【作者】 郜洁；

【导师】 罗颂平；

【作者基本信息】 广州中医药大学 ， 中医妇科学， 2009， 博士

【摘要】 目的:本研究以临床药效确切的补肾安胎复方寿胎丸为研究对象,以肾虚-黄体抑制大鼠流产模型（病-证结合模型）为研究载体,以流产率、雌二醇（E₂）、孕激素（P）、孕激素受体（PR）的表达为评价追踪指标,对寿胎丸进行提取方法的筛选,并通过均匀设计对寿胎丸进行复方组成及配伍规律的研究,经药效验证筛选出补肾安胎的优选配比方,总结并验证了肾虚-黄体抑制大鼠流产造模的标准流程,初步建立了寿胎丸补肾安胎的药效学评价体系。方法:实验一:购置组成复方的各道地药材,并进行生药鉴定。在前期研究基础结合文献研究,确定复方寿胎丸溶液的不同提取方法并制备成为寿胎丸水提液（A药液）、乙醇提取液（B药液）、醇提后药渣水提液（C药液）、醇提+醇提后药渣水提液（B+C药液）。妊娠第1日至第9日以450mg/kg/d羟基脲灌胃,第10天时予以米非司酮（RU486）4.0mg/kg建立肾虚自然流产模型。将70只SPF级SD孕鼠随机分为7个组:①正常对照组;②模型组对照组（羟基脲+米非司酮）;③阳性对照组（羟基脲+米非司酮+地屈孕酮,简称阳性组）;④寿胎丸A组（羟基脲+米非司酮+寿胎丸水提液）;⑤寿胎丸B组（羟基脲+米非司酮+乙醇提液）;⑥寿胎丸C组（羟基脲+米非司酮+寿胎丸乙醇提取后药渣水提液）;⑦寿胎丸B+C组（羟基脲+米非司酮+寿胎丸乙醇提取液与醇提后药渣水提液的混合液）。24小时后无菌低温条件下处死大鼠,取子宫经滤纸吸干、称重、计算胚胎数、流产率、测量胚胎直径。用电化学发光法测定血清E₂、P水平。无菌低温条件下取出同侧大鼠子宫新鲜的蜕膜组织,30分钟内采用qRT-PCR方法检测蜕膜组织PR mRNA的表达。实验二:通过均匀设计得到8个不同剂量配比的寿胎丸组方,将110只SPF级SD孕鼠随机分为11个组:正常对照组（简称对照组）、模型组对照组（羟基脲+米非司酮）、阳性对照组（羟基脲+米非司酮+地屈孕酮,简称阳性组）、8个不同配比的寿胎丸用药组。造模方法、检测方法同实验一。分别进行多元回归分析、典型相关分析、直接最优化分析、综合加权分析,并结合均匀设计软件预报设计及临床用药经验,筛选出理论优选方。实验三:在实验二得出的理论优选方的基础上,将60只SPF级SD孕鼠随机分为6个组:①模型组对照组（羟基脲+米非司酮）;②阳性对照组（羟基脲+米非司酮+地屈孕酮,简称阳性组）;③寿胎丸临床常用量组（菟丝子:桑寄生:续断:阿胶=20:10:10:5）;④寿胎丸优选1号方（菟丝子:桑寄生:续断:阿胶=15:21:21:0）;⑤寿胎丸优选2号方（菟丝子:桑寄生:续断:阿胶=20:10:10:0）;⑥寿胎丸优选3号方（菟丝子:桑寄生:续断:阿胶=30:21:21:5）。造模方法与检测方法同实验一。经统计分析得出剂量配比优选方。结果:建立肾虚-黄体抑制大鼠流产模型的标准流程,孕鼠表现出中医肾虚证侯、脏器指数下降,流产率升高,分泌E₂、P水平降低,PRmRNA表达下调。经寿胎丸治疗后能够改善上述流产状态,抑制流产率、增加E₂、P水平,上调PRmRNA表达,来实现保护胚胎的作用。实验一:1寿胎丸A组、B组、C组和（B+C）组大鼠的体重、饮食量、卵巢指数、连胎子宫指数与模型组比较,差异无显著性（P＞0.05）。2在流产率方面,寿胎丸A组、B组、C组和（B+C）组大鼠的流产率与模型组比较,差异有显著性（P＜0.05）。A组、B组、C组、B+C组大鼠的流产率与阳性对照组比较差异无显著性（P＞0.05）。A组、B组、C组、B+C组大鼠的流产率各组间比较,差异无显著性（P＞0.05）。3在雌激素水平方面,寿胎丸A组、B组、C组和B+C组大鼠的雌二醇水平与模型组比较,差异无显著性（P＞0.05）。4在孕激素水平方面,寿胎丸A组、B组、C组大鼠的孕激素水平与模型组比较,差异无显著性（P＞0.05）。B+C组大鼠的孕激素水平与模型组比较,差异有显著性（P＜0.05）。A组、B组、C组、B+C组大鼠的孕激素水平与阳性对照组比较,差异无显著性（P＞0.05）。5在孕激素受体表达方面,寿胎丸A组、C组大鼠的孕激素受体的表达与模型组比较,差异无显著性（P＞0.05）。寿胎丸B组、B+C组大鼠孕激素受体的表达与模型组比较,差异有显著性（P＜0.05）。B组大鼠的孕激素受体的表达与B+C组比较,差异无显著性（P＞0.05）。6通过等值加权综合计算各指标与模型组的相对百分率,在寿胎丸各个提取方法中,B+C组与模型组的相对百分率最高,推测B+C组防治流产的作用最强。7综合各指标进行多元方差分析时,4个指标平衡等值加权处理后比较,差异有显著性（P＜0.05）,在降低流产率方面,B+C组＞B组＞C组＞A组;在提高E₂水平方面,B+C组＞A组＞C组＞B组;在提高P水平方面,B+C组＞A组＞C组＞B组;在上调PR表达方面,B+C组＞B组＞A组＞C组。综上B+C组防治流产的作用最强。与寿胎丸各提取部位与模型组的相对百分率的推断一致。实验二、三:1模型组大鼠的连胎子宫指数与对照组比较,差异有显著性（P＜0.05）。阳性组、组二、组四、组五、组六组大鼠的连胎子宫指数与模型组比较,差异有显著性（P＜0.05）。2模型组大鼠的流产率与对照组比较,差异有显著性（P＜0.05）。阳性组、组一、组二、组三、组四、组五、组六、组七和组八大鼠的流产率与模型组比较,差异有显著性（P＜0.01）。3模型组大鼠血清中雌激素水平与对照组比较,差异有显著性（P＜0.05）。阳性组、组一、组二、组三的大鼠血清中雌激素水平与模型组比较,差异有显著性（P＜0.05）。4模型组大鼠血清中孕激素水平与对照组比较,差异有显著性（P＜0.05）。阳性组、组一、组二、组三、组四、组六大鼠血清中孕激素水平与模型组比较,差异有显著性（P＜0.05）。5模型组大鼠子宫蜕膜中孕激素受体表达与对照组比较,差异有显著性（P＜0.05）。组一、组二、组三、组四、组五、组六子宫蜕膜中孕激素受体与模型组比较,差异有显著性（P＜0.05）。6寿胎丸组成药物的关系:6.1各单味药的补肾安胎贡献度的定性分析:菟丝子＞桑寄生=续断＞阿胶。6.2交互作用中:菟丝子与桑寄生、菟丝子与阿胶的交互作用与流产率有相关性;菟丝子与桑寄生、桑寄生与续断的交互作用与E₂有相关性;菟丝子与桑寄生、菟丝子与阿胶、桑寄生与续断、桑寄生与阿胶、续断与阿胶的交互作用与P有相关性;菟丝子与桑寄生、菟丝子与阿胶、桑寄生与续断交互作用与PR有相关性。7将药效学指标与不同剂量配比的寿胎丸相关联,经二次回归得到的回归方程为（R²=0.9832,P＜0.05）:（?）=35.0123-0.2204*X1*X3-0.02321*X1*X4+0.2165X2X3+0.01078*X1²得到理论优选寿胎丸方（菟丝子:桑寄生:续断:阿胶=15:21:21:0）。8由验证实验得出,寿胎丸优选1号方（菟丝子:桑寄生:续断:阿胶=15:21:21:0）、寿胎丸优选2号方（菟丝子:桑寄生:续断:阿胶=20:10:10:0）、寿胎丸常量方（菟丝子:桑寄生:续断:阿胶=20:10:10:5）与模型组比较,差异有显著性（P＜0.05）。但组间比较,差异无统计学意义（P＞0.05）。结论:1罗颂平教授创立的肾虚-黄体抑制（病证结合）流产模型,成功模拟了临床上肾虚型自然流产的状态。本研究在总结前期研究的基础上,初步制定肾虚-黄体抑制（病证结合）流产模型的SOP。2初步确立了寿胎丸的提取流程,并通过药效学实验筛选得到经醇提+醇提后水提的寿胎丸对肾虚流产大鼠模型的补肾安胎药效最优。3验证了寿胎丸可明显降低流产率,寿胎丸具有拮抗米非司酮,促进妊娠黄体和合体滋养细胞分泌功能,提高血清E₂、P水平,通过增加孕激素受体mRNA的转录来上调PR的表达,通过改善孕鼠的肾虚状态来实现保护胚胎的作用。4寿胎丸组成的药物关系:①各单味药的贡献度的定性分析:菟丝子＞桑寄生=续断＞阿胶;②交互作用的定性分析:菟丝子与桑寄生交互作用与各个指标均有相关性;桑寄生与续断的交互作用与E₂、P、PR有相关性;菟丝子与阿胶的交互作用与流产率、P、PR有相关性;续断与阿胶的交互作用与P有相关性。③定性研究初步证明了寿胎丸配伍关系,菟丝子与桑寄生交互作用（君臣关系）＞桑寄生与续断的交互作用（臣佐关系）＞续断与阿胶的交互作用（佐使关系）;菟丝子与桑寄生交互作用（君臣关系）＞菟丝子与阿胶的交互作用（君使关系）＞续断与阿胶的交互作用（佐使关系）,与原方的配伍关系基本一致。5验证了阿胶在原方中处于使药的地位,起调和药性的作用,在本实验研究中阿胶的补肾安胎作用不显著。6将药效学指标与不同剂量配比的寿胎丸相关联,经二次回归得到的回归方程为（R²=0.9832,P＜0.05）:（?）=35.0123-0.2204*X1*X3-0.02321*X1*X4++0.2165X2X3+0.01078*X1²经最优分析得出理论优选寿胎丸方（菟丝子:桑寄生:续断:阿胶=15:21:21:0）。7经过药效验证实验,寿胎丸优选1号方（菟丝子:桑寄生:续断:阿胶=15:21:21:0）、寿胎丸优选2号方（菟丝子:桑寄生:续断:阿胶=20:10:10:0）与寿胎丸常量方（菟丝子:桑寄生:续断:阿胶＝20:10:10:5）均具有较好的补肾安胎作用,与阳性对照药物抑制流产作用相当。寿胎丸优选2号方＞阳性对照药物＞寿胎丸优选1号方＞常量方,均可以保护胚胎、降低流产率。8本研究初步建立了系统的寿胎丸补肾安胎作用的药效学评价体系,规范改良了造模方法、筛选了药效评价的指标,运用数学方法与计算机技术相结合研究复方组方关系和优选配比,并审慎地分析了实验结果,为寿胎丸深层次的配伍规律及药效物质基础研究奠定了基础。更多还原

【Abstract】 Objective:Pharmacological experiments,the effect of Shou Tai Wan（STW） on abortion animal models of corepus luteum inhibition due to renal deficiency,were performed in order to bring out the optimal extration method,the optimal proportion and the compatibility rule of STW.Methods:Experiment 1:Collection of Genuine Medicinal Materials and Pharmacognostic identification of Semen Cuscutae,Herba Taxilli,Radix Dipsaci,Colla Corii asini were prepared in accordance with Chinese Pharmacopoeia.SA rats model of corepus luteum inhibition due to renal deficiency was made by intragsstric administration of hydroxycarbamide 450mg/kg/D in dioestrus.Mifepristone（RU 486） was used by intragsstric administration in 10^th day.SPF 70 pregnant SD rats,10 weeks old,weight 280～300g were randomly divided into 7 groups:①normal control group;②model group（Hydroxyurea + mifepristone）;③positive control group（Hydroxyurea + mifepristone + dydrogeterone）;④STW A（aqueous extract solution of STW）;⑤STW B（ethanol-extracts of STW）;⑥STW C（aqueous extract solution of guffs after alcohol extracting）;⑦STW B+C（ethanol-extracts of STW + aqueous extract solution of guffs after alcohol extracting）. Generalized case of rats in every group was observered.Rats were killed after 24 hours,the following data was measured:embryos number,abortion rats sand diameter of embryos.Decidua tissue on one side was collected in 30 minutes after rats death in asepsis condition to detect expression of PR by quantitative RT-PCR.Serum estradiol（E₂）,progestogen（P） were measured by chemiluminescence.Experiment 2:8 combinations of STW were obtained by uniform design.SPF 110 pregnant SD rats,10 weeks old,weight 280～300g were randomly divided into 11 groups:①normal control group;②model group（Hydroxyurea + mifepristone）;③positive control group（Hydroxyurea + mifepristone + dydrogeterone）;④8 combinations of STW by uniform design.Animal model building and detection Methods were similar to Experiment 1.The optimal proportion of STW in theory was obtained by multiple regression analysis,canonical correlation analysis,optimum analysis,integrated weighting analysis.Experiment 3:SPF 60 pregnant SD rats,10 weeks old,weight280～300g were randomly divided into 6groups:①model group（Hydroxyurea + mifepristone）;②positive control group（Hydroxyurea + mifepristone + dydrogeterone）;③clinical routine dose of STW;④STW1（Semen Cuscutae:Herba Taxilli:adix Dipsaci:Colla Corii=15: 21:21:0）;⑤STW 2（Semen Cuscutae:Herba Taxilli:adix Dipsaci:Colla Corii=20: 10:10:0）;⑥STW 2（Semen Cuscutae:Herba Taxilli:adix Dipsaci:Colla Corii=30: 21:21:5）.Animal model building and detection Methods were similar to Experiment 1.Results:Experiment 1:1.Intake of food/water,changes of pregnant rat body weight in 4^th and 9^th day,organ index in pregnant rats:compared with model group,there was no significant difference in all groups（P＞0.05）.2.Abortion rate of pregnant rat:compared with model group,there was significant difference on abortion rate between model group and other groups （P＜0.05）.Compared with positive control group,there was no significant difference in STW A group,STW B group,STW C group and STW B+C group（P＞0.05）.3.The mean levels of serum E₂:compared with model group,there was no significant difference in STW A group,STW B group,STW C group and STW B+C group（P＞0.05）.4.The mean levels of serum P:compared with model group,there was no significant difference in STW A group,STW B group and STW C group（P＞0.05）. The mean levels of serum P in STW B+C group was significantly different from model group（P＜0.05）.Compared with positive control group,there was no significant difference in STW A group,STW B group,STW C group,STW B+C group （P＞0.05）.5.Expression of PR:compared with model group,there was no significant difference in STW A group,STW C group（P＞0.05）.Expression of PR in STW B group and STW B+C group was significantly different from model group （P＜0.05）.There was no significant difference between STW A group and STW C group（P＞0.05）.6.The mean relative percentages compared with model group,which was calculated by Weighted Average Method,indicated that STW B+C group was better than other groups on SA rats model model7.Multivariate analysis of variance on 4 indexes of pharmacodynamic experiment indicated that STW B+C group＞STW B group＞STW C group＞STW A group in abortion rate of pregnant rat;STW B+C group＞STW A group＞STW C group＞STW B group in the mean levels of serum E₂;STW B+C group＞STW A group＞STW C group＞STW B group in the mean levels of serum P;STW B+C group＞STW B grou＞STW A group＞STW C group in expression of PR.It is suggested STW B+C group was better than other groups on SA rats model model.Experiment 2:1.Intake of food/water,changes of pregnant rat body weight in 4^th day:compared with model group,there was no significant difference in all groups（P＞0.05）.2.Organ index in pregnant rats:there was significant difference between control group and model group（P＜0.05）.Organ index of pregnant rats in positive control group,group2,group4,group5,group6 was significantly different from model group（P＜0.05）.3.Abortion rate of pregnant rat:there was significant difference on abortion rate between control group and model group（P＜0.05）.Abortion rate of pregnant rat in positive control group,group1,group2,group4, group5,group6,group7,group8 was significantly different from model group （P＜0.01）.4.The mean levels of serum E₂:there was significant difference on the levels of serum E₂ between control group and model group（P＜0.05）. The mean levels of serum E₂ in positive control group,group1,group2,group3 was significantly different from model group（P＜0.05）.5.The mean levels of serum P:there was significant difference on the levels of serum P between control group and model group（P＜0.05）. The mean levels of serum P in positive control group,group1,group2, group3,group4,group6 was significantly different from model group（P＜0.05）.6.Expression of PR:there was significant difference on expression of PR between control group and model group（P＜0.05）.Expression of PR in group1,group2,group3,group4,group5,group6 was significantly different from model group（P＜0.05）.7.The Compatibility Rule of STW:7.1 Qualitative Analysis of contribution degrees of single medicine in STW:Semen Cuscutae＞Herba Taxilli=Radix Dipsaci＞Colla Corii7.2 Qualitative Analysis of interactions of single medicine in STW:It is suggested that abortion rate of pregnant rat is associated with interactions between Semen Cuscutae and Herba Taxilli,interactions between Semen Cuscutae and Colla Corii.The mean levels of serum E₂ is related to interactions between Semen Cuscutae and Herba Taxilli,interactions between Herba Taxilli and Radix Dipsaci.The mean levels of serum P is associated with interactions between Semen Cuscutae and Herba Taxilli, interactions between Herba Taxilli and Colla Corii,interactions between Radix Dipsaci and Colla Corii.Expression of PR is associated with 125interactions between Semen Cuscutae and Herba Taxilli,interactions between Semen Cuscutae and Colla Corii,interactions between Herba Taxilli and Radix Dipsaci.8.Amount of each crude drug in 8 combinations of STW and the mean levels of serum E₂ and P,expression of PR results of 8 combinations of STW on abortion rat models are interrelated by quadratic regression. The equation（R²=0.9832,P＜0.05） （?）=35.0123-0.2204*X1*X3-0.02321*X1*X4++0.2165X2X3+0.01078*X1² was obtained. The optimal proportion of STW are（Semen Cuscutae:Herba Taxilli:adix Dipsaci:Colla Corii=15:21:21:0）.9 It was concluded from authentication experiment that compared with model group,there was significant difference in STW 1 group（Semen Cuscutae:Herba Taxilli:adix Dipsaci:Colla Corii=15:21:21:0）,STW 2 group（Semen Cuscutae:Herba Taxilli:adix Dipsaci:Colla Corii=20:10:10:0）,STW 3 group （Semen Cuscutae:Herba Taxilli:adix Dipsaci:Colla Corii=20:10:10:5）.Conclusion:1.Abortion animal models of corepus luteum inhibition due to renal deficiency,which was established by Professor Luo Song Ping,successfully analogue renal deficiency of spontaneous abortion.更多还原