【作者】 梁娜；

【导师】 刘瑞芬；

【作者基本信息】 山东中医药大学 ， 中医妇科学（专业学位）， 2013， 博士

【摘要】 目的:本课题旨在通过临床流行病学调查，总结出IUD出血副反应的发病特点、影响因素、中医证候分布规律；同时在评价无创法、开腹法IUD避孕大鼠模型优劣的基础上，应用祛瘀清热、止血调经的方剂宫宁颗粒对IUD避孕大鼠模型进行实验研究，试以阐明宫宁颗粒对子宫血管平滑肌细胞（VSMC）增殖凋亡的影响及调节机理，从而进一步揭示放置IUD后子宫VSMC增殖凋亡与血管重塑的关系，探讨宫宁颗粒作用机制，为中医药防治本病的作用机理研究奠定分子生物学基础。方法:制定IUD出血副反应中医证候调查表，并进行临床调查，采用频数分析、2检验、多因素分析、主成分分析等方法对本病发病特点、影响因素、证候分布规律进行统计分析。实验研究以SD雌性已生育大鼠为受试对象，首先对无创法、开腹法建立IUD避孕大鼠模型的优劣进行评价，进而采用无创法建立IUD避孕大鼠模型，将100只大鼠随机分为空白对照组、假手术组、模型组、消炎痛组和宫宁颗粒组，应用透射电镜对各组大鼠子宫VSMC超微结构进行观察；应用免疫组化法及原位杂交法对各组大鼠子宫VSMC中Bcl-2、Bax、Fas、FasL蛋白及相关基因Bcl-2mRNA、BaxmRNA、FasmRNA、FasLmRNA表达进行检测。结果:1.本次临床调查中IUD出血副反应的高发年龄是31-35岁；学历以高中/中专为主；单因素分析得出，劳动强度，劳动姿势、分娩方式、子宫位置、环形均是其发病的影响因素，多因素分析纳入方程模型的危险因素有：劳动强度、分娩方式、子宫位置及流产次数；本病异常出血时间多发生在置IUD后3个月内，6个月次之，12个月及以上明显减少；395例IUD出血副反应患者可归纳为五个证型，其中瘀热互结证最为常见。本病常见症状有放置IUD后经量增多、经期延长、小腹刺痛、经色黯红、经血有块等。其中，瘀热互结证主成份分别是：主成分1(34.944%)：小腹刺痛、口渴欲饮冷、经血有块；主成分2(10.809%)：经期延长、心胸烦闷、便结；主成分3(10.076%)：舌红、脉弦数；主成分4(8.437%)：经量增多。肝郁血瘀证主成份分别是：主成分1(41.450%)：小腹刺痛、精神抑郁、乳房胀痛；主成分2(12.601%)：经期延长、经血有块；主成分3(10.436%)：舌黯红、脉弦涩；主成分4(9.023%)：经间期出血。阴虚血瘀证主成份分别是：主成分1(39.629%)：经期延长、小腹刺痛、咽干口燥；主成分2(12.461%)：经量增多、五心烦热、脉细涩；主成分3(9.664%)：经间期出血、经血有块；主成分4(8.881%)：经血黯红、少苔。气虚血瘀证主成份分别是：主成分1(33.409%)：经量增多、小腹刺痛、经质清稀；主成分2(13.287%)：经期延长、神疲懒言；主成分3(11.983%)：舌淡、脉缓弱；主成分4(9.194%)：气短、面色淡白；主成分5(8.535%)：经间期出血。肾阳虚证主成份分别是：主成分1(37.531%)：经量增多、腰膝酸软；主成分2(13.669%)：经期延长、小腹冷痛、畏寒肢冷；主成分3(10.335%)：经间期出血、经质清稀；主成分4(9.687%)：脉沉细。2.动物实验方面，无创法在放置IUD成功率方面，不低于开腹法，且避孕效果良好，同时，较之开腹法，无创法对大鼠一般状况、行为、子宫损伤影响更小；子宫VSMC超微结构观察，宫宁颗粒可使VSMC表型转化减轻，并促进子宫合成型VSMC凋亡，其效果明显优于消炎痛组；对子宫VSMC凋亡相关蛋白及基因表达进行检测，得出宫宁颗粒可通过上调BaxmRNA、FasmRNA、FasLmRNA及下调Bcl-2mRNA基因表达量，启动相应的细胞信号转导通路，调节相关蛋白Bcl-2、Bax、Fas、FasL表达。结论:临床调查分析表明，瘀热互结为本病的主要证候，“血瘀”在本病发病中起重要作用。因此祛瘀清热、活血调经为本病基本治法。实验研究证明，无创法更符合临床IUD的放置过程及避孕特征。置IUD后模型大鼠子宫VSMC以合成表型为主，存在增殖与凋亡失衡状况，宫宁颗粒可通过调节凋亡基因表达，启动相应的细胞信号转导通路，调节相关蛋白表达，进而起到促进子宫VSMC凋亡,调节子宫VSMC增殖和凋亡之间的平衡,逆转病理性重塑作用,这可能也是其防治置环后异常出血的机制之一。更多还原

【Abstract】 Objective: This subject aimed to sum up the characteristics influencing factors andthe distribution regularity of TCM syndromes of the bleeding side-effect induced by IUDby clinical epidemiological investigation；At the same time，on the basis of evaluating theadvantages and disadvantages of IUD contraceptive rats model established by noninvasivemethod or operation method, we developed experimental study by using GongNinggranule on the IUD contraceptive model rats, which have the functions of clearing heat、dispelling stasis、hemostasis and regulating menstruation, for the aim of clarifing the effectand regulation mechanism of GongNing granule on the proliferation apoptosis of uterinevascular smooth muscle cell (VSMC), further revealing the relationship between theproliferation apoptosis of uterine VSMC and vascular remodeling after IUD insertion,discussing the mechanism of GongNing granule, and providing molecular biological basefor the study of mechanism of traditional Chinese medicine preventing and treating thisdisease.Methods: First, We established a TCM syndrome questionnaire of the bleedingside-effect induced by IUD, then, launched the clinical investigation, last, statisticallyanalyzed the characteristics, influencing factors and the distribution regularity of TCMsyndromes of this disease by using the methods of frequency analysis、chi-square test、multivariate analysis and principal component analysis factor analysis.The experimentalresearch based on the SD female infertile rats as subjects. Firstly, we evaluated theadvantages and disadvantages of IUD contraceptive rats model established by noninvasive method or operation method, then using the noninvasive method to establish IUDcontraceptive model rats,100rats were randomly divided into blank control group、 modelgroup、 sham operation group、 indomethacin group and GongNing granule group.Transmission electron microscopy was applied to each group of rats for observing theultrastructure of uterine VSMC; immunohistochemical method and in situ hybridizationmethod were respectively applied to each group of rats to detect the protein expression ofBcl-2, Bax, Fas, FasL and related gene expression of Bcl-2mRNA、 BaxmRNA、FasmRNA、FasLmRNA in the uterine VSMC.Results:1.In the clinical investigation of the bleeding side-effect induced by IUD,31-35years old was the age vulnerable; the women with educational backgrounds of seniorhigh school and polytechnic school account for the majority; Through the Single factoranalysis, we can get the conclusion that the intensity of labor, labor posture, delivery mode,uterine position, and the shape of the IUD were the influencing factors of the disease. Inthe Multi-factor analysis, the intensity of labor, delivery mode, uterine position, abortiontimes were included in the model of logistic regression; the abnormal uterine bleedingmostly occurred in3months after IUD insertion,6month taked the second place,12months and over12months significantly reduced;395patients of bleeding side-effectinduced by IUD could be summarized as five syndrome types, among them, syndrome ofstagnation of blood-stasis and heat was most common. The main clinical symptoms ofthis disease were menorrhea、menostaxis、stabbing pain in lower abdomen、purple and darkmenstrual color, menstrual blood with blots after IUD insertion and so on. Among them,the principal components of syndrome of stagnation of blood-stasis and heat blood stasisrespectively were: the first principal component (34.944%): stabbing pain in lowerabdomen、thirsty and want to drink cold、 menstrual blood with clots; the second principalcomponent(10.809%): menostaxis、cardiothoracic heart annoyance、coprostasis; the thirdprincipal component(10.076%):red tongue、wiry and rapid pulse; the fourth principalcomponent(8.437%): menorrhea. The principal components of the syndrome of liverstagnation and blood stasis respectively were: the first principal component (41.450%):stabbing pain in lower abdomen、mental depression、mammary swelling pain depression;the second principal component(12.601%): menostaxis、menstrual blood with clots；thethird principal component(10.436%): dull red tongue、wiry and intermittent pulse; thefourth principal component(9.023%): ovulation bleeding. The principal components of syndrome of yin deficiency with blood stasis respectively were: the first principalcomponent (39.629%): menostaxis、stabbing pain in lower abdomen、dry mouth andthroat；the second principal component(12.461%): menorrhea、burning sensation of fivecentres、thready and intermittent pulse；the third principal component(9.664%): ovulation、bleeding menstrual blood with clots; the fourth principal component(8.881%): purple anddark menstrual color、few fur. The principal components of syndrome of qi deficiency withblood stasis respectively were: the first principal component(33.409%): menorrhea、stabbing pain in lower abdomen、thin menses; the second principal component(13.287%):menostaxis、languor and laziness to speak; the third principal component (11.983%): paletongue、slow and feeble pulse；the fourth principal component (9.194%): shortness ofbreath、pale white complexion; the fifth principal component (8.535%): ovulation bleeding.The principal components of kidney-yang deficiency syndrome respectively were: the firstprincipal component (37.531%): menorrhea、soreness-tired of waist and knee；the secondprincipal component (13.669%): menostaxis、lower abdominal pain with cold sensationintolerance to cold, cold hands and feet; the third principal component (10.335%):ovulation bleeding、thin menses; the fourth principal component (9.687%): deep and thinpulse.2. In animal experiment, the noninvasive method was no less than the operationmethod in the success rate of inserting IUD. Meanwhile, its effect of contraception wasgood and expulsion rate was lower than the operation method. Compared with theoperation method, the noninvasive method resulted in less damages to the rats’ generalcondition, behavior and uterine; by observing the uterine’s VSMC ultrastructure,we couldfind that GongNing granule had the function of relieving phenotypic transition of uterineVSMC and promoting apoptosis of synthetic uterine VSMC, whose effect was better thanindomethacin group; by detecting the expression of apoptosis-related proteins and gene inuterine VSMC, we could find that GongNing granule could start the cell signaltransduction pathway by raising the gene expression of BaxmRNA, FasmRNA,FasLmRNA and reducing Bcl-2mRNA to adjust the related protein expression.Conclusion: The clinical investigation showed that the stagnation of blood-stasisand heat was the main syndrome of this disease.“Blood stasis”played an important role inthe incidence of the disease. So clearing heat、dispelling stasis、hemostasis and regulating menstruation were the basic treatments. Experiment research proved that noninvasivemethod was more suitable to describe the clinical process of IUD inserting and thecharacteristics of contraceptive method. Synthetic uterine VSMC was the main part afterinserting IUD. There was an imbalance in the proliferation and apoptosis, GongNinggranule could start the cell signal transduction pathway by regulating apoptosis-relatedgene expression to adjust related protein expression, which could inhibit the uterineVSMC’s proliferation and promote apoptosis, and regulate the balance betweenproliferation and apoptosis, further to reverse pathological remodeling. This might be oneof the mechanisms of prevention and treatment of abnormal bleeding after inserting IUD.更多还原