【作者】 孟君；

【导师】 黄健玲；

【作者基本信息】 广州中医药大学 ， 中医妇科学， 2011， 博士

【摘要】 研究背景多囊卵巢综合征(polycystic ovary syndrome, PCOS),是以持续性无排卵、高雄激素或胰岛素抵抗为特征的内分泌紊乱的症候群。卓于1935年Stein和Leventhal首次报道,故又称为Stein-Leventhal综合征。常见临床表现有无排卵型月经失调、不孕、多毛、痤疮、肥胖、黑棘皮症和卵巢多囊性改变等。近年来的研究发现该疾病的功能紊乱远超出生殖轴,由于胰岛素抵抗,常发展为代谢综合征,PCOS代谢综合征的患病率高于正常人群的4-11倍。PCOS不仅对生殖内分泌功能影响,远期可增加代谢综合症、糖尿病、子宫内膜癌及心血管疾病的发病率。近年的研究认为,胰岛素抵抗是PCOS病理生理过程中的中心环节,多数PCOS患者具有胰岛素抵抗。胰岛素抵抗不仅出现在肥胖的PCOS患者中,正常体重的PCOS患者亦有胰岛素抵抗。我院名老中医李丽芸教授根据前人经验结合临床经验提出PCOS中医病机以肾虚痰湿为主,属虚实夹杂之证,研制出灵术颗粒制剂,治疗胰岛素抵抗,临床上取得较好疗效。研究目的本课题通过动物实验研究,探讨灵术颗粒对PCOS大鼠卵泡基底膜,促进卵泡发育的可能作用靶点,阐明其疗效的可能分子机制,为临床治疗PCOS胰岛素抵抗的基础研究提供实验依据,以期进一步揭示其可能的疗效机理。为中医药治疗PCOS赋予新的内涵,为灵术颗粒临床应用提供科学依据。并通过临床观察研究灵术颗粒与二甲双胍治疗PCOS胰岛素抵抗疗效的对比,研究灵术颗粒治疗PCOS胰岛素抵抗的临床疗效及安全性。研究方法本研究本课题通过应用Poresky方法制造PCO大鼠模型。采用随机对照实验的方法,分为灵术颗粒组、二甲双胍组、模型对照组及空白对照组,围绕PCOS卵泡发育及排卵障碍的主要环节,通过观察卵巢形态学、血清T、INS及糖耐量试验、卵泡基底膜厚度和其蛋白表达及颗粒细胞、卵泡内膜细胞数量等的影响等指标,以探讨灵术颗粒可能作用机制。本研究采用临床随机对照研究方法,进行临床疗效评价及安全性评价,将广东省中医院妇科2008年9月-2011年1月在门诊就诊符合纳入标准的患者随机分为治疗组(35例)和对照组(35例),选用灵术颗粒为治疗组,二甲双胍为对照组,采用中医证候评分表,结合女性激素、血脂、血糖及胰岛素、B超等监测。将观察的数据输入SPSS17.0建立数据库并进行统计学分析。研究结果1动物实验结果：(1)空白对照组与造模成功组比较分析：造模成功组糖耐量、体重、睾酮、胰岛素均数显著高于空白对照组(P<0.05)。(2)造模成功率分析：Poresky方法造模组大鼠排卵障碍率为80.36%,大鼠死亡率11.67%,但是造模成功率仅有43.33%。(3)4组治疗后各项指标比较：4组睾酮、胰岛素均数比较,空白对照组与其余3组分别比较有显著差异(P<0.05),其余组别比较无显著差异(P>0.05)。经药物治疗后,体重均数4组间无显著差异(P=-0.309>0.05)。治疗后,体重均数4组间无显著差异(P＞0.05)。治疗后睾酮、胰岛素均数4组比较,有差异性(P>0.05)。(4)4组治疗前后差值的比较：①体重增长值方面,空白对照组与模型对照组比较无显著差异(P=-0.511>0.05),灵术颗粒组与二甲双胍组比较无显著差异(P=-0.905>0.05),其余组别比较均有显著差异性(P<0.05),灵术颗粒组与二甲双胍组体重增长值,明显低于空白对照组与模型对照组,有统计学意义。②睾酮降低幅度方面,空白对照组与模型对照组比较无显著差异(P=-0.962>0.05),灵术颗粒组与二甲双胍组比较无显著差异(P=-1.000>0.05),其余组别比较均有显著差异性(P<0.05),灵术颗粒组与二甲双胍组睾酮下降幅度,均明显高于空白对照组与模型对照组,有统计学意义。③胰岛素降低幅度方面,空白对照组与模型对照组比较无显著差异(P=-0.596>0.05),灵术颗粒组与二甲双胍组比较无显著差异(P=-1.000>-0.05),其余组别比较均有显著差异性(P<0.05),灵术颗粒组与二甲双胍组胰岛素下降幅度,均明显高于空白对照组与模型对照组,有统计学意义。(5)子宫湿重与总卵巢湿重的比较：①子宫湿重比较方面,空白对照组与模型对照组比较,可信区间为(-258.0085,-18.3665),可信区间不包括0,表示在α=0.05水准有显著差异性,其他组无显著差异性,但从均数比较,模型对照组(676.68)最高,二甲双胍组(621.01)高于灵术颗粒组(567.87),空白对照组(538.50)最低。②大鼠双侧卵巢总重量比较,表示4组组间均数比较无显著差异,根据均数比较,模型对照组高于二甲双胍组,再次为灵术颗粒组,空白对照组最低。上述数据虽无统计学意义,根据均数比较,有一定临床意义,可见治疗后模型对照组子宫湿重、总卵巢湿重明显高于其他3组,空白对照组最低。由此可见肾虚痰湿型大鼠的子宫及总卵巢湿重明显重于空白组,而二甲双胍组与灵术颗粒组对于降低其重量有一定效果。(6)大鼠卵泡基底膜3种主要蛋白表达的评级比较：采用Kruskal-WallisH检验,①Ⅳ型胶原(CollagenⅣ)蛋白表达的4组比较,以模型对照组Ⅳ型胶原表达最多,灵术颗粒组次之,空白对照组再次之,二甲双胍组表达最少。②层连接蛋白(Laminin)蛋白表达的4组比较,以模型对照组Ⅳ型层连接蛋白表达最多,灵术颗粒组与二甲双胍组效果无差异,空白对照组层连接蛋白表达最少。③串珠养蛋白聚糖蛋白(Perlecan)的表达的4组比较有显著差异,以模型对照组串珠养蛋白聚糖表达最多,灵术颗粒组与二甲双胍组效果无差异,空白对照组串珠养蛋白聚糖表达最少。(7) HitachiH—7500型透射电镜观察卵泡基底膜厚度的比较：秩和检验,P=O.261>0.05,表示4组组间均数比较无显著差异。但均数比较,模型对照组基底膜厚度(707.52nm)显著高于其他三组,而灵术颗粒组与二甲双胍组基底膜厚度接近空白对照组。(8)颗粒细胞、卵泡内膜细胞层数的比较：采用Kruskal-Wallis H检验,颗粒细胞与卵泡膜细胞计数4组比较,P>0.05,4组间均无显著差异。(9) HitachiH-7500型透射电镜观察,空白对照组、灵术颗粒组均有较多黄体细胞,提示大鼠有排卵。2临床研究结果：(1)体重指数及腰臀比：治疗组(灵术颗粒组)与对照组(二甲双胍组)在降低PCOS胰岛素抵抗患者的体重指数及腰臀比均有显著疗效,P＜0.05,提示治疗前后有差异性,有统计学意义。(2)灵术颗粒与二甲双胍可均显著降低PCOS胰岛素抵抗患者体内胰岛素水平并改善胰岛素抵抗。经配对t检验,对照组治疗后降低空腹血糖有显著差异,P＜0.05,有统计学意义。治疗组降低餐后2小时血糖、餐后2小时胰岛素及HOMA—β有显著差异,P<0.05,有统计学意义。2组均在降低空腹胰岛素及HOMA-IR有显著差异,P<0.05,有统计学意义。(3)灵术颗粒组降低TC、升高HDL-C有显著疗效,二甲双胍组降低TG、ApoB有显著疗效。均可降低LDL-C。配对t检验,治疗组治疗后TC显著低于治疗前(P=0.003<0.05), HDL-C显著高于治疗前(P=0.021<0.05)；对照组TG治疗后显著低于治疗前(P=0.008<0.05),治疗后ApoB显著低于治疗前(P=0.037<0.05)；2组治疗后LDL-C均显著低于治疗前(P<0.05)。2组ApoA/ApoB均无统计学意义。(4)灵术颗粒与二甲双胍均可降低PCOS胰岛素抵抗患者T水平,二甲双胍降低LH/FSH效果显著。治疗前后2组女性激素水平的均数比较配对t检验,对照组治疗后LH/FSH水平显著低于治疗前(P=0.025<0.05)；2组治疗后T均显著低于治疗前(P＜0.05),提示治疗前后有显著差异性。在升高E2、降低LH方面,治疗组与对照组治疗前后无显著差异(P>0.05),无统计学意义。(5)灵术颗粒与二甲双胍在提高BBT双相周期方面无显著差异。卡方检验2组治疗期间BBT双相周期数P=1.563>0.05,2组无显著差异。(6)在改善中医症候评分方面,灵术颗粒与二甲双胍均有显著效果。配对t检验,2组治疗后中医症候评分显著低于治疗前(P<0.05)提示2组均能改善PCOS患者中医症候。(7)本研究的PCOS胰岛素抵抗患者并非全部是肥胖者,非肥胖患者占所有研究对象的43.55%(27/62)；治疗组占51.61%(16/31)；对照组占35.48%(11/31)。研究结论本院名老中医李丽芸教授根据前人论述同时结合自己的临床经验提出PCOS中医病机以肾虚痰湿为主,并研究出灵术颗粒剂,全方共奏补肾化痰之功,临床疗效较佳。本研究表明灵术颗粒治疗肾虚痰湿型PCOS疗效确切,与二甲双胍组比较,PCOS大鼠在体重增长及降低大鼠睾酮、胰岛素方面无差异(P>0.05),2组疗效相似。在降低子宫湿重、总卵巢湿重有一定临床意义。灵术颗粒与二甲双胍均可减少Ⅳ型胶原、层连接蛋白与串珠养蛋白聚糖的表达,对于改善基底膜分子筛方面有着明显作用。在Ⅳ型胶原的表达方面,二甲双胍组优于灵术颗粒组。在层连接蛋白与串珠养蛋白聚糖的表达方面,二甲双胍组与灵术颗粒组无显著差异。灵术颗粒与二甲双胍均可改善基底膜厚度,对于改善基底膜分子筛功能方面有着明显的作用。综上所述,从分子生物学方面,补肾化痰中药灵术颗粒可改善PCOS卵泡基底膜分子筛功能,调节两细胞间信息交换,维持“LH—卵泡内膜细胞轴”和“FSH—颗粒细胞轴”两轴功能平衡,为中医补肾化痰治疗PCOS赋予新的内涵,也为其临床应用提供科学依据。本研究提示灵术颗粒治疗PCOS胰岛素抵抗疗效显著,与二甲双胍相比较,在治疗该病方面既有相似,又各有所长。降低体重指数、腰臀比,改善胰岛素抵抗方面,二者均有显著疗效。2组在降低空腹胰岛素及HOMA-IR效果无差异,二甲双胍降低空腹血糖较好,灵术颗粒降低餐后2小时血糖、餐后2小时胰岛素及HOMA—β效果较好。灵术颗粒组降低TC、升高HDL-C有显著疗效,二甲双胍组降低TG、ApoB有显著疗效。二者均可降低LDL-C。二者对患者内分泌改善方面,各有千秋,灵术颗粒与二甲双胍均可降低PCOS胰岛素抵抗患者T水平,二甲双胍降低LH/FSH效果显著。降低LH方面,治疗组与对照组治疗前后无显著差异。在改善中医症候评分方面,灵术颗粒与二甲双胍均有显著效果。灵术颗粒与二甲双胍在提高BBT双相周期方面无显著差异。另在确诊PCOS时,不论胖瘦,均应排除胰岛素抵抗,以免遗漏。更多还原

【Abstract】 BackgroundPolycystic ovarian syndrome(PCOS) is one of most common endocrine disease, The syndrome encompasses a broader spectrum of signs and symptoms, such as elevated serum hyperandrogenism and insulin resistance. In 1935, Stein and Leventhal reported this disease for the first time, so the disease is named the syndrome of Stein-Leventhal too. PCOS is with different phenotypic aspects, Clinical manifestation of PCOS includes anovulatory menoxenia, infertility, hirsutism, acne, obesity, acanthosis nigricans and PCO. The later-day researches covers that PCOS dysfunction oversteps reproductive axis. Insulin resistance is evolving to metabolic syndromes generally. The probability of PCOS patients suffering from metabolic syndromes is 4-11 times than ordinary persons. Not only it impairs function of procreation and incretion, but also is associated with metabolic syndromes, type 2 diabetes, endometrial carcinoma, cardiovascular and cerebrovascular disease。Latest studies reports that insulin resistance is the physiological and pathological key link of PCOS. Most PCOS sufferers are with insulin resistance. The famous professor Li LiYun of GuangDong Province Hospital Of Traditional Chinese Medicine, according to experiences of the ancients and her clinical experiences, created Ling-Zhu granule for PCOS, especially insulin resistance. It is very availability in the clinic.ObjectivesThe aim of this empirical study is to discuss the mechanism target of Ling-Zhu granule ameliorating the follicle basement membrane of PCOS rat, and accelerate the development of the follicle, to illuminate the molecular mechanism of the effect. It could be provide evidences for the clinical treatment and new contents of Traditional Chinese Medicine. The aim of this clinical study is to discuss the clinical curative effect and security of Ling-Zhu granule. The research is according to the comparison of Ling-Zhu granule and Metformin in treatment of patients with polycystic ovary syndrome and insulin resistance.MethodsThis study is basis of Poresky Methods to make the formers of PCO rats. The rats were divided into four groups by randomization and comparison, study group, control group, former group and blank group. This study is to discuss the main mechanism of the development of the follicle and the ovulation failure, to discuss the possible mechanism of Ling-Zhu granule, by observing the targets, such as ovarian configuration, T, insulin, OGTT, the thickness of the follicle basement membrane, the expression of the protein of the follicle basement membrane, the quantity of granular cells and theca cells. According to the RCT and the diagnosis criterion, The clinical study collected 70 patients of from Province Hospital Of Traditional Chinese Medicine, from 2008.9-2011.1 They are divided into two groups by 1:1 randomly, Ling-Zhu granule is given to treatment group including 35 cases, and at the same time Metformin is given to the control group also including 35 cases, two groups are inspected by using Chinese medicine syndromes graded tables and other observe variable index, such as female hormone, blood-fat, blood glucose, insulin, type-B ultrasonic. Lastly, the study data collected to make the data-base is analyzed by the software of SPSS17.0.Results1 Results of empirical study(1)The comparison of the blank group and the succeeded animal model group:The latter surpass the former in these mean observe index, such as OGTT, avoirdupois, T, insulin.(2)The analysis of the animal model succeeded: The rats of ovulation failure account for 80.36%, the dead rats account for 11.67%, and the rats succeeded account for 43.33%.(3)The comparison of the four groups indexes:To the mean of T and insulin, the blank group is distinct statistical differences between the other groups (P<0.05). There is no distinct statistical differences between the others (P>0.05). After the therapy, There is no distinct statistical differences between the four groups of the mean of weight (P>0.05). There is distinct statistical differences between the four groups the mean of insulin and T (P<0.05)(4)The difference of the four groups After the therapy:①weight:There is no distinct statistical differences between the blank group and the former group (P=0.511>0.05), the study group and the control group (P=0.905>0.05), except between other groups (P<0.05). The increase of weight of the study group and the control group is under the others, it has the statistical significance.②T:There is no distinct statistical differences between the blank group and the former group (P=0.962>0.05), the study group and the control group (P=1.000>0.05), except between other groups (P<0.05). The decrease of T of the study group and the control group is under the others, it exists the statistical significance.(5)The comparison of the green weight of uteruses and ovaries:①the green weight of uteruses:the comparison of the blank group and the former group:confidence interval (CI) (-258.0085,-18.3665), except 0, a =0.05, There is distinct statistical differences, there is no distinct statistical differences between other groups. But from The comparison of the mean, the first is the former group, the second is the control group, the third is the study group, the last is the blank group.②the green weight of ovaries: There are no distinct statistical differences between the four groups. But from The comparison of the mean, the first is the former group, the second is the study group, the third is the control group, the last is the blank group. Although it does not exist statistical significance to the data, it exist clinical significance. After the therapy, the green weight of uteruses and ovaries of the former group are more overweight than the others. The last is the blank group. So the green weight of uteruses and ovaries of the rats of "deficiency of kidney with sputum", are distinct overweight than the blank rats. The Ling-Zhu granule and Metformin are effective to lose weight.(6)The comparison of the three proteins of the follicle basement membrane of PCOS rat:With Kruskal-Wallis H method;①CollagenⅣ:the former group is the most of the expression of CollagenⅣ, the second is the study group, the third is the blank group, the last is the control group.②Laminin:The former group is the most of the expression of Laminin, the second is the study group and the control group, the last is the blank group.③Perlecan:There are distinct differences between the four groups. The former group is the most of the expression of Perlecan, the second is the study group and the control group, the last is the blank group.(7)The mean thickness of the follicle basement membrane:There is no distinct statistical differences between the four groups (P=0.261>0.05). The former group is the most thickness of the follicle basement membrane (707.52nm), the study and the control groups are close to the blank group.(8)The quantity of granular cells and theca cells:With Kruskal—Wallis H method; there is no distinct statistical difference between the four groups (P>0.05).(9)Ransmission electron microscope (HitachiH-7500):There are many yellow bodies of the blank and the study groups. It shows ovulation.2 Results of Empirical study(1)BMI、WHR:There is distinct statistical difference between the study group and the control group (P<0.05).(2) Ling-Zhu granule and Metformin can reduce insulin and insulin resistance of the patient of PCOS and insulin resistance.Paired t-test, In fasting blood-glucose, there is distinct statistical difference in the control group (P<0.05), there is statistical significance too. In reducing 2-hour postprandial blood glucose,2-hour postprandial insulin and HOMA—β, the study group has distinct statistical difference (P <0.05) and statistical significance. In reducing fasting insulin and HOMA-IR, they both have distinct statistical difference (P<0.05) and statistical significance.(3)Ling-Zhu granule can reduce TC and increase IIDL-C, Metformin can reduce TG、ApoB. They both can reduce LDL-C.Paired t-test, in reducing TC (P=0.003<0.05), increasing HDL-C (P =0.021＜0.05), there is distinct statistical difference in the study group than before. In reducing TG (P=0.008<0.05) and ApoB (P=0.037<0.05), there is distinct statistical difference in the study group than before. In reducing LDL-C, there is distinct statistical difference in the both groups than before (P<0.05).There is no distinct statistical difference in the both groups to ApoA/ApoB.(4)Ling-Zhu granule and Metformin can reduce free testosterone. Metformin can reduce LH/FSH.In reducing LH/FSH, there is distinct statistical difference in the control group than before (P=0.025<0.05).In reducing free testosterone, there is distinct statistical difference in the both groups than before (P <0.05).In reducing LH and increasing E2, there is no distinct statistical difference in the both groups (P>0.05)(5)There is no distinct statistical difference in the both groups in basal body temperature (BBT) with two-phase.Chi-square test, in the BBT with two-phase during the treatment, there is no distinct statistical differences between the two groups (P=1.563> 0.05).(6)Paired t-test, the point of Chinese medicine syndromes graded tables decline in the two groups is no distinct statistical difference (P<0.05). Fat and not-fat:(7)The patient of PCOS and insulin resistance in this study are not all fat.The not-fat patient account for 43.55%(27/62),the study group 51.61% (16/31);the control group 35.48%(11/31)ConclusionThe famous professor Li LiYun of GuangDong Province Hospital of Traditional Chinese Medicine, according to experiences of the ancients and her clinical experiences, declared that "deficiency of kidney with sputum" is the pathogenesis of PCOS, and created Ling-Zhu granule of restoring kidney and eliminating sputum for PCOS. Ling-Zhu granule is very availability in the clinic.This study shows that Ling-Zhu granule is effective to treatment the patient of PCOS of "deficiency of kidney with sputum". Compare to Metformin, there is (P>0.05) in the changes of the weight, free testosterone and insulin of rats. In reducing the green weight of uteruses and ovaries, Metformin is more effective than Ling-Zhu granule. There is clinical significance to the both groups. The both medi distinct statistical differences between the two groups caments are effective to reduce the expression of the three proteins, and to ameliorate the molecular sieve of the follicle basement membrane of PCOS rat. The Metformin is more effective to reduce the expression of the Collagen IV than the study group. There is no distinct statistical difference between the both medicaments in reducing the expression of Laminin and Perlecan. The both medicaments can ameliorate the thickness of the follicle basement membrane, so to ameliorate the molecular sieve of the follicle basement membrane of PCOS rat.In molecular biology, Insulin resistance of restoring kidney and eliminating sputum, can ameliorate the membrane molecular sieve of the follicle basement membrane, accommodate the communication of the two cells, and maintain the balance the function of "LH-theca cells axes and FSH-granular cell axes". It gives new content to the restoring kidney and eliminating sputum of Chinese Medicine, and provides the scientific evidence of the clinical application.This study shows that Ling-Zhu granule is very effective to treat the patient of PCOS and insulin resistance. Compare to Metformin, they are both effective, but have some dissimilarity. In reducing BMI、WHR and Insulin resistance, they both effective. There are no distinct statistical differences between the two groups in reducing fasting insulin and HOMA-IR. In reducing fasting blood glucose, Metformin is more effective. In reducing 2-hour postprandial blood glucose,2-hour postprandial insulin and HOMA—β, Ling-Zhu granule is more effective. Ling-Zhu granule is effective in reducing TC and increasing HDL-C. Metformin is effective in reducing TG and ApoB. They are both effective to reduce LDL-C. They are both effective to incretion, but have some dissimilarity. Ling-Zhu granule is more effective to reduce LH. They both can reduce free testosterone. The point of Chinese medicine syndromes graded tables decline in the two groups is no distinct statistical difference. There is no distinct statistical difference in the both groups in basal body temperature (BBT) with two-phase. A patient is diagnosed, without reference to fat or not, insulin resistance is needed to diagnose.更多还原