【作者】 王占利；

【导师】 李坤寅；

【作者基本信息】 广州中医药大学 ， 中医妇科学（专业学位）， 2012， 博士

【摘要】 目的：1.通过对症状性子宫肌瘤患者的临床资料进行采集、归纳、整理,以分析子宫肌瘤患者的中医证候、证型、饮食习惯等特点,以期指导临床,为临床辨证治疗提供依据。2.根据子宫肌瘤为性激素依赖性肿瘤的发病机理,对长期和短期两种雌、孕激素联合造模法进行比较,探索子宫肌瘤的动物模型制造方法,为进一步实验提供依据；并对大鼠血液粘度进行检测,分析造模后大鼠血液流变学的改变。3.采用橘荔散结丸剂和汤剂和对雌孕激素负荷子宫肌瘤模型大鼠进行干预,并设米非司酮为西药对照,桂枝茯苓胶丸为中药对照,观察对大鼠子宫的大体形态学、全血粘度、血清白细胞介素6(IL-6)及肿瘤坏死因子α(TNF-α)水平和子宫肌层组织雌激素受体(ER)、孕激素受体(PR)及表皮生长因子受体(EGFR)的影响,探讨橘荔散结丸治疗子宫肌瘤的作用机理。方法：1.收集2010年1月～2012年2月期间在广州中医药大学第一附属医院妇科住院及妇科门诊诊断为子宫肌瘤的患者。通过采集患者一般资料及四诊资料,分析归纳其年龄、病程、饮食习惯、中医证型分布等及常见症状的发生率。2.将48只健康雌性SD大鼠随机分为短期对照组、短期模型组、长期对照组、长期模型组4组。短期模型组每天腹腔注射苯甲酸雌二醇注射液,0.05mg/100g,每鈤1次,连续25d后改为腹腔注射黄体酮注射液0.4mg/100g,每日1次,连续5d；短期对照组每天腹腔注射等体积高压灭菌花生油,连续30d。长期模型组隔日肌注苯甲酸雌二醇注射液0.05mg/100g,3次/周,12周后加肌注黄体酮注射液0.5mg/100g,2次/周,连续4周；长期对照组于同时间肌注等体积高压灭菌花生油。3.将100只健康雌性SD大鼠随机分为10组,每组10只,分别为：空白组(A组)、模型对照组(B组)、西药对照组(C组)、中药对照组(D组)、丸剂低剂量组(E组)、丸剂中剂量组(F组)、丸剂高剂量组(G组)、汤剂低剂量组(H组)、汤剂中剂量组(Ⅰ组)、汤剂高剂量组(J组)。除空白组外其余各组均隔日肌注苯甲酸雌二醇注射液0.05mg/100g,3次/周,12周后加肌注黄体酮注射液0.5mg/100g,2次/周,连续4周；空白组同时间肌注等体积高压灭菌花生油。从造模开始第5周起,除正常对照组和模型对照组灌服等量生理盐水外,其余各组灌服相应药物至第16周末。结果：1.本研究共纳入子宫肌瘤患者505例,平均年龄(37.66±7.12)岁。其中>36-≤40岁发病率较高,其次为>30-≤35岁与>40-≤45岁,30-50岁发病者共79.41%,占总病例数的绝大多数；而25岁以下者有8例,占1.58%,说明子宫肌瘤的发病有逐渐年轻化趋势。对178例子宫肌瘤患者饮食情况调查结果表明,其中有肉食习惯者占多数,占53.37%,而素食习惯者占比例较少,共4.49%。经统计学分析,各饮食习惯构成问有统计学意义(P<0.05)。下腹包块、经量增多为子宫肌瘤最常见症状,发生率分别为77.62%、33.66%；舌淡黯,苔薄白,脉弦细为子宫肌瘤患者最常见舌脉,分别占30.10%、61.39%、32.08%；气滞血瘀证为子宫肌瘤最常见证型,占总例数的45.5%,其次为痰湿瘀结证和肾虚血瘀证,分别占17.4%、14.3%；患者不同年龄、孕次、产次、饮食习惯间六种证型分布有显著性差异(P<0.05或P<0.01),而病程、人流次数证型分布无明显差异。2.经长期和短期两种双模法造模后,长期模型组及短期模型组大鼠子宫体积均有增大,子宫系数、肌层厚度显著增加,以长期模型组增加明显,长期模型组与长期对照组相比,差别有统计学意义(P<0.05或P<0.01)；HE染色观察显示长期模型组子宫平滑肌排列紊乱明显,部分呈栅栏状改变。长期模型组及短期模型组大鼠卵巢系数均显著降低(P<0.05或P<0.01)。长期造模后大鼠血液粘稠度高切、中切、低切均较对照组显著降低,差异有统计学意义(P<0.05或P<0.01)；短期模型组虽较对照组血液粘稠度降低,但差异无统计学意义(P>0.05)。短期模型组、长期模型组血浆粘度均有所增高,与对照组对比,差别均无统计学意义(P>0.05)。3.橘荔散结丸对子宫肌瘤模型大鼠子宫形态学的影响：造模后,模型对照组大鼠子宫体积明显增大,分角下横径及竖径、左右分角横径、子宫系数、镜下子宫平滑肌厚度均显著增加,差异有统计学意义(P<0.05或P<0.01),模型对照组大鼠卵巢系数明显下降(P<0.05)。进行药物干预后,各组大鼠子宫体积明显减小,各子宫指标及子宫系数均有所下降,卵巢系数上升。西药对照组大鼠各子宫指标下降明显,但卵巢系数上升不明显。中药对照组各子宫指标有所下降,橘荔散结丸剂中剂量组及汤剂低、中、高剂量组各子宫指标明显下降,其中以汤剂中剂量组子宫系数下降明显,与模型对照组相比,差异有统计学意义(P<0.05),中药对照组、丸剂中剂量组、汤剂高剂量组卵巢系数升高显著,与模型对照组相比,差异有统计学意义(P<0.01或P<0.05)。各组子宫平滑肌厚度明显下降,西药对照组、橘荔散结丸剂中剂量组及各汤剂组与模型对照组相比,差异显著(P<0.01或P<0.05)。4.橘荔散结丸对子宫肌瘤模型大鼠血液流变学的影响：模型对照组全血粘度(高、中、低切)均较空白组下降,差异极显著(P<0.01)；而血浆粘度上升,与空白组相比,差异无统计学意义(P>0.05)。药物干预后,各组血液粘度均有不同程度的改变,以橘荔散结丸剂及各汤剂治疗组变化明显,其中橘荔散结丸汤剂中剂量组全血粘度明显升高,汤剂低剂量组血浆粘度显著下降,与模型对照组相比,差异有统计学意义(P<0.01或P<0.05),而西药对照组及中药对照组全血粘度的改善不明显。5.橘荔散结丸对子宫肌瘤大鼠模型血清IL-6的影响：模型对照组血清工L-6明显下降,与空白组相比,差异有统计学意义(P<0.05)；用药治疗后除西药对照组外各组血清IL-6均有所上升,橘荔散结丸剂及各汤剂治疗组血清IL-6升高明显,其中汤剂低剂量组及汤剂中剂量组与模型对照组相比,差异显著(P<0.05)。6.橘荔散结丸对子宫肌瘤大鼠模型血清TNF-α的影响：模型对照组血清TNF-α明显升高,与空白对照组相比,差异极显著(P<0.01)；药物干预后,各组大鼠血清TNF-α明显降低,与模型对照组相比,差异极显著(P<0.01),以橘荔散结丸剂、汤剂各剂量组下降明显。7.橘荔散结丸对子宫肌瘤模型大鼠子宫肌层ER的影响：模型对照组与空白组相比,子宫肌层ER表达明显升高,差异有显著统计学意义(P<0.05)；汤剂中剂量组子宫肌层ER表达明显下降,与模型对照组相比,差异显著(P<0.05)；西药对照组、中药对照组、丸剂中剂量组、汤剂低剂量组、汤剂高剂量组ER表达较模型对照组均有所下降,与模型对照组相比,差异无统计学意义(P>0.05)8.橘荔散结丸对子宫肌瘤模型大鼠子宫肌层PR的影响：模型对照组子宫肌层PR表达明显升高,与空白组相比,差异有显著统计学意义(P<0.05)；药物干预后,各组子宫肌层PR表达明显下降,西药对照组及汤剂中剂量组与模型对照组相比,差异显著(P<0.05),中药对照组、丸剂中剂量组、汤剂低剂量组及汤剂高剂量组子宫肌层PR表达水平较模型对照组均有所下降,但与模型对照组相比,差异无统计学意义(P>0.05)。9.橘荔散结丸对子宫肌瘤模型大鼠子宫肌层EGFR的影响：模型对照组EGFR表达明显高于空白组,差异有显著统计学意义(P<0.05)；药物干预后,各组大鼠子宫肌层EGFR表达明显下降,其中以汤剂中剂量组下降显著,与模型对照组相比,差异有统计学意义(P<0.05)；西药对照组、汤剂高剂量组较中药对照组、丸剂中剂量组表达降低,但各组与模型对照组相比,差异无统计学意义(P>0.05)。结论：1.症状性子宫肌瘤患者36-40岁发病率较高。嗜食肉食可能损伤脾肾,导致痰湿阻滞,肾虚血瘀,增加子宫肌瘤发生的危险性。下腹包块、经量增多为症状性子宫肌瘤最常见症状；舌淡黯,苔薄白,脉弦细为子宫肌瘤患者最常见舌脉；气滞血瘀证为子宫肌瘤最常见证型,其次为肾虚血瘀证、痰湿瘀结证,且随着年龄段的增加、孕产次的增多,子宫肌瘤的肾虚血瘀证逐渐增多。2.采用雌、孕激素长期双模法,可成功造成SD大鼠子宫肌瘤模型,可作为子宫肌瘤动物实验的理想模型。3.橘荔散结丸及汤剂可以显著抑制子宫肌瘤模型大鼠子宫平滑肌增生,促进子宫肌瘤模型大鼠卵巢恢复,其综合效果优于米非司酮和桂枝茯苓胶囊,证实橘荔散结丸具有治疗子宫肌瘤的作用。其作用机理可能是通过降低子宫肌层局部ER、PR、EGFR的表达,抑制子宫平滑肌的增生；调节机体免疫功能,抑制炎症反应,增强机体杀伤肿瘤细胞的作用,并能促进血细胞的生成,改善贫血及血瘀状态。更多还原

【Abstract】 Objective:1. The clinical symptoms in patients with symptomatic uterine leiomyomas (UL) were collected and organized, then to analyze the distributive regulation of TCM, the syndrome influential factors and the eating habits on UL, so as to guide the clinical application, establish foundation of diagnostic standardization on syndromes and Chinese medicine treatment.2. Based on the pathogenesis of uterine leiomyoma that were hormone dependent tumors, to compare the diffierence between the two constructing methods rat model with uterine leiomyoma, and to explore the method of the rat model of uterine leiomyoma, to provide the basis for further experiments; and to test the blood viscosity and analyze the hemorheological of the model rats.3. To study the effect of Juli Sanjie Pills and Decoction on the rat model of UL induced by estrogen and progesterone, then to observe the general morphology of the rats’uterus, the viscosity of whole blood, inerleukin-6(IL-6) and tumor necrosis factor-α (TNF-α) in the rats’serum, the expression of estrogen receptor (ER), progesterone receptor (PR) and epidermic growth factor receptor (EGFR). And to explore the treatment mechanisms of Juli Sanjie Pills on uterine leiomyoma.Methods:1. This study collected the basic information and the four diagnostic data through the observation of the in-patients and out-patients from the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine from January2010to February2012, then analyzed and summarized the distribution of each factors:their age, duration, syndrome distribution, eating habits and the incidence of the common symptoms.2.48healthy female SD rats were randomly divided into short-term control group, short-term model group, long-term control group and long-term model group. The rats of short-term model group were given a daily intraperitoneal injection of estradiol benzoate injection with the dose of0.05mg/100g, and after25days changed for progesterone injection (0.4mg/100g) once daily for5days; The rats of short-term control group were given a daily intraperitoneal injection of peanut oil for30days. The rats of long-term model group were given an intramuscular injection of estradiol benzoate injection with the dose of0.05mg/100g,3times/week.12weeks later, they were given together progesterone injection0.5mg/100g,2times/week for4weeks. The rats of long-term control group were gave an intramuscular injection of peanut oil in the same time.3.100healthy female SD rats were randomly divided into10groups (n=10):group A, group B, group C, group D, group E, group F, group G, group H, group I, group J. In addition to group A, the rats were given an intramuscular injection of estradiol benzoate injection0.05mg/100g,3times/week.12weeks later, they were given together progesterone injection0.5mg/100g,2times/week for4weeks. The rats of group A were given an intramuscular injection of peanut oil in the same time. From the fifth weeks, except the rats of group A and group B, the rest rats were fed with corresponding drugs until the16th weeks.Results:1.505cases with final diagnosis of uterine leiomyoma had been collected, whose average age was37.66±7.12.>36-≤40years old have a higher prevalence, followed by>30-≤35years old and>40-≤45years old;30-50years old in a total incidence of79.41%. The number of under the age of25was8, with the incidence of1.58%, which explained that uterine leiomyoma had young tendency. The result of dietary survey of178cases with uterine leiomyoma showed that most patients addicted to meat, accounted for53.37%, while the patients with vegetarian habits accounted for less proportion, a total of4.49%. The difference of diet composition have statistical significance(P<0.05). Abdominal mass and large volume of menses were the common symptoms, whose occurrence rate respectively was77.62%and33.66%respectively. The common tongue proper, the tongue coating and pulse was dark pale tongue, thin and white tongue coating and string and thin pulse, accounting for30.10%,61.39%,32.08%respectively. The most common syndrome was syndrome of Qi stagnation and blood stasis, whice accounted for45.5%of the total cases, and followed by syndrome of phlegm and blood stasis and blood stasis and kidney deficiency, accounted for17.4%and14.3%respectively. The correlation between patients’age, gravidity, parity, dietary habits and syndromes had significant difference (P<0.05or P<0.01). And there was no correlation for patients’course of disease, the number of abortion and syndrome distribution.2. The uterus volume, uterus coefficient and uterine muscle’s thickness of rats from short-term model group and long-term model group increased after long-time and short-time modeled, and the long-term model group increased significantly. The difference between the long-term model group and the long-term control group was statistically significant (P<0.05or P<0.01). HE staining showed the the uterine muscles were disorde, some looked like palisade. The ovarians’coefficient of rats from short-term model group and long-term model group was lowered significantly (P<0.05or P<0.01). The whole blood viscosity at high shear, middle shear and lower shear of the rats from long-term model group was decreased significantly compared with the long-term control group. The whole blood viscosity was reduced too, but there was no difference between the short-term control group (P>0.05). The rat’plasma viscosity was increased in short-term model group and long-term model group, but there was no difference compared with the short-term control group and the long-term control group (P>0.05).3. Uterine morphology:The uterus vertical diameter and transverse diameter over the cervical, the width of left cornua uteri, the width of right cornua uteri, uterus coefficient, uterine muscle’s thickness of rats of group B increased significantly (P<0.05or P<0.01). And the ovarians’coefficient of rats from group B decreased significantly (P<0.05). After treated by medicine, the uterus coefficient, uterus volume and quota decreased, and the ovarians’coefficient increased. The uterus quota of rats from group C decreased significantly, but the ovarians’coefficient didn’t increase. The uterus quota of rats from group D decreased too, the group F, H, I, J decreased significantly, and the group I decreased most significantly; the difference was significantly compared with group B (P<0.05). The ovarians’coefficient of rats from group D, F and J increased significantly compared with group B (P<0.05or P<0.01). The uterine muscle’s thickness of rats from D, F, H, I and group J increased significantly compared with group B (P<0.05or P<0.01).4. Blood viscosity:The whole blood viscosity at high shear, middle shear and lower shear of the rats from group B was decreased extremely significantly compared with group A (P<0.01). The plasma viscosity of rats from group B was increased, but there was no significant difference compared with the group A (P>0.05). After treated by medicine, The whole blood viscosity was changed. The whole blood viscosity of the rats from group I was increased significantly and the plasma viscosity of rats from group B was decreased significantly compared with group B (P<0.05or P<0.01). The change of group C and D was not significant.5. IL-6:The serum IL-6of group B was decreased significantly compared with group A (P<0.05). After treated by medicine, the serum IL-6increased except group C. The serum IL-6of group H and I was increased significantly compared with group B (P<0.05).6. TNF-a:The serum TNF-a of group B was increased extremely significantly compared with group A (P<0.01). After treated by medicine, the serum TNF-a decreased extremely significantly compared with group B (P<0.01) and group F, H, I and J decreased more significantly.7. ER:The ER expression in myometrial of group B increased significantly compared with group A (P<0.05). ER expression in myometrial of I group decreased significantly compared with group B (P<0.05). ER expression in myometrial of group C, D, F, H and J decreased too, but the difference was not significant compared with group B (P>0.05).8. PR:The PR expression in myometrial of group B increased significantly compared with group A (P<0.05). After treated by medicine, The PR expression in myometrial decreased, group C and I decreased significantly compared with group B (P<0.05). PR expression in myometrial of group D, F, H and J decreased too, but the difference was not significant compared with group B (P>0.05).9. EGFR:The EGFR expression in myometrial of group B increased significantly compared with group A (P<0.05). After treated by medicine, The EGFR expression in myometrial decreased, group I decreased significantly compared with group B (P<0.05). EGFR expression in myometrial of group C and J decreased more than group D and F, but the difference was not significant compared with group B (P>0.05).Conclution:1. High prevalence in patients with uterine leiomyoma was from36to40years old. Addicted to meat might damage the function of spleen and kidney, resulting in phlegm stasis, blood stasis and kidney deficiency, which increased the risk of uterine leiomyoma. Abdominal mass and large volume mense were the common symptoms. The common tongue proper, the tongue coating and pulse respectively was dark pale tongue, thin and white tongue coating and string and thin pulse. The most common syndrome was syndrome of Qi stagnation and blood stasis. And followed by syndrome of phlegm and blood stasis, blood stasis and kidney deficiency. With the growth of the age, the increase of the number of abortion and syndrome, the rate of blood stasis and kidney deficiency increased gradually.2. The method of Estrogen and progesterone load with long time could successfully establish rat model of uterine leiomyoma. The method could be used as the ideal model of uterine leiomyoma animal experiments.3. Juli Sanjie Pills and Decoction could significantly inhibit hyperplasia of uterine smooth muscle and promote the recovery of ovarian. The comprehensive effect of Juli Sanjie Pills and Decoction was better than mifepristone and Guizhifuling capsule and it was a good approach in treating uterine leiomyoma. The mechanism of Juli Sanjie Pills was probably by reducing ER, PR and EGFR, inhibiting hyperplasia of uterine smooth muscle, modulating the immunity, inhibitting inflammatory response, enhancing the effect of body killing tumor cells, promoting the formation of blood cells and ameliorating anemia and blood stasis state.更多还原