蒲灵化瘀止痛方调控子宫腺肌病小鼠病灶血管生成的雌激素效应靶点的研究

【作者】 付先芸；

【导师】 魏绍斌；

【作者基本信息】 成都中医药大学 ， 中医妇科， 2013， 博士

【摘要】 目的：以雌激素效应相关因子、血管生成相关因子及其相互作用为靶点,探讨在AM小鼠疾病发生发展的不同时间段及子宫在位及异位内膜不同空间部位,疾病的主导机制,蒲灵化瘀止痛方治疗AM的作用机制,不同治疗剂量的横向对照,以及与现有中西医治疗方法的纵向比较。方法：对8周龄ICR雌鼠施行雄鼠垂体宫腔内移植手术,术后分别饲养3月及6月,形成AM小鼠模型3月组及6月组。选择导师行气化瘀法临床验方(蒲灵化瘀止痛方)为实验药物,分为蒲灵化瘀止痛方高剂量组、中剂量组及低剂量组,同时将丹莪妇康煎膏作为中药对照组、孕三烯酮组为西药对照组、未造模同期小鼠为正常组。各组分别给药3月。运用免疫组化技术观察雌激素效应相关因子(p450、COX-2、ERa)及血管生成相关因子(VEGF、ENS、CD31、MMP-2、TIMP)在各模型组、各用药组随疾病发生发展的不同时间段及子宫在位及异位内膜不同空间部位的变化。结果：1.P450在位内膜：在位内膜p450在形成AM模型后随病程进展逐渐升高,6月组显著高于3月组及正常组(P<0.01)。经治疗后,各组p450值均显著下降,造模6月后的模型组治疗后下降显著显著高于3月模型组治疗后。孕三烯酮组显著低于丹莪妇康煎膏组治疗组(P<0.05),中药的剂量对p450的影响无显著统计学差异。异位内膜：模型组异位内膜p450值显著高于正常组(P<0.01)。治疗后均显著下降,各组间比较无显著统计学差异。在疾病不同时间段(3月、6月),异位内膜P450始终显著高于在位内膜(P<0.01)。2.ERa在位内膜：形成AM模型后3月,在位内膜ERa稍低于正常组,形成模型后6月,ERa显著高于3月模型组(P<0.05)。3月模型组治疗前后ERa值无明显变化,6月模型组治疗后有显著下降(P<0.01)。与6月模型组比较,除蒲方中低剂量治疗组外,各治疗组均显著下降(P<0.05)异位内膜：形成AM模型后6月,异位内膜ERa显著高于正常组(P<0.01)。3月模型组治疗前后ERa值无明显变化。与6月模型组比较,各组治疗后下降显著(P<0.01),高剂量组下降显著优于中、低剂量组。3.COX-2在位内膜：形成AM模型后随病程进展逐渐升高,6月组高于3月组及正常组(P<0.01)。3月模型组治疗前后COX-2值无明显变化,与6月模型组比较,各组治疗后均显著下降,中药的高剂量组对COX-2的影响显著优于中、低剂量组(P<0.01)、(P=0.054)。异位内膜：异位内膜COX-2值6月组显著高于3月组(P<0.01)。异位内膜3月模型组治疗前后COX-2值无明显变化,与6月模型组比较,各组治疗后均显著下降,中药的高剂量组对COX-2的影响显著优于中、低剂量组(P<0.01)、(P=0.054)。4.CD31在位内膜：形成模型后,各时间段在位内膜CD31与正常组比较均显著下降(P<0.05),CD313月模型组治疗后显著升高(P<0.01),6月模型组与3月模型组比较无显著差异；高剂量组显著高于其他各治疗组。异位内膜：各组治疗后均显著下降(P<0.01),各组间无统计学差异。而异位内膜CD31均显著高于同期在位内膜。5.VEGF在位内膜：与正常组比较,在位内膜VEGF在形成模型后显著上调,因子的显著变化均发生于形成模型后3月组,6月组与3月组比较,无显著差异。VEGF在各治疗组后均呈显著下降趋势(P<0.01),各组间无明显差异。异位内膜：异位内膜显著下调(P<0.01)。因子的显著变化均发生于形成模型后3月组,6月组与3月组比较,无显著差异。3月组模型治疗后优于6月组治疗后。高剂量组显著优于低剂量组(P<0.05)。6.MMP-2及TIMP在位内膜：与正常组比较,形成AM模型后在位内膜MMP-2均显著升高(P<0.05)。在位内膜MMP-2、MMP-2/TIMP,3月模型组治疗前后显著下降(P<0.05),显著优于6月模型组治疗后。与6月模型组比较,治疗后各组均显著下降,组间比较无显著差异。异位内膜：形成AM模型后6月,异位内膜MMP-2显著高于3月组异位内膜值(P<0.05)。与6月模型组比较各治疗组均显著下降(P<0.01),各组间无显著差异；MMP-2/TIMP蒲方高剂量组组及孕三烯酮组均显著下降(P<0.01),高剂量组显著优于低剂量组(P<0.05)。形成模型后,在位内膜MMP-2值上调显著高于同期异位内膜(P<0.01)。模型后3月MMP-2/TIMP比值在位内膜显著高于异位内膜(P<0.01)。结论：1.本实验研究认为,雌激素效应因子异常、血管生成相关因子异常及其相互作用,促成了AM的发生和发展,但在疾病的不同阶段,其主导机制有所不同。疾病早期，血管生成相关因子为疾病的主导方面：在位内膜发生的微血管密度下降,局部发生缺血缺氧,从而诱发快速反应因子COX-2的急剧增高,触发COX-—PGE2—P450ar om正反馈环路,导致p450在位内膜及异位内膜随着病程进展逐渐升高。P450的增高,使局部雌激素水平增高,早期因主要通过膜受体机制,在位内膜核受体ERa并未增高。疾病中晚期，雌激素效应相关因子成为疾病的主导方面：雌激素可通过雌激素受体调节机体的多种信号途径,上调VEGF,导致异常血管增生。在位内膜及异位ERa均显著增高,雌激素主要通过核受体机制产生效应。2.蒲灵化瘀止痛方可多靶点治疗AM,其治疗靶点可因疾病的发展阶段而不同。病程早期的中药治疗主要以血管生成相关因子为靶点,改善早期在位内膜的缺血缺氧状况,直接截断由此触发的雌激素生成的正反馈链。疾病的后阶段,血管相关因子已不再是疾病的主要环节,雌激素效应的变化主导了疾病的发展,此时,中药治疗靶点主要是在位及异位内膜雌激素效应相关因子,通过雌激素效应的变化影响血管生成因子的变化。3.蒲灵化瘀止痛方剂量对在位内膜的CD31、VEGF、COX-2、MMP-2、MMP-2/TIMMP的影响正相关,而对雌激素效应其他因子,如P450、ERa,无显著相关性。因此,可在疾病早期运用大剂量行气活血类中药,有效改善在位内膜微循环,阻断疾病的早期触发机制；在疾病的中晚期,可以持续中低剂量使用行气活血类药物,改善雌激素效应异常,在保证治疗疗效的同时减少用药剂量,从而减少药物毒副作用,降低费用,提高患者的依从性。4.孕三烯酮不仅有效降低了异位内膜局部雌激素及ERa水平,同时也显著降低了在位内膜的雌激素及ERa水平。与西药治疗相比,中药治疗可以避免在位内膜雌激素水平的下调。更多还原

【Abstract】 Objective:take Estrogen effect correlator and angiogenesis correlator as target point, investigate the mean mechanism of the disease in different time and in different space of the uterus endomembrane; approach the mechanism of Chinese medicinal prescription of promoting qi to activate blood; compare the distinction between different therapeutic(al) does of Chinese medicinal formulae、and between the chinese medical science and Western medicineMethods:carry out an operation to transplant the Male mice pituitary gland into the uterine cavity of the female mice in order to construct the AM mice model. breed the mice for3and6months to form the the model of3months and which of6months. select clinical effective medical prescription (Chinese medicinal prescription of promoting qi to activate blood) as the experimental medicine, divide into high dose group、medium dose group and low-dose group, meanwhile take soft extract of DANEFUKANG as the control group of traditional Chinese medicine, take Gestrinone as the control group of western medicine, take mice without operation as blank group, give different drugs for3months in each group. then observe the change of Estrogen effect correlator (p450、COX-2、Era) and angiogenesis correlator(VEGF、 ENS、CD31、IMMP-2、TIMMP) by immunohistochemisty technique in different time and in different space of the uterus endomembrane.Results:1. p450:in the uterus endomembrane, the the value of p450gradually increase after the construction of mice model of AM, the group of3months is slightly higher the the blank group (P=0.258),the group of6months is exceed the group of3months (P=0.006); in dystopia endomembrane, the the value of p450in the group of6months notable higher than the group of3months. In the deferent period of the diease, the value of p450in dystopia endomembrane is dramatics higher than in the uterus endomembranThe value of p450is dramatics decrease after treatment in each group no matter in uterus endomembrane or in dystopia endomembrane. in uterus endomembrane, the value is significant lower in Gestrinone group than in soft extract of DAFK (P=0.038), slightly lower than which in high dose group. However in dystopia endomembrane,,there is no statistics difference between each group.after treatmentm, the value of p450decrease in each group contrast to the model group in uterus endomembrane, which is more higher in6months group (P=0.007) than3months group. in dystopia endomembrane, the value of p450is notable decrease in each group after treatment. The drease is dramatics in6months group (P=0.001), and in3months group as well (P=0.012), the decrease is more higher in6months group than in3months group.there is no statistics difference between different dose group wether i n uterus endomembrane or i n dystopia endomembrane2. COX-2:in the uterus endomembrane, the value of COX-2gradually increase after the construction of mice model of AM, the group of3months is slightly higher the the blank group (P=0.212),the group of6months is exceed the group of3months (P<0.001). in dystopia endomembrane, the the value of p450is slightly higher than in the uterus endomembrane, there is not statistically significant between the groups.in dystopia endomembrane,, the value of COX-2is notable higher in the group of6months than in the group of3months (P=0.001) The value of COX-2is dramatics decrease after treatment in each group no matter in uterus endomembrane or in dystopia endomembrane. in dystopia endomembrane, the value is significant lower in Gestrinone group than in high dose group (P=0.052) after treatment, in uterus endomembrane, the value of COX-2has no notable change in3months group (P=0.992), however, there is a dramatics decrease in6months group.in dystopia endomembrane, the value of COX-2has no notable change in3months group after treatment(P=0.677), however, there is a dramatics decrease in6months group. wether in uterus endomembrane or in dystopia endomembrane, after treatment, the value of COX-2decrease dramatics in high dose group than in medium、low dose group (P=0.005)(P=0.054)。3. ERa:in the uterus endomembrane, the ERa in3months is slightly lower than the blank group (P=0.470), in the6months group, the value of ERa is significant exceed the value in the group of3months (P=0.029). in dystopia endomembrane, the value in the group of6months is slightly than which in group of3months (P=0.107)。In the deferent period of the diease, the value of p450in dystopia endomembrane is dramatics higher than in the uterus endomembraneAfter treatment, the value of Era is decrease wether in uterus endomembrane or in dystopia endomembrane. in uterus endomembrane, the value is slightly lower in Gestrinone group than which in high dose group (P=0.236, P=0.309), there is no statistics difference between the two groups, there is no statistics difference between each group in dystopia endomembrane.after treatment, in uterus endomembrane, the value of ERa has no notable change in3months group, however, there is a dramatics decrease in6months group. in dystopia endomembrane, the value of ERa has a dramatics decrease in each group after treatment, which is especially dramatics in6months group.in uterus endomembrane, after treatment, the value of ERa decrease in each group, but there is no statistics difference between different dose group. in dystopia endomembrane, there is a dramatics decrease in high dose group than in medium、low dose group after treatment。4. CD31与VEGFMVD CD31is significant decrease in group of3months contrast to the group of blank group (P=0.006), after6months of the operation, CD31is slightly increase, but still significant lower than blank group (P=0.011)In the deferent period of the diease, the value of p450in dystopia endomembrane is dramatics higher than in the uterus endomembrane raise up not statistically significant significant exceed;to surpassThe value of CD31is increase after treatment in each group in dystopia endomembrane. i n dystopia endomembrane, the value is significant higher in high dose group than in Gestrinone group (P<0.001) and in soft extract of DAFK group (P<0.001). in uterus endomembrane, there is no statistics difference between each group. The value of VEGF and CD31decrease in each group after treatment in dystopia endomembrane, there is no statistics difference between he chinese medical science and Western medicine. after treatment, in uterus endomembrane, the value of VEGF dramatics decrease in each model group (P<0.001); in3months group, the value of CD31has a dramatics increase in3months group (P<0.001), however, there is no significant change in6months group (P=0.945)。. in dystopia endomembrane, the value of VEGF is significant decrease wether in3months group (P=0.018) or in6months group (P=0.102); the value of CD31has downtrend wether in3months group(P=0.238) or in6months group (P=0.087)i n uterus endomembrane, after treatment, the value of VEGF decrease in each group, but there is no statistics difference between different dose group. the value of CD31significant increase (P<0.001) after treatment which is higher in high dose group than in medium and low dose group (P<0.001) in dystopia endomembrane, the value of VEGF has a downtrend in each dose group, which is more lower in high dose group than in low dose group. The value of CD31has a downtrend in each dose group, but there is no statistics difference between different dose group.5. MMP-2、MMP-2/TIMP:in the uterus endomembrane, the value MMP-2significant increase after the construction of mice model of AM, the group of3months is higher the the blank group (P=0.012),the group of6months is exceed the group of3months (P=0.698). in dystopia endomembrane, the value in the group of6months is slightly than which in group of3months (P=0.027)。 after the operation, the value of MMP-2in each group is notable increase wether in the uterus endomembrane or in the dystopia endomembrane contrast to the blank group, which is more higher in the uterus endomembrane than in the dystopia endomembrane. the value of MMP-2/TIMP is higher in the uterus endomembrane than in the dystopia endomembrane, especially in the group of3months.Contrast to the mice model group, the value of MMP/TIMMP decrease in each group wether in uterus endomembrane or in dystopia endomembrane. Which in Gestrinone group is slightly lower than the other two group, however, there is no statistics difference between the three groups. Contrast to the mice model group, the value of MMP-2significant decrease in each group wether in uterus endomembrane or in dystopia endomembrane, the value is significant lower in Gestrinone group than in high dose group (0.001) and in soft extract of DAFK group (0.042) in dystopia endomembrane. after treatment, in uterus endomembrane, the value of MMP-2、MMP-2/TIMMP, has no notable change in6months group, however, there is a dramatics decrease in3months group. in dystopia endomembrane, there is no statistics difference after treatment in each model group.in uterus endomembrane, after treatment, the value of MMP-2decrease in group (P=0.041), which is lower in high dose group than in medium and low dose gmnp, but there is no statistics difference between different dose group. in dystopia endomembrane, there is a dramatics decrease in each dose group (P=0.002, P=0.001, P=0.001), but there is no statistics difference between different dose group. The value of MMP-2/TIMMP decrease dramatics in high dose group than in medium、low dose group after treatment。Conclusions:1. It can be presumed that the abnormality of Estrogen effect correlator angiogenesis correlator and the interaction of the two correlators bring about the development the AM, in defferent phase of the disease, the main mechanism is different.In early phase, angiogenesis correlator is the main mechanism to manage the disease. In uterus endomembrane, the descend of the MVD lead to the ischemia and anoxia in local position, and then induce the pullulation of the fast reaction factor COX-2, triggering the positive feed back loop of COX-2-PGE2-P450arom, induce the value of p450gradually increase with the progress the disease. The increase of p450lead to the raise of the value of estrogen, because of the degenerative, there is a slightly decrease of the value of ERa in uterus endomembrane.I n advanced stage, Estrogen effect correlator is the main mechanism to manage the disease. Estrogen can up-regulation the express of VEGF by receptor regulation, induce the disorder Vascular proliferation. The sensitivity of receptor descend by the continuously stimulation of the high concentration of the estrogen, which is tolerance of the estrogen. Then the level of the receptor compensatory increase, the level of ERa increase wether in uterus endomembrane or in dystopia endomembrane.2. Chinese medicinal prescription of promoting qi to activate blood treat AM by multitarget. The target of the treatment can change along with the different stage of the disease. In early phase, angiogenesis correlator is sthe main target. The Chinese medicine can improve the situation of the anoxemia, interrupt the positive feedback link directly. In advanced stage, the angiogenesis correlator is not the main mechanism any more, Estrogen effect correlator manages the progress of the disease. At the time, the main target of the Chinese medicine is Estrogen effect correlator, the change of Estrogen effect correlator conduct the change of angiogenesis correlator.3. The dose of the Chinese medicine is positive correlate to the express of CD31、VEGF、COX-2、MMP-2、MMP-2/TIMMP in uterus endomembrane, which has no notable correlation to the Estrogen effect correlator,such as p450、ERa. It can be presumed that using high dose of Chinese medicinal prescription of promoting qi to activate blood in early stage of the disease impeoves the microcirculation effectively, interrupt the early trigger mechanism. In adevanced stage of the disease, continuely using low-media dose Chinese medicine change the abnormity of estrogen effect, guarantee the healing,effect,, at the same time, reduce the dose of the medicine, responently reduce the toxic and side-effect, decrease the cost of the patient.4. Gestrinone decrease the the level of ERa in not only ectopia endomembrane, but also in uterus endomembrane. Compared to the western medicine, Chinese medicine avoids the side effect of the low estrogen symptom effectly更多还原