促性腺激素及雌激素(E2)的影響。由此推測,M-CSF可能在維持卵子的發(fā)育及促進顆粒細胞的增殖分化、雌激素的合成分泌過程中起了重要作用本研究通過測定顆粒細胞表面FSH受體mRNA及M-CSF受體mRNA的表達,進一步探討FSH、M-CSF和E2之間錯綜復雜的交互關系,同時研究M-CSF調節(jié)顆粒細胞功能的可能信號通路機制,從而更深刻地揭示M-CSF與顆粒細胞相互調控的全貌。目的:探討巨噬細胞集落刺激因子(M-CSF)對卵泡顆粒細胞的功能調節(jié)及其分子機制。方法:2014年7月至2014年10月間,在浙江大學醫(yī)學院附屬婦產科醫(yī)院接受體外受精-胚胎移植的30例因男性因素不孕婦女,平均年齡(30.8±2.1)歲,經肌內注射人絨毛膜促性腺激素(hCG)后34-36h在陰道超聲探頭引導下經陰道取卵,從剩余卵泡液中提取顆粒細胞培養(yǎng)24小時后,分為空白對照組、M-CSF組(10、25、50、100ng/mlrhM-CSF)、M-CSF+來曲唑組(0、10、25、50、100ng/mlrhM-CSF+10-65mol/l 來曲唑)、FSH 組(10、25、50、100IU/mlrhFSH)、FSH+來曲唑組(0、10、25、50、100IU/ml rhFSH+10-6mol/l來曲唑),繼續(xù)培養(yǎng)24小時,留取上清液,用ELISA法測定培養(yǎng)液E2濃度,RT-PCR法測定顆粒細胞FSH受體mRNA及M-CSF受體mRNA的表達。取指數生長期的人卵巢腫瘤顆粒細胞系COV434,給予不同濃度(0、10、25、50、100ng/ml)的人重組M-CSF,于37℃,5%CO2溫箱內培養(yǎng)24小時,MTS比色實驗測定細胞增殖率。用Western Blot法檢測rhM-CSF(50ng/ml)處理后不同時間點(0,15,30分鐘1,2,6小時)COV434細胞絲裂原活化蛋白激酶(MAPK)通路相關蛋白:細胞外調節(jié)的蛋白激酶(ERKs)、c-JunN末端蛋白激酶(JNKs)、p38蛋白激酶(p38kinase)的表達。結果:顆粒細胞培養(yǎng)液中E2濃度與M-CSF或FSH濃度呈正相關(P0.05)。rhFSH在一定濃度范圍(25IU/ml)內可促進顆粒細胞M-CSF受體mRNA表達(p0.05)。與來曲唑聯合作用后,不同濃度組顆粒細胞M-CSF受體mRNA的表達水平與對照組相比均顯著升高(p0.05)。rhM-CSF單獨或與來曲唑聯合作用,均可促進顆粒細胞FSH受體mRNA表達(p0.05)。隨著rhM-CSF濃度的升高,COV434細胞的增殖能力也呈上升趨勢。50ng/ml濃度的rhM-CSF能以時間依賴的方式瞬間激活JNK及P-38信號通路,使其磷酸化水平增高,但總蛋白表達水平未受影響,對ERK1/2通路則無明顯激活作用。分別使用JNK抑制劑SB203580,P38抑制劑SP600125預處理后,并不影響各組雌激素水平。結論:M-CSF可能在促進顆粒細胞的增殖分化以及雌激素的合成分泌過程中起了重要作用。
[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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R714.8
本文編號：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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R714.8
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