【作者】 夏天；

【导师】 韩冰；

【作者基本信息】 天津中医学院 ， 中医妇科学， 2005， 博士

【摘要】 目的:补肾调冲方是导师韩冰教授根据中医理论及多年的临床实践所创立的,具有滋补肝肾、平衡阴阳、疏达肝气、调理冲任气血等功能,适用于肾虚、冲任失调所致的一系列卵巢功能失调性疾患的方药。本研究即以补肾调冲方作为干预手段,以体外培养的大鼠卵巢颗粒细胞作为研究对象,运用直接给药法及血清药理学两种方法观察补肾调冲方对卵巢颗粒细胞增殖与甾体激素分泌功能的影响,并进一步研究中药对卵泡刺激素受体（FSHR）、类固醇激素合成急性调节蛋白（StAR）、类胰岛素样生长因子-1（IGF-1）等基因表达的影响,从而在卵巢一级水平探讨补肾调冲方调节卵巢功能的分子生物学机制,为补肾调冲方在临床上的推广应用提供理论依据。 方法:制备原代培养的大鼠卵巢颗粒细胞,预培养24小时后,直接加入FSH（50ng/ml）和/或不同浓度的补肾调冲方（100、10、1、0.1 μg/ml）,同时根据血清药理学方法,用6周龄的未成年SD大鼠制备FSH及大、中、小剂量补肾调冲方含药血清,并设空白血清组,分别加入颗粒细胞培养体系中。细胞培养48小时后,用³H—TdR掺入法及流式细胞术测定颗粒细胞的增殖变化及细胞周期分布,用放免法检测细胞培养液中E₂、P的含量以及细胞内cAMP的含量,并用半定量RT-PCR及实时荧光定量（Real-time）RT-PCR（SYBR GreenI）两种方法检测颗粒细胞FSHR、StAR、IGF-1 mRNA的表达量。 结果:1.补肾调冲方直接给药对颗粒细胞的作用:①可明显增加³H—TdR掺入颗粒细胞DNA中的数量;②促使颗粒细胞由G₀/G₁期向S期转变,并可提高PI指数;③增加颗粒细胞E₂、P的分泌量;④增加颗粒细胞中cAMP的含量;⑤补肾调冲方对颗粒细胞增殖与分泌的作用在一定范围内随药物浓度的增大而增加,高浓度组与FSH作用相似;⑥补肾调冲方与FSH共同作用可进一步增强FSH促颗粒细胞增殖与分泌的作用,并进一步提高细胞内cAMP的含量。 2.补肾调冲方含药血清对颗粒细胞的作用:①明显促进基础状态下颗粒细胞的增殖与E₂、P的分泌,提高细胞中cAMP的含量;②大、中剂量补肾调冲方含药血清促颗粒细胞增殖与分泌的作用相似,与FSH血清组均无明显差别,小剂量中药血清组作用稍弱;③高雄激素状态下,颗粒细胞增殖与E₂、P分泌功能均受更多还原

【Abstract】 Objective: Bushen Tiaochong Recipy （BSTCR） is established by my tutor, professor Hanbing according to Chinese traditional medical theory and his clinical practice, which has the effect of tonifying and nourishing the liver and kidney, balancing Yin and Yang, dispersing the depressed liver-Qi as well as regulating Qi and blood in the Chong and Ren channels. It is applicable to a series of ovarian dysfunctional diseases caused by renal deficiency and the Chong and Ren channels disorder. The present study was undertaken to observe the effect of BSTCR on rats ovarian granulosa cell proliferation and steroidogenesis with both methods of direct regimen and serum pharmacology. Furthermore we examined the molecular mechanism at the level of ovary, by which BSTCR regulated the expression of follicle stimulating hormone receptor （FSHR）, steroidogenic acute regulator protein （StAR） and Insulin-like growth factor-1 （IGF-1） mRNA in order to provide the theoretical basis for the application of BSTCR in clinic.Methods: Granulosa cells was isolated from rats ovaries and cultured in medium containing 20% calf serum for 24 hours preincubation, followed by culture in medium containing FSH （50ng/ml） and/or varying dosage of BSTCR （100,10,1, 0.1 u g/ml） for a further 48 hours. The serum contained FSH or varying dosage of BSTCR were prepared with 6-weeks-old Sprague Dawley rats using the method of serum pharmacology, with which granulosa cells were incubated for 48 hours. 3H-Thymidine incorporation assay were performed using a scintillation counter. Cell cycle analysis was carried out by flow cytometer（FCM）. Quantification of Estrodial, progestogen and cAMP were examined by radio-immunity. FSHR StAR IGF-1 mRNA expression were measured by semiquantitative and real-time RT-PCR（SYBR GreenI）.Results: 1. Effect of BSTCR on granulosa cell proliferation and steroidogenesis: （1） BSTCR increased [³H]thymidine incorporation into DNA of granulosa cells significantly; （2）BSTCR promoted cell conversion from G₀ phase to S and G2M phase and increased proliferation index （PI）; （3）BSTCR improved the content of cAMP;（4）The result indicated a dose-dependent increase in granulosa cell proliferation and steroidogenesis at a certain extent. And the effect was maximum in the highest dosage（100 u g/ml） group, which was similar to FSH;（5）BSTCR reinforced FSH-induced increase of proliferation, steroidogenesis and cAMP.2. Effect of the serum contained BSTCR on granulosa cells function:（1）The serum contained BSTCR increased significantly granulosa cells proliferation, steroidogenesis and the content of cAMP; （2）There were no marked difference among the three groups of granulosa cells incubated by the serum contained large, medium dosage BSTCR and FSH, while the effect of the serum contained small dosage BSTCR were lower than those three serum. （3）Excessive androgen-treated granulosa cells showed reduced proliferation, steroidogenesis and cAMP. While the serum contained BSTCR rivaled excessive androgen-induced depressant effect and improved granulosa cells function.3. Effect of BSTCR on granulosa cells FSHR mRNA expression: （1）FSH treatment stimulated the expression of FSHR mRNA in FSH-exposed granulosa cells, whereas BSTCR had no significant effect on the expression of FSHR mRNA; （2） BSTCR increased FSH-induced expression of FSHR mRNA; （3）The serum contained BSTCR had a certain dosage-dependent effect on FSHR mRNA.4.Effect of BSTCR on granulosa cells StAR mRNA expression: （1）Both of BSTCR and BSTCR-treated serum stimulated the expression of StAR mRNA with a certain dose-dependent manner; （2）BSTCR-treated Granulosa cells showed increased expression of StAR mRNA in response to forskolin stimulation.5. Effect of BSTCR on granulosa cells IGF-1 mRNA expression: （1）Both of BSTCR and BSTCR-treated serum stimulated the expression of IGF-1 mRNA with a certain dose-dependent manner; （2）BSTCR-treated Granulosa cells showed increased expression of IGF-1 mRNA in response to forskolin stimulation.Conclusion: The present study indicated defferent mechanism of BSTCR to regulate ovarian granulosa cells proliferation and steroidogenesis in defferent conditions: 1. In culture of absence of FSH, BSTCR regulates granulosa cells更多还原