护卵汤对GnRHa超排卵大鼠卵泡发育的影响研究
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摘要
目的：研究护卵汤对GnRHa超排卵大鼠卵泡发育及卵子质量的影响,从细胞和分子水平探讨相关的作用机制,为护卵汤参与西医辅助生殖技术提供有效的依据。
方法：
模仿人类辅助生殖中的长周期,对大鼠进行GnRHa垂体降调节预处理后再用HMG等药物超排卵(模型组),同时随机给予不同剂量的中药护卵汤辅助治疗(中药低、中、高剂量组),以及自然周期未使用任何药物的大鼠为正常对照(正常组)。在HCG日通过光镜及电镜等方法观察各组大鼠的卵巢形态结构；TUNEL法检测卵巢细胞凋亡现象；放射免疫法检测血清FSH、LH、E2、P含量;抗体芯片法检测卵巢微环境细胞因子及受体的表达；免疫组织化学法观察卵巢细胞FSHR、LHR蛋白的表达。在孕12天观察各组大鼠的妊娠胚胎数。
结果：
(1)与正常组相比：模型组卵巢指数增加,差异有统计学意义(P<0.05)；模型组优质卵泡率下降,差异有显著统计学意义(P<0.01)；模型组卵母细胞超微结构异常；模型组卵巢体细胞凋亡增多,差异有显著统计学意义(P<0.01)；模型组卵巢微环境细胞因子及受体ACTH、BDNF、basic-FGF、CSK、GM-CSF、GH、GHR、IL-1β、 IL-12/IL-23p40、TIMP-1、TIMP-2、TIMP-3、NGF-β、EG-VEGF/PK1、 PDGF-AA表达减少(下调1.5倍以上),Leptin (OB)、IFN-γ、Fas/TNFRSF6、TRAIL表达增多(上调1.5倍以上)；与正常组相比,模型组卵巢FSHR蛋白表达减少,差异有统计学意义(P<0.05)。
(2)与模型组相比,中药各剂量组的卵巢指数均增高,中剂量组的增高有显著统计学意义(P<0.01),低、高剂量组的增高无统计学意义(P>0.05)；与模型组相比,中药各剂量组的卵巢细胞凋亡均减少,中剂量组的减少有统计学意义(P<0.05),低、高剂量组的减少有显著统计学意义(P<0.01)；与模型组相比,中药各剂量组的优质卵泡率均增高,中、高剂量组的增高有统计学意义(P<0.05),低剂量组的增高无统计学意义(P>0.05)；与模型组相比,中药各剂量组血清E2含量均增加,中剂量组的增加有显著统计学意义(P<0.01),高剂量组的增加有统计学意义(P<0.05),低剂量组的增加无统计学意义(P>0.05)；与模型组相比,中药高剂量组卵巢FSHR表达增加,差异有统计学意义(P<0.05)；与模型组相比,中药中、高剂量组卵巢LHR表达增加,中剂量组的增加有显著统计学意义(P<0.01),高剂量组的增加有统计学意义(P<0.05)；与模型组相比,中药中剂量组卵巢微环境细胞因子及受体ACTH、BDNF、basic-FGF、CSK、EG-VEGF/PK1、GFR-α1、GM-CSF、GHR、IL-1β、IL-12/IL-23p40、 NGF-β、PDGF-AA、TGF-β1、TGF-β3、TIMP-3表达增多(上调1.5倍以上),Fas/TNFRSF6、IFN-γ、Leptin (OB)、TNF-α表达减少(下调1.5倍以上)。与模型组相比,中药中剂量组妊娠总活胎数增多(超过1.5倍)。
结论：
(1)与自然周期相比,GnRHa超排卵大鼠排卵前的卵巢体细胞凋亡增多、优质卵泡率及卵子质量下降、卵巢微环境发生不良改变,提示GnRHa超排卵对卵泡发育成熟产生某些不利影响。
(2)护卵汤参与GnRHa超排卵,可增加大鼠卵巢微环境GM-CSF、 IL-1β、TGF-β1等多种细胞因子及受体的含量,减少卵巢微环境IFN-γ、 TNF-α等细胞凋亡因子的含量,提示护卵汤参与超排卵能改善大鼠的卵巢微环境。
(3)护卵汤参与GnRHa超排卵,能明显减少大鼠卵巢体细胞凋亡。这可能与护卵汤减少卵巢微环境Fas/TNFRSF6、IFN-γ、TNF-α等细胞凋亡因子的含量有关。
(4)护卵汤参与GnRHa超排卵,能增加大鼠血清E2含量,提示护卵汤能影响超排卵大鼠的生殖内分泌环境。这可能与护卵汤改善超排卵大鼠卵巢局部细胞及分子环境相关。
(5)护卵汤参与GnRHa超排卵,能增加大鼠卵巢FSHR和LHR蛋白表达,提示护卵汤可提高超排卵大鼠的卵巢反应性。这可能与护卵汤改善体内生殖内分泌环境及卵巢局部环境有关。
(6)护卵汤通过以上作用机制相互关联、共同促进GnRHa超排卵大鼠的卵泡发育,增加卵巢重量,提高卵子质量,从而获得更多的优质排卵前卵泡及妊娠活胎数。提示护卵汤参与GnRHa超排卵可能提高ART的成功率
Objective:The objective of this thesis is to observe the effects of Huluantang on the follicular development and the quality of occytes in rats under GnRHa controlled ovarian hyperstimulation, to explore the related mechanism in both cellular and molecular levels, and to provide scientific basis for effectiveness of Huluantang by assisted reproductive technology.
Methods:After receiving GnRHa pituitary down regulation pretreatment which imitates the human long period of COH, a number of rats were separated into a so-called model group treated with HMG/HCG superovulation drugs and so-called Chinese medicine groups treated with HMG/HCG superovulation drugs and different doses of Chinese medicine of Huluantang. The natural cycle of the rats were compared to a normal group receiving no medicine. The following indicators were observed on the day of HCG injection:morphology of ovary by means of light microscope and electron microscope; cell apoptosis of ovary through TUNEL; contents of FSH, LH, E2, P in serum by radioimmunoassay; contents of cytokines in ovary by antibody array; expression of FSHR and LHR in ovary through immunohistochemistry.
Results:
1. Compared with those in the normal group:the increase of the ovary index in the model group had statistical significance (P<0.05); the decrease of the rate of high quality follicular had statistical significance obviously (P<0.01); the ultrastructure of oocyte was abnormal; the increase of cell apoptosis of ovary had statistical significance obviously (P<0.01); the levels of ACTH, BDNF, basic-FGF, CSK, GM-CSF, GH, GHR、IL-1β, IL-12/IL-23p40, EG-VEGF/PK1,TIMP-1, TIMP-2, TIMP-3, NGF-β, PDGF-AA in ovary were decreased above1.5times, the levels of Leptin (OB), IFN-y, Fas/TNFRSF6, TRAIL in ovary were increased above1.5times; the decrease of the expression of FSHR in ovary had statistical significance (P<0.05).
2. Compared with those in the model group, the increase of the ovary index in the middle dose group had statistical significance obviously (P<0.01); the increase of the ovary index in the low dose and high dose groups had no statistical significance (P>0.05); the decrease of cell apoptosis of ovary in the middle dose group had statistical significance (P <0.05); the decrease of cell apoptosis of ovary in the low dose and high dose groups had statistical significance obviously (P<0.01); the increase of the rate of high quality follicular in the middle close and high dose groups had statistical significance (P<0.05); the increase of the rate of high quality follicular in the low dose group had no statistical significance (P>0.05); the increase of the content of E2in serum in the middle dose group had statistical significance obviously (P<0.01); the increase of the content of E2in serum in the high dose groups had statistical significance (P<0.05); the increase of the content of E2in serum in the low dose group had no statistical significance (P>0.05); the increase of the expression of FSHR in ovary in the high dose group had statistical significance (P<0.05); the increase of the expression of LHR in ovary in the middle dose group had statistical significance obviously (P<0.01); the increase of the expression of FSHR in ovary in the high dose group had statistical significance (P<0.05); the levels of ACTH, BDNF, basic-FGF, CSK, EG-VEGF/PK1, GFR-α1, GM-CSF, GHR, IL-1β, IL-12/IL-23p40, NGF-β, PDGF-AA, TGF-β1, TGF-β33, TIMP-3in ovary increased above1.5times in the middle dose group; the levels of Fas/TNFRSF6, IFN-γ, Leptin (OB), TNF-a in ovary were decreased above1.5times in the middle dose group; the total live fetus in the middle dose group increased above1.5times.
Conclusions:
1. Compared with those in normal natural cycle, the cell apoptosis of ovary increased and the rate of high quality follicular and quality of occytes decreased, and the contents of cytokines in ovarian microenvironment were abnormal for rats under GnRHa superovulation. The results suggest some of the adverse effects of GnRHa superovulation on follicular development.
2. The Chinese medicine of Huluantang can increase the expression of some cytokines in ovarian microenvironment which can promote follicular development and the quality of occytes, such as IL-1β,GHR, TGF-β1, and decrease the expression of some cytokines such as IFN-γ, TNF-α and Leptin. The results suggest that Huluantang can improve the ovarian microenvironment in rats under GnRHa superovulation.
3. The Chinese medicine of Huluantang can decrease the ovarian cell apoptosis in rats under GnRHa superovulation. The reason may be the decrease of the expression of IFN-y and TNF-a in the ovarian microenvironment.
4. The Chinese medicine of Huluantang can increase the content of E2in serum, which suggests Huluantang can improve the endocrine environment in rats under GnRHa superovulation. The reason may be the improvement of the cellular and molecular environment in ovary by Huluantang.
5. The Chinese medicine of Huluantang can increase the expression of FSHR and LHR in ovary, which may improve the ovarian response in rats under GnRHa superovulation. The reasons may be the improvement of ovarian microenvironment and endocrine environment.
6. Through the above mechanism, the Chinese medicine of Huluantang can promote the follicular development, the weight of ovary and the quality of occytes in rats under GnRHa superovulation, through which more high quality occytes and live fetus can be obtained. The results suggest that Huluantang may increase the success rate of assisted reproductive technology (ART).
方法：
模仿人类辅助生殖中的长周期,对大鼠进行GnRHa垂体降调节预处理后再用HMG等药物超排卵(模型组),同时随机给予不同剂量的中药护卵汤辅助治疗(中药低、中、高剂量组),以及自然周期未使用任何药物的大鼠为正常对照(正常组)。在HCG日通过光镜及电镜等方法观察各组大鼠的卵巢形态结构；TUNEL法检测卵巢细胞凋亡现象；放射免疫法检测血清FSH、LH、E2、P含量;抗体芯片法检测卵巢微环境细胞因子及受体的表达；免疫组织化学法观察卵巢细胞FSHR、LHR蛋白的表达。在孕12天观察各组大鼠的妊娠胚胎数。
结果：
(1)与正常组相比：模型组卵巢指数增加,差异有统计学意义(P<0.05)；模型组优质卵泡率下降,差异有显著统计学意义(P<0.01)；模型组卵母细胞超微结构异常；模型组卵巢体细胞凋亡增多,差异有显著统计学意义(P<0.01)；模型组卵巢微环境细胞因子及受体ACTH、BDNF、basic-FGF、CSK、GM-CSF、GH、GHR、IL-1β、 IL-12/IL-23p40、TIMP-1、TIMP-2、TIMP-3、NGF-β、EG-VEGF/PK1、 PDGF-AA表达减少(下调1.5倍以上),Leptin (OB)、IFN-γ、Fas/TNFRSF6、TRAIL表达增多(上调1.5倍以上)；与正常组相比,模型组卵巢FSHR蛋白表达减少,差异有统计学意义(P<0.05)。
(2)与模型组相比,中药各剂量组的卵巢指数均增高,中剂量组的增高有显著统计学意义(P<0.01),低、高剂量组的增高无统计学意义(P>0.05)；与模型组相比,中药各剂量组的卵巢细胞凋亡均减少,中剂量组的减少有统计学意义(P<0.05),低、高剂量组的减少有显著统计学意义(P<0.01)；与模型组相比,中药各剂量组的优质卵泡率均增高,中、高剂量组的增高有统计学意义(P<0.05),低剂量组的增高无统计学意义(P>0.05)；与模型组相比,中药各剂量组血清E2含量均增加,中剂量组的增加有显著统计学意义(P<0.01),高剂量组的增加有统计学意义(P<0.05),低剂量组的增加无统计学意义(P>0.05)；与模型组相比,中药高剂量组卵巢FSHR表达增加,差异有统计学意义(P<0.05)；与模型组相比,中药中、高剂量组卵巢LHR表达增加,中剂量组的增加有显著统计学意义(P<0.01),高剂量组的增加有统计学意义(P<0.05)；与模型组相比,中药中剂量组卵巢微环境细胞因子及受体ACTH、BDNF、basic-FGF、CSK、EG-VEGF/PK1、GFR-α1、GM-CSF、GHR、IL-1β、IL-12/IL-23p40、 NGF-β、PDGF-AA、TGF-β1、TGF-β3、TIMP-3表达增多(上调1.5倍以上),Fas/TNFRSF6、IFN-γ、Leptin (OB)、TNF-α表达减少(下调1.5倍以上)。与模型组相比,中药中剂量组妊娠总活胎数增多(超过1.5倍)。
结论：
(1)与自然周期相比,GnRHa超排卵大鼠排卵前的卵巢体细胞凋亡增多、优质卵泡率及卵子质量下降、卵巢微环境发生不良改变,提示GnRHa超排卵对卵泡发育成熟产生某些不利影响。
(2)护卵汤参与GnRHa超排卵,可增加大鼠卵巢微环境GM-CSF、 IL-1β、TGF-β1等多种细胞因子及受体的含量,减少卵巢微环境IFN-γ、 TNF-α等细胞凋亡因子的含量,提示护卵汤参与超排卵能改善大鼠的卵巢微环境。
(3)护卵汤参与GnRHa超排卵,能明显减少大鼠卵巢体细胞凋亡。这可能与护卵汤减少卵巢微环境Fas/TNFRSF6、IFN-γ、TNF-α等细胞凋亡因子的含量有关。
(4)护卵汤参与GnRHa超排卵,能增加大鼠血清E2含量,提示护卵汤能影响超排卵大鼠的生殖内分泌环境。这可能与护卵汤改善超排卵大鼠卵巢局部细胞及分子环境相关。
(5)护卵汤参与GnRHa超排卵,能增加大鼠卵巢FSHR和LHR蛋白表达,提示护卵汤可提高超排卵大鼠的卵巢反应性。这可能与护卵汤改善体内生殖内分泌环境及卵巢局部环境有关。
(6)护卵汤通过以上作用机制相互关联、共同促进GnRHa超排卵大鼠的卵泡发育,增加卵巢重量,提高卵子质量,从而获得更多的优质排卵前卵泡及妊娠活胎数。提示护卵汤参与GnRHa超排卵可能提高ART的成功率
Objective:The objective of this thesis is to observe the effects of Huluantang on the follicular development and the quality of occytes in rats under GnRHa controlled ovarian hyperstimulation, to explore the related mechanism in both cellular and molecular levels, and to provide scientific basis for effectiveness of Huluantang by assisted reproductive technology.
Methods:After receiving GnRHa pituitary down regulation pretreatment which imitates the human long period of COH, a number of rats were separated into a so-called model group treated with HMG/HCG superovulation drugs and so-called Chinese medicine groups treated with HMG/HCG superovulation drugs and different doses of Chinese medicine of Huluantang. The natural cycle of the rats were compared to a normal group receiving no medicine. The following indicators were observed on the day of HCG injection:morphology of ovary by means of light microscope and electron microscope; cell apoptosis of ovary through TUNEL; contents of FSH, LH, E2, P in serum by radioimmunoassay; contents of cytokines in ovary by antibody array; expression of FSHR and LHR in ovary through immunohistochemistry.
Results:
1. Compared with those in the normal group:the increase of the ovary index in the model group had statistical significance (P<0.05); the decrease of the rate of high quality follicular had statistical significance obviously (P<0.01); the ultrastructure of oocyte was abnormal; the increase of cell apoptosis of ovary had statistical significance obviously (P<0.01); the levels of ACTH, BDNF, basic-FGF, CSK, GM-CSF, GH, GHR、IL-1β, IL-12/IL-23p40, EG-VEGF/PK1,TIMP-1, TIMP-2, TIMP-3, NGF-β, PDGF-AA in ovary were decreased above1.5times, the levels of Leptin (OB), IFN-y, Fas/TNFRSF6, TRAIL in ovary were increased above1.5times; the decrease of the expression of FSHR in ovary had statistical significance (P<0.05).
2. Compared with those in the model group, the increase of the ovary index in the middle dose group had statistical significance obviously (P<0.01); the increase of the ovary index in the low dose and high dose groups had no statistical significance (P>0.05); the decrease of cell apoptosis of ovary in the middle dose group had statistical significance (P <0.05); the decrease of cell apoptosis of ovary in the low dose and high dose groups had statistical significance obviously (P<0.01); the increase of the rate of high quality follicular in the middle close and high dose groups had statistical significance (P<0.05); the increase of the rate of high quality follicular in the low dose group had no statistical significance (P>0.05); the increase of the content of E2in serum in the middle dose group had statistical significance obviously (P<0.01); the increase of the content of E2in serum in the high dose groups had statistical significance (P<0.05); the increase of the content of E2in serum in the low dose group had no statistical significance (P>0.05); the increase of the expression of FSHR in ovary in the high dose group had statistical significance (P<0.05); the increase of the expression of LHR in ovary in the middle dose group had statistical significance obviously (P<0.01); the increase of the expression of FSHR in ovary in the high dose group had statistical significance (P<0.05); the levels of ACTH, BDNF, basic-FGF, CSK, EG-VEGF/PK1, GFR-α1, GM-CSF, GHR, IL-1β, IL-12/IL-23p40, NGF-β, PDGF-AA, TGF-β1, TGF-β33, TIMP-3in ovary increased above1.5times in the middle dose group; the levels of Fas/TNFRSF6, IFN-γ, Leptin (OB), TNF-a in ovary were decreased above1.5times in the middle dose group; the total live fetus in the middle dose group increased above1.5times.
Conclusions:
1. Compared with those in normal natural cycle, the cell apoptosis of ovary increased and the rate of high quality follicular and quality of occytes decreased, and the contents of cytokines in ovarian microenvironment were abnormal for rats under GnRHa superovulation. The results suggest some of the adverse effects of GnRHa superovulation on follicular development.
2. The Chinese medicine of Huluantang can increase the expression of some cytokines in ovarian microenvironment which can promote follicular development and the quality of occytes, such as IL-1β,GHR, TGF-β1, and decrease the expression of some cytokines such as IFN-γ, TNF-α and Leptin. The results suggest that Huluantang can improve the ovarian microenvironment in rats under GnRHa superovulation.
3. The Chinese medicine of Huluantang can decrease the ovarian cell apoptosis in rats under GnRHa superovulation. The reason may be the decrease of the expression of IFN-y and TNF-a in the ovarian microenvironment.
4. The Chinese medicine of Huluantang can increase the content of E2in serum, which suggests Huluantang can improve the endocrine environment in rats under GnRHa superovulation. The reason may be the improvement of the cellular and molecular environment in ovary by Huluantang.
5. The Chinese medicine of Huluantang can increase the expression of FSHR and LHR in ovary, which may improve the ovarian response in rats under GnRHa superovulation. The reasons may be the improvement of ovarian microenvironment and endocrine environment.
6. Through the above mechanism, the Chinese medicine of Huluantang can promote the follicular development, the weight of ovary and the quality of occytes in rats under GnRHa superovulation, through which more high quality occytes and live fetus can be obtained. The results suggest that Huluantang may increase the success rate of assisted reproductive technology (ART).
引文
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