益气血法通过调节GDF9分泌对大鼠超排卵卵丘细胞凋亡的影响及其作用机制研究

发布时间：2018-05-06 11:10

本文选题：益气血法 + 超排卵大鼠　；参考：《广州中医药大学》2017年博士论文

【摘要】：控制性超排卵(controlled ovary hyperstimulation,COH)是体外受精-胚胎移植(In vitro fertilization and embryo transfer,IVF-ET)周期的重要组成部分,已有研究证实COH可能破坏卵细胞中染色体的结构,影响染色体的功能,导致细胞中某些基因的表达缺陷,对卵细胞的发育起负面影响,降低胚胎的质量和其着床能力。因此COH能导致胚胎着床率和妊娠率处于明显低下的水平。中医药介入IVF-ET周期收效显著,我们前期研究证实益气血补肝肾中药能提高胚胎移植成功率和妊娠率。本文主要研究经后增殖方对COH卵巢卵丘细胞(cumulus cells,CCs)凋亡的影响。人CCs与卵母细胞有密切的关系,CCs能直接影响卵母细胞的发育和成熟,影响胚胎的质量,而卵母细胞也可以分泌卵细胞分泌因子(oocyte secreted factors,OSFs),通过旁分泌的作用来调节周围CCs的生长和分化。在卵母细胞的成熟过程中,两者互为影响,缺一不可。生长分化因子9(growth differentiation factor 9,GDF9)是转化生长因子β(transforming growth factor β,TGFβ)超家族中一个重要的成员,作为卵母细胞分泌的细胞因子,GDF9与卵细胞的发育和发展具有密切关系,是与卵泡发育有关的重要调节因子,卵泡液中的GDF9已经作为一项重要指标,用于对卵母细胞和胚胎质量的预测。Bim(Bcl-2 interacting mediator of cell death)是 Bcl-2 家族中的成员,隶属于BH3-only亚家族。Bim在机体多种组织包括生殖细胞中有不同程度的表达,在启动凋亡和调节凋亡上具有重要的作用,是一种重要的凋亡调节蛋白。本文主要探讨益气血法经后增殖方通过调节GDF9分泌和Bim表达抑制COH卵巢CCs凋亡的作用机制,同时对其可能的信号通路进行验证,为其广泛的临床应用提供分子理论基础。期望能找到改善IVF-ET中卵母细胞质量及发育潜能的途径,进一步提高IVF-ET临床胚胎种植率与妊娠率。目的:观察益气血法经后增殖方对COH大鼠卵巢GDF9和Bim分泌的影响,探讨经后增殖方在抑制COH卵巢CCs凋亡中的作用机制,并对其可能的信号通路进行验证。方法:1动物实验研究通过建立COH大鼠模型,同时应用经后增殖方进行干预,以自然排卵大鼠作为空白对照,以COH大鼠作为阳性对照,采用TUNEL和qPCR技术检测不同剂量经后增殖方干预的COH大鼠卵巢颗粒细胞凋亡率及卵巢组织中Bim基因的表达水平。2细胞实验研究2.1通过制备不同浓度经后增殖方含药血清,,对COH大鼠卵巢颗粒细胞体外培养进行干预,采用qPCR技术检测颗粒细胞中Bim基因的表达水平,选出含药血清最佳作用浓度和时间,应用于后续实验。2.2建立COH大鼠模型,获取卵巢COCs和CCs进行体外培养,用经后增殖方含药血清和GDF9受体阻断剂进行干预,采用qPCR和Westernblot技术检测COCs和CCs中GDF9基因和蛋白及Bim基因的表达水平。2.3建立COH大鼠模型,获取卵巢COCs进行体外培养,用经后增殖方含药血清和PI3K、PKA、p38MAPK、SMAD和NF-κ B通路阻断剂进行干预,采用qPCR和Westernblot技术检测COCs中GDF9基因和蛋白及Bim基因的表达水平。结果:1经后增殖方对COH大鼠卵巢颗粒细胞凋亡率及卵巢组织中Bim基因表达的影响1.1经后增殖方可降低COH大鼠卵巢颗粒细胞凋亡率中药低、中、高剂量组均明显低于阳性对照组(均为P0.05),中药高剂量组与空白对照组比较无显著差异(均为P0.05),说明高、中、低剂量经后增殖方均可降低COH大鼠卵巢颗粒细胞凋亡率,且高剂量经后增殖方能使COH大鼠卵巢颗粒细胞凋亡率达到接近自然排卵大鼠的水平;中药低、中、高剂量组卵巢颗粒细胞凋亡率逐渐下降,且各剂量组之间均有显著差异(P0.01),说明COH大鼠卵巢颗粒细胞凋亡率凋亡率与经后增殖方剂量呈负相关。1.2经后增殖方可降低COH大鼠卵巢组织Bim基因的表达中药低、中、高剂量组均明显高于空白对照组(均为PM0.05),明显低于阳性对照组(均为P0.05),说明高、中、低剂量经后增殖方均可降低COH大鼠卵巢组织Bim基因的表达水平;中药低、中、高剂量组Bim mRNA相对表达水平逐渐下降,但无显著差异(均为P0.05),说明高、中、低剂量经后增殖方降低COH大鼠卵巢组织Bim基因的表达水平的作用相当。2经后增殖方含药血清经p38MAPK和NF-κ B通路的调控调节GDF9和Bim表达抑制COH大鼠卵巢CCs凋亡2.1经后增殖方含药血清抑制COH大鼠卵巢颗粒细胞Bim基因表达的最佳作用浓度和时间24小时中药低、中、高剂量组明显低于空白血清组(均为P0.01),说明高、中、低剂量经后增殖方含药血清干预24小时后Bim基因的表达水平均有显著下降;中药低、中、高剂量组BimmRNA相对表达水平逐渐下降,且各组间均有显著差异(均为P0.01),说明经后增殖方含药血清降低Bim基因表达水平的效果与浓度呈正相关,高剂量的效果最优。48小时中药低、中、高剂量组Bim mRNA相对表达水平逐渐下降,中药高剂量组明显低于其他3组(均为P0.05),其他3组之间无明显差异(均为P0.05),说明仅高剂量经后增殖方含药血清在干预48小时仍能维持Bim基因表达的低水平,而中、低剂量经后增殖方含药血清48小时干预效果并不理想。结合以上结果,筛选出经后增殖方含药血清抑制卵巢颗粒细胞凋亡的最佳作用时间为24小时,最佳作用浓度为高剂量。2.2经后增殖方含药血清对COH大鼠COCs和CCs中GDF9和Bim表达的调节2.2.1经后增殖方含药血清能提高COH大鼠COCs和CCs中GDF9基因和蛋白的表达COCs-含药血清组GDF9基因相对表达量明显高于COCs-空白血清组和COCs-含药血清+GDF9受体阻断剂组(均为P0.05),CCs-含药血清组GDF9基因相对表达量明显高于CCs-空白血清组和CCs-含药血清+GDF9受体阻断剂组(均为P0.05),说明经后增殖方含药血清能提高COH大鼠COCs和CCs中GDF9基因的表达。COCs-含药血清组GDF9蛋白相对表达量明显高于COCs-空白血清组(P0.05),高于COCs-含药血清+GDF9受体阻断剂组,但差异无统计学意义(P0.05);CCs-含药血清组GDF9蛋白相对表达量明显高于CCs-空白血清组和CCs-含药血清+GDF9受体阻断剂组(均为P0.05),说明经后增殖方含药血清能提高COH大鼠COCs和CCs中GDF9蛋白的表达。COCs-含药血清组GDF9基因相对表达量明显高于CCs-含药血清组(P0.05),说明COCs较去除卵母细胞的CCs更有利于GDF9的表达。2.2.2经后增殖方含药血清能抑制COH大鼠COCs和CCs中Bim基因的表达COCs-含药血清组和COCs-含药血清+GDF9受体阻断剂组Bim基因相对表达量均明显低于COCs-空白血清组(均为PO.05),CCs-含药血清组和CCs-含药血清+GDF9受体阻断剂组Bim基因相对表达量均明显低于CCs-空白血清组(均为P0.05),说明经后增殖方含药血清能抑制COH大鼠COCs和CCs中Bim基因的表达。COCs-含药血清组Bim基因相对表达量低于COCs-含药血清+GDF9受体阻断剂组,但差异无统计学意义(P0.05),CCs-含药血清组Bim基因相对表达量明显低于CCs-含药血清+GDF9受体阻断剂组(P0.05),说明由于受GDF9受体阻断剂作用,GDF9的表达明显降低,从而无法维持Bim表达的低水平,验证了 GDF9有维持CCs中Bim表达的低水平的作用。2.3经后增殖方含药血清对COH大鼠COCs中GDF9和Bim表达的调节是通过p38MAPK和NF-κB通路介导的含药血清+PI3K inhibitors组、含药血清+PKA inhibitors组、和含药血清+SMAD inhibitors组GDF9基因和蛋白的相对表达量均明显高于空白血清组(均为P0.05),与含药血清组比较,差异均无统计学意义(均为P0.05);含药血清+p38MAPK inhibitors组和含药血清+NF-κ B inhibitors组GDF9基因和蛋白的相对表达量均明显低于含药血清组(均为P0.05),与空白血清组比较,差异均无统计学意义(均为P0.05)。说明经后增殖方含药血清能提高COH大鼠COCs中GDF9基因和蛋白的表达水平,该作用是通过p38MAPK和NF-κ B通路介导的。含药血清+PI3K inhibitors组、含药血清+PKA inhibitors组和含药血清+SMAD inhibitors组Bim基因相对表达量明显低于空白血清组(均为P0.05),与含药血清组比较,差异无统计学意义(均为P0.05);含药血清+p38MAPK inhibitors组和含药血清+NF-κ B inhibitors组Bim基因相对表达量明显高于含药血清组(均为P0.05),与空白血清组比较,差异无统计学意义(均为P0.05)。说明经后增殖方含药血清能降低COH大鼠COCs中Bim基因的表达水平,该作用可能是通过p38MAPK信号通路和NF-κ B信号通路介导的。结论:①益气血法经后增殖方能通过促进COH大鼠卵巢COCs中卵母细胞分泌GDF9,而GDF9通过旁分泌作用周围的CCs,维持CCs的低凋亡率而改善COCs的功能,从而提高卵细胞质量;②益气血法经后增殖方能通过多途径抑制COH大鼠卵巢COCs和CCs中Bim的表达水平,途径之一是通过促进GDF9的分泌以维持Bim表达的低水平,从而抑制CCs凋亡;③益气血法经后增殖方对COH大鼠卵巢COCs和CCs中GDF9和Bim表达的调节是通过p38MAPK和NF-κB信号转导通路介导的。
[Abstract]:Controlled ovary hyperstimulation (COH) is an important part of the cycle of in vitro fertilization and embryo transfer (In vitro fertilization and embryo transfer, IVF-ET). It has been proved that COH may destroy the structure of chromosomes in the egg cells, affect the function of the chromophore, and lead to the defects in the expression of some genes in the cells, The development of the egg cell has a negative effect on the quality of the embryo and its implantation ability. Therefore, COH can lead to a lower level of embryo implantation rate and pregnancy rate. The effect of TCM Intervention on IVF-ET cycle is remarkable. The effect of post proliferation on the apoptosis of COH ovarian cumulus cells (cumulus cells, CCs). Human CCs has a close relationship with oocyte. CCs can directly influence the development and maturation of oocyte, affect the quality of embryo, and oocyte can also secrete the secretory factor of oocyte (oocyte secreted factors, OSFs), and regulate by the role of paracrine. The growth and differentiation of the surrounding CCs. During the maturation of oocytes, the two are mutually beneficial, and the growth differentiation factor 9 (growth differentiation factor 9, GDF9) is an important member of the transforming growth factor beta (transforming growth factor beta, TGF beta) superfamily, as the cytokine secreted by oocyte, GDF9 and egg thin. Cell development and development are closely related to the development of follicle development. The GDF9 in follicular fluid is an important indicator. The prediction of the quality of oocyte and embryo.Bim (Bcl-2 interacting mediator of cell death) is a member of the Bcl-2 family, which is subordinate to the BH3-only subfamily.Bim in the body. Species, including different levels of expression in germ cells, plays an important role in initiating apoptosis and regulating apoptosis. It is an important apoptosis regulating protein. This paper mainly discusses the mechanism of inhibiting the apoptosis of COH ovarian CCs by regulating the GDF9 secretion and Bim expression by the beneficial Qi and blood method, and the possible signaling pathway. To provide the basis of molecular theory for its extensive clinical application. We hope to find a way to improve the quality and development potential of oocyte in IVF-ET, and to further improve the implantation rate and pregnancy rate of IVF-ET clinical embryo. Objective: To observe the effect of the recipe of Supplementing Qi and blood on the secretion of GDF9 and Bim in the egg nests of COH rats, and to explore the post proliferative side. The mechanism of inhibiting the apoptosis of COH ovarian CCs and its possible signal pathways were verified. Methods: 1 animal experiments were conducted by establishing a COH rat model and using the post proliferation prescription to intervene with the natural ovulation rats as the blank control, and the COH rats were used as the positive control, and the TUNEL and qPCR techniques were used to detect the different doses of the rats. The apoptosis rate of ovarian granulosa cells and the expression level of Bim gene in the ovarian tissue of COH rats with the intervention of the posterior proliferative prescription (.2 cells) experimental study 2.1 by preparing the serum containing different concentrations of the post proliferating prescription, the expression level of Bim gene in the granulosa cells of the COH rats was detected by qPCR technique. The optimal concentration and time of the drug containing serum were used to establish the COH rat model in the follow-up experiment.2.2, and the ovarian COCs and CCs were cultured in vitro. The serum and GDF9 receptor blockers were used to intervene. The qPCR and Westernblot techniques were used to detect the expression level of GDF9 genes and proteins and Bim genes in COCs and CCs. OH rat model was used to obtain ovarian COCs for culture in vitro. The serum and PI3K, PKA, p38MAPK, SMAD and NF- kappa B pathway blockers were intervened by the post proliferation prescription, and the expression level of GDF9 genes and proteins in COCs was detected by qPCR and Westernblot techniques. The effect of Bim gene expression in ovarian tissue 1.1 can reduce the apoptosis rate of ovarian granulosa cells in COH rats lower than that of the positive control group (all P0.05), and there is no significant difference between the high dose group and the blank control group (all P0.05), indicating that the high, middle, and low dose of the post proliferative side can reduce the COH big. The apoptosis rate of rat ovarian granulosa cells, and the high dose of after proliferation can make the apoptosis rate of ovarian granulosa cells in COH rats reach the level of the rats with natural ovulation. The apoptosis rate of ovarian granulosa cells in the low, middle and high dose groups gradually decreased, and there were significant differences between each dose group (P0.01), indicating that the apoptosis rate of ovarian granulosa cells in COH rats withered. The negative correlation between the death rate and the volume of the post proliferating agent.1.2 could reduce the expression of the Bim gene in the ovarian tissue of COH rats, and the high dose group was significantly higher than that of the blank control group (all PM0.05), which was significantly lower than the positive control group (all P0.05), indicating that the high, middle and low dose of the post proliferative side could reduce the Bim base in the ovarian tissue of COH rats. The relative expression level of Bim mRNA decreased gradually in low, medium and high dose groups, but there was no significant difference (all P0.05). The effect of high, medium and low doses on the expression level of Bim gene in the ovarian tissue of COH rats was equivalent to the regulation of.2 and Bi by the p38MAPK and NF- kappa B pathway in the serum containing.2 after the proliferation. M expression inhibits the apoptosis of CCs in the ovary of COH rats 2.1 to inhibit the optimal concentration of Bim gene expression in the ovarian granulosa cells of COH rats and the time 24 hours of Chinese medicine low, and the high dose group is significantly lower than that of the blank sera group (P0.01), indicating that the high, middle, low dose of the Bim gene after the intervention of the serum containing drug serum after 24 hours. The expression level of BimmRNA decreased significantly, and the relative expression level of the low, middle and high dose groups decreased gradually, and there were significant differences between each group (all P0.01). The results showed that the effect of reducing the expression level of Bim gene in the post proliferating prescription serum was positively correlated with the concentration of Bim gene. The best effect of high dose.48 hours was low, middle and high dose group Bim mRNA. The relative expression level decreased gradually. The high dose group of traditional Chinese medicine was significantly lower than the other 3 groups (all P0.05), and there was no significant difference between the other 3 groups (all P0.05). It indicated that the low dose of the serum containing the high dose of the posterior proliferative side still maintained the low level of Bim gene expression in the 48 hour intervention, and the intervention effect of the serum containing the low dose of the post proliferation prescription was 48 hours. Not ideal. Combined with the above results, the best action time of inhibiting the apoptosis of ovarian granulosa cells was 24 hours. The optimum concentration was the regulation of the expression of GDF9 and Bim in COCs and CCs of COH rats by the high dose.2.2 serum containing serum of the proliferating prescription of the proliferative prescription of 2.2.1 and the COCs and CCs of COH rats could be improved. The expression of GDF9 gene and protein in COCs- containing serum group GDF9 gene was significantly higher than that of COCs- blank sera group and COCs- containing serum +GDF9 receptor blocker group (P0.05). The relative expression of GDF9 gene in CCs- containing serum group was significantly higher than that of CCs- blank sera group and CCs- drug serum +GDF9 blocker group. The expression of serum containing GDF9 in COH rats after Ming Jing can improve the expression of GDF9 gene in COH rats, and the relative expression of GDF9 protein in serum group of.COCs- was higher than that of COCs- blank sera group (P0.05), which was higher than that of COCs- containing serum +GDF9 receptor blocker group, but the difference was not statistically significant (P0.05). Higher than CCs- blank sera group and CCs- containing serum +GDF9 receptor blocker group (all P0.05), the expression of serum containing GDF9 protein in COCs and CCs in COH rats was significantly higher than that of CCs- drug serum group (P0.05) in COH rats. The expression of DF9 expression.2.2.2 can inhibit the expression of Bim gene in COCs and CCs of COH rats, COCs- containing serum group and COCs- containing serum +GDF9 receptor blocker group, the relative expression of Bim gene in the +GDF9 receptor blocker group is significantly lower than that of COCs- blank sera group (all PO.05). The relative expression was significantly lower than that of the CCs- blank sera group (P0.05), indicating the expression of Bim gene in COCs and CCs in COH rats could inhibit the expression of Bim gene in the serum of COH rats. The relative expression of Bim gene in the serum group of.COCs- was lower than that of the COCs- containing serum +GDF9 receptor blocker group, but the difference was not statistically significant (P0.05). The relative expression of the gene was significantly lower than that of the +GDF9 receptor blocker group (P0.05), which indicated that the expression of GDF9 was obviously reduced because of the effect of the GDF9 receptor blocker. Thus, the low level of Bim expression could not be maintained. It was proved that the GDF9 has a low level of Bim expression in the CCs, and the serum containing the.2.3 via the proliferating side of the.2.3 is used in COH rat COCs. The expression of 9 and Bim was mediated by p38MAPK and NF- kappa B pathway in the drug serum +PI3K inhibitors group. The relative expression of GDF9 gene and protein in the serum containing +PKA inhibitors and the serum +SMAD inhibitors group of the drug serum were significantly higher than that in the blank sera group (all P0.05), and the difference was not statistically significant compared with the drug serum group. P0.05): the relative expression of the GDF9 gene and protein in the serum +p38MAPK inhibitors group and the serum containing +NF- kappa B inhibitors group were significantly lower than that in the drug containing serum group (all P0.05), and the difference was not statistically significant (all P0.05) compared with the blank sera group (all P0.05). The expression level of white was mediated by the p38MAPK and NF- kappa B pathway. The drug serum +PI3K inhibitors group, the relative expression of the Bim gene in the serum +PKA inhibitors group and the serum containing +SMAD inhibitors group was significantly lower than that in the blank sera group (all P0.05), and the difference was not statistically significant (all P0.05), compared with the drug serum group. The relative expression of the Bim gene in the drug serum +p38MAPK inhibitors group and the drug serum +NF- kappa B inhibitors group was significantly higher than that in the drug serum group (all P0.05). The difference was not statistically significant (all P0.05) compared with the blank sera group (all P0.05). It was suggested that the serum level of the posterior proliferative prescription could reduce the expression level of Bim gene in COCs of COH rats, which may be the effect of this effect. Through the p38MAPK signaling pathway and the NF- kappa B signaling pathway. Conclusion: (1) the supplementing qi and blood method can improve the function of COCs by promoting the secretion of GDF9 in the oocytes of COCs in the ovarian COCs of COH rats, while GDF9 passes the paracrine CCs around the paracrine, and improves the function of COCs so as to improve the quality of the egg cell. One way is to inhibit the expression level of Bim in the COCs and CCs of COH rats by multiple pathways. One way is to maintain the low level of Bim expression by promoting the secretion of GDF9, and thus inhibit the apoptosis of CCs. Guide.

【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285.5

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