【作者】 曾蕾；

【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】 目的本研究是在硫酸脱氢表雄酮-多囊卵巢综合征(DHEA-PCOS)大鼠模型的基础上,加用HCG和高脂饲料,建立一种新的改良青春期PCOS-IR大鼠模型,通过观察加味苍附导痰汤对该模型体重、脏器湿重、内分泌激素及代谢指标、组织形态学与超微结构的影响,探讨温肾健脾与理气化痰中药对PCOS的治疗机理。方法实验一:改良青春期PCOS-IR大鼠模型的建立将52只21日龄SPF级雌性SD大鼠(体重控制在35～45克)随机分为5组:A T组:第23日龄开始每日颈背皮下注射硫酸普拉睾酮纳针9mg/100g(相当于DHEA 6mg/100g),连续40天;B T+HCG组:同A组,并于第27日龄开始腹部皮下注射绒毛膜促性腺激素(HCG)1.5单位,bid,共35天;C HCG组:第23日龄开始每日颈背部皮下注射绒毛膜促性腺激素针1.5单位,bid,连续40天;D空白组:第23日龄开始每日皮下注射0.2ml生理盐水,连续40天;E高脂组:同D组。每组10—11只,喂以颗粒饲料,自由饮水,适应性喂养2d。D组全程喂以普通颗粒饲料。第23日龄开始,其余四组于前20天予普通饲料,后20天予高脂饲料喂养。观察记录各种动物体重、活动、进食、粪便及阴道涂片等一般情况。第40天晚8pm禁食不禁水,次日处死。10%水合氯醛麻醉后眼眶后静脉丛取血,保留血清,检测内分泌激素(总睾酮T、雄烯二酮A2、黄体生成素LH)、代谢指标(胰岛素INS、甘油三脂TG、总胆固醇CHOL);电子天平称取双侧离体卵巢、子宫及生殖器周围脂肪湿重;光镜下计算囊性变卵泡、成熟卵泡、黄体数量,电镜下观察卵巢超微结构的变化。实验二:加味苍附导痰汤拆方对PCOS-IR大鼠模型的影响加味苍附导痰汤拆方为温肾健脾方及理气化痰方两部分。将90只21日龄SPF级雌性SD大鼠(体重控制在35～45克)随机分为9组,所有组别均予20天灌胃,并根据大鼠体重的增加而改变灌胃剂量。A全方高剂量组灌服加味苍附导痰汤全方水煎剂,按48.4g/kg/d剂量灌胃;B全方低剂量组按12.1g/kg/d剂量灌胃;C温肾健脾高剂量组予该拆方水煎剂23.6g/kg/d剂量灌胃;D温肾健脾低剂量组予该拆方水煎剂5.9g/kg/d剂量灌胃;E理气健脾高剂量组予该拆方水煎剂24.8g/kg/d剂量灌胃;F理气健脾低剂量组予该拆方水煎剂6.2g/kg/d剂量灌胃;G二甲双胍组予格华止0.19g/kg/d剂量灌胃;H模型组及I高脂组予等量生理盐水灌胃。造模方法及观察项目同实验一B组。结果一、本实验一的动物模型具有以下特征1、体重:T+HCG模型组与空白组比较,虽未有显著性差异(P＞0.05),但三个类PCOS模型及高脂组体重均较空白组增长快。2、卵巢及生殖器周围脂肪积聚情况:单用雄激素造模导致卵巢体积较小,甚至小于空白对照组,未出现人类PCOS的卵巢白膜增厚,而加用HCG可令卵巢体积及湿重明显增加,T+HCG组、HCG组卵巢湿重与空白组比较,有统计学意义(p＜0.05)。T+HCG组与空白组比较,生殖器周围脂肪湿重有统计学意义(p＜0.05)。说明T+HCG联合应用可放大高脂饲料增重的效应,并改变体脂分布,使腹部脂肪增加明显,尤其是生殖器周围脂肪增加。3、内分泌改变:T+HCG组总睾酮及雄烯二酮含量均较空白组明显增高,比较有统计学差异(p＜0.05)。说明HCG与T联合应用,可放大雄激素的效应及增加雄激素的生物利用度。且以升高雄烯二酮为主。4、代谢改变:T+HCG组PCOS特征明显。T+HCG联合应用可使胰岛素显著升高,与空白组比较差异有统计学意义(p＜0.05)。由此推断高胰岛素血症是PCOS病理过程的重要环节。HCG联合高脂饲料能引起代谢异常,而以总胆固醇的增高为特征。5、形态学改变:从统计分析得出结论,三个类PCOS模型均可达到抑制排卵的目的,说明雄激素对于模型形态学改变的主要作用在于抑制排卵方面,而加用HCG对于模型卵巢囊性改变的作用更大。6、单纯高脂饲料并不引起卵巢湿重及多囊改变的增加,也不引起内分泌激素的明显改变。硫酸普拉睾酮纳+HCG及高脂饮食的联合应用,可起到互补作用,使动物模型在内分泌、代谢及形态学上更接近临床PCOS的表现。二、加味苍附导痰汤对青春期PCOS-IR模型的干预作用主要表现在以下方面:1、大鼠体重及生殖器周围脂肪积聚情况:加味苍附导痰汤对于模型体重的影响无显著性差异(P＞0.05),提示PCOS超重的问题,应通过运动及控制饮食等方法进行干预。而在实验一中,T组、T+HCG组及HCG组与空白组相比,生殖器周围脂肪湿重的差异有显著性(p＜0.05);而在实验二中,虽然各组比较无显著性差异(P＞0.05),但各组脂肪湿重均较模型组降低,说明药物的提前干预,对调整腹部脂肪的分布及脂肪内细胞因子的代谢有一定意义。上方对于子宫湿重无影响(P＞0.05)。2、卵巢湿重:用药的各组与空白组比较,对于卵巢的重量均有影响(p＜0.05),考虑有两个原因:第一,由于造模的同时用药,有预防与治疗的双重作用;第二,可能是促进排卵,使囊内卵泡液排出,从而促进卵巢体积及重量的下降。3、内分泌改变:①高剂量组别与低剂量组别比较,在降调总睾酮的作用更大,差异有统计学意义(p＜0.05),说明中药治疗作用具有一定的量效关系。②在降调LH方面,并未总结出明显的规律。理气化痰剂量组不仅未能降调LH,反而导致升高,是否与促排卵,诱发LH峰有关还有待进一步证实。整体而言,全方高剂量组在内分泌激素的降调效应最强,说明中药相须而用,药物的协同治疗作用的优势。4、代谢改变:格华止可提高胰岛素敏感性。本实验的理气高剂量组数值较格华止组稍低,且两组与空白组比较均有统计学意义(p＜0.05)考虑理气化痰中药在降调胰岛素,提高胰岛素兴奋性中的作用更明显。甘油三脂在实验一及实验二均未见显著变化(P＞0.05),考虑该高脂模型并未引起甘油三脂域值的改变。全方、理气化痰方及格华止在降调总胆固醇的疗效方面优于温肾健脾方,提示理气化痰成分在整首加味苍附导痰汤中,可能在调节机体代谢,尤其是降调总胆固醇方面有突出疗效。5、形态学改变:对于卵巢囊性改变及成熟卵泡数量、黄体数量的改变不是太明显,中药治疗的疗效主要体现在促进囊性扩张卵泡功能的恢复——即颗粒细胞层数增加,卵丘及透明带的重现,黄体成熟度等方面。全方高剂量组及温肾健脾高剂量组有助于促进囊性卵泡功能恢复、促排卵及黄体功能的健全与维持。结论本研究成功建立一种新的改良青春期PCOS-IR大鼠模型。通过观察加味苍附导痰汤对该模型体重、内分泌激素及代谢指标的影响、组织形态学与超微结构的影响,来探讨温肾化痰中药对PCOS的治疗机理。一、改良青春期PCOS-IR大鼠模型具有以下特征:1、保留了高雄激素血症和/或高LH血症等内分泌特征;同时有胰岛素抵抗,代偿性高胰岛素血症以及总胆固醇升高的糖脂代谢异常。2、具有腹部肥胖的特征——尤其是生殖器周围脂肪垫明显增加;3、卵巢明显增大,皮质增厚,卵巢内囊性扩张卵泡数目显著增加,排卵抑制。造模成功率高达100%。二、加味苍附导痰汤可改善青春期PCOS-IR大鼠模型内分泌及代谢异常。全方高剂量组对降调雄激素效果突出;理气化痰组分在调节机体代谢,降调胰岛素、总胆固醇方面作用明显;可能是改善卵巢内环境,促进排卵的关键因素;三、全方高剂量组在综合疗效方面显著优于拆方,说明中药相须而用,协同作用的优势;高、低剂量组之间有一定的量效关系;四、加味苍附导痰汤可促进已发生卵巢囊性扩张的卵泡功能恢复——使颗粒细胞层数增加,卵丘及透明带的重现,因造模药物引起的卵巢超微结构异常改变亦可逐渐修复。五、在造模期间提前予以加味苍附导痰汤进行干预,对调整腹部脂肪的分布有一定意义。更多还原

【Abstract】 ObjectiveThe study is based in the traditional PCOS rat model induced by Dehydroeplandrosterone（DHEA）,to establish the improved Puberty PCOS-IR rate model.While used the Chinese Herbal Jia Wei Cang Fu Dao Tan Tang as the treatment,to investigate the effect on weight,organ weight,hormonal, metabolized and morphology changes in the ovary of PCOS rat model.In Addition, the study could explore the mechanism of action towards reproductive system after using a herbal formula for supplementing the kidney and lustrating Tan.MethodExperiment 1:Establishment of improved Puberty PCOS-IR rate model52 21^st day SPF SD female rats which weight controlled between 35-45g were randomized into five groups.Group A（Sodium Prasterone Sulfate of injection shorted for T）:Begin in 23^rd day,hypodermic injection with Sodium Prasterone Sulfate of injection 9mg/100g（equal to DHEA6mg/100g ） for 40 days.Group B（T+HCG）:The same as group A,and from 27^th day hypodermic injection with HCG 1.5u twice a day for 35 days.Group C（HCG）:From 23^th day hypodermic injection with HCG 1.5u twice a day for 40 days.Group D（Blank control group）:From 23^th day hypodermic injection with Physiological saline 0.2ml for 40 days.Group E（High Fat Feed group）:The same as group D.Feed adaptively on normal feedstuff for 2 days.On 23rd day,except Group D only feed on normal feedstuff for 40 days,the other groups will be feed on normal feedstuff for the first 20 days and High Fat Feedstuff for the late 20 days.To observe the general condition such as weight、eating、excrement、Vagina smear and so on.Fasting but not endure the water on the 40^th day night,all rats were killed the next day.Anaesthesia by 10%Chloral hydrate,draws blood from the vein of eye socket,conserved the blood serum to test hormonal and metabolized index;weight ovary、uterus and fat around reproductive organ in by electronic balance.The histological and morphological alteration of ovarian were studied with microscope.Ovary ultrastrusture was detected by electric microscope.Experiment 2:The Influence of the Jia Wei Cang Fu Dao Tan Tang Decoction on PCOS-IR Rat ModelJia Wei Cang Fu Dao Tan Tang Decoction were divided into 2 parts—Wen Shen Jian Pi Decoction and Li Qi Hua Tan Decoction.90 21^st day SPF SD female rats were randomized into 9 groups.All groups will be orally fed for 21 days and have to change dosage when rats weight gained,group A were treated with Jia Wei Cang Fu Dao Tan Tang Decoction at 48.4g/kg/d.Group B Jia Wei Cang Fu Dao Tan Tang Decoction at 12.1g/kg/d.Group C were treated with Wen Shen Jian Pi Decoction at 23.6g/kg/d.Group D were treated with Wen Shen Jian Pi Decoction at 5.9g/kg/d.Group E were treated with Li Qi Hua Tan Decoction at 24.8g/kg/d. Group F were treated with Li Qi Hua Tan Decoction at 6.2g/kg/d.Group G were treated with Metformin at 0.19g/kg/d.Group H（model group） and GroupⅠ（High Fat Feed group） were treated with Physiological saline.The establishment of rat model and the observation projects were the same as experiment 1 group B.Result1.This experimental rat model has following characteristic:（1） Body weight:,Although there was no significant difference between the model group and the blank group,but compared with the blank group,the weight of rats in the three kind of PCOS models and High Fat Feed group grown more quickly.（2） Fat agglomeration around ovary and reproductive organ:Used androgen only would cause the ovary volume of rat model too small,even smaller than the blank control group;and it has not appeared ovary sclera accumulation as human PCOS.But if added with HCG may increases obviously the volume and the wet weight of ovary.Compared with the blank group,the T+HCG group and the HCG group ovary wet weight has statistics significance（p＜0.05）.The fat of the T+HCG group around the reproductive organ was significantly heavier than the blank group（p＜0.05）.The application of T+HCG union may enlarge the effect which the high fat feed could increase the weight,and changed the bodily fat distribution,causes the abdomen fat increase to be obvious,in particular around reproductive organ fat increase.（3） Endocrine changes:The androgen and the androstenedione of the T+HCG group were significantly higher than the blank group（p＜0.05）.The application of T and HCG union may enlarge the effect of the androgen and increase androgen biological exploitability.Also elevates the androstenedione primarily.（4） Metabolism changes:PCOS characteristic is obvious in the T+HCG group. Application of the T+HCG union may cause the insulin remarkable ascension, compared with the blank group it has statistics significance（p＜0.05）.It infers that the high insulin blood syndrome is the important link of PCOS pathology process.HCG united the high fat feed could cause unusual metabolism, and took the cholesterol markup as the characteristic.（5） Morphology changes:Draw the conclusion from the statistical analysis, three kinds of PCOS models may achieve that the ovulation had been suppressed. It showed that the main function of the androgen lied in the suppression to the ovulation in morphology change,added with HCG may enlarge the effect of the multi-pouches change of the model.（6） The pure high fat feed does not cause the increase of the ovary wet weight and the multi-pouches changed,also does not cause the endocrine hormone obvious changed.The application of Sodium Prasterone Sulfate of injection +HCG and the high fat diet union,may play the supplementary role,caused the performance of animal model much closer to clinical PCOS in the endocrine,metabolism and the morphology changes.2.The Influence of the Jia Wei Cang Fu Dao Tan Tang Decoction on PCOS-IR Rat Model（1） Body weight and fat agglomeration around ovary and reproductive organ: The Jia Wei Cang Fu Dao Tan Tang Decoction has no significance influence on the body of the rat model（P＞0.05）.It showed that other methods such as sports and diet control would be a good intervention on over-weight problem of PCOS. In the experiment 1,compared with the blank group,the fat around the reproductive organ of T group、T+HCG group and the HCG group were significantly heavier（p＜0.05）;while in the experiment 2,although there were no significantly difference in each group（P＞0.05）,but compared with the model group,the fat around the reproductive organ of each group reduced.It showed that there were certain significance of decoction intervention ahead of time which could adjust the abdomen fat in the distribution and the fat cell factor metabolism.The decoction did not have the influence regarding to the wet weight of uterus.（2） Wet weight of ovary:Compared with the blank group,the ovary weight of other groups were significantly lighter（p＜0.05）.There is two reasons:First, because of decoction intervention ahead of time,it may had the prevention and the treatment dual function;Second,it possibly promoted the ovulation, and caused the liquid discharged from the pouch of the ovary,thus promoted ovary volume and weight decline.（3） Endocrine changes:①The content of androsterone in high dose groups were significantly lower than the low dose groups（p＜0.05）,Chinese herbal had certain dose-effect relationship.②There were no obvious rule in decreasing LH.Instead of reducing but elevating LH,whether LiQiHuaTan group with acceleration of the ovulation,induced the LH peak or not still under confirmation.For the whole,high dose of JiaWeiCangFuDaoTanTang group was the strongest in decreasing the endocrine hormone.The superiority function of coordination treatment of Chinese herb was conformed.（4） Metabolism changes:Metformin enhanced the insulin sensitivity.Insulin in high does of LiQiHuaTan group was lower than the metgormin group,both two groups were significantly lower than the blank group（p＜0.05）.It considered that it was more effective for LiQiHuaTan herb in reducing insulin and enhances the excitability of insulin.The glycerin had no significantly change in experiment 1 and 2（P＞0.05）.It may be considered that the high fat model did not cause the glycerin territory value change by no means. JiaWeiCangFuDaoTanTang decoction,LiQiHuaTan decoction and metgormin were more effective in decreasing cholesterol than WenSenJianPi decoction,it showed that WenSenJianPi decoction could possibly in the adjustment organism metabolism,in particular falling tone cholesterol.（5） Morphology changes:The quantity of bursa ovarica change、the mature graafian follicle and the corpora luteum were not obvious,the curative treatment effect of Chinese native medicine mainly manifests was promoting the function of pouch expansion graafian follicle restoration----Namely promoting the granular cell layer increased,oviferous tubercle and transparent belt reproduced and the corpora luteum matured and so on. JiafeiCangFuDaoTanTang decoction and WenSenJianPi decoction were helpful in promoting the pouch graafian follicle function to restore,to press the ovulation and to perfect and maintain the function of the yellow body.ConclusionWhile used the Chinese Herbal Jia Wei Cang Fu Dao Tan Tang as the treatment, to investigate.In Addition,the study could after using a herbal formula for supplementing the kidney and lustrating Tan.This research successfully improved the puberty PCOS-IR rat model. Through the observation of the effect on weight,organ weight,hormonal, metabolized and morphology changes in the ovary of PCOS rat model,we concluded that JiaWeiCangFuDaoTanTang decoction could explore the mechanism of action towards reproductive system.1.The improved puberty PCOS-IR rat model has following characteristic:Retained the high androgen syndrome and/or the high LH syndrome as the endocrine characteristics;Simultaneously it had sugar fat metabolism unusual, such as the insulin resistance,the redemptive high insulin blood sickness as well as cholesterol ascension.Has the abdomen obese characteristic----In particular fat pad which around reproductive organ obvious increased;The ovary increased obviously,Ovarian cortex accumulated,the number in ovary pouch expansion graafian follicle were remarkable increased, ovulation suppression.The ratio of PCOS model reached as high as 100%.2.JiaWeiCangFuDaoTanTang decoction could possibly improve the unusual of internal secretion and metabolism of the puberty PCOS-IR rat model.The high does of JiaWeiCangFuDaoTanTang decoction was prominent effectively in the falling tone androgen;LiQiHuaTan component was good at adjusting organism metabolism,the falling tone insulin and the cholesterol obviously;Possibly the key aspect was improved the environment of ovary and promoted ovulation;3.The high does of JiaWeiCangFuDaoTanTang decoction obviously surpasses in the synthesis curative effect compared with the other two components,it conformed that the superiority function of coordination treatment of Chinese herb.The high and low does group of Chinese herbal had certain dose-effect relationship; 4.The curative treatment effect of JiaWeiCangFuDaoTanTang decoction mainly manifested that it could promote the function of pouch expansion graafian follicle restoration----Namely promoting the granular cell layer increased, oviferous tubercle and transparent belt reproduced and the corpora luteum matured.The decoction could gradually reduce and repair abnormal changes in the ultrastructure of ovarian grafts;5.It showed that there were certain significance of decoction intervention ahead of time which could adjust the abdomen fat in the distribution and the fat cell factor metabolism.更多还原