川夏宁坤汤治疗痰湿瘀结型子宫腺肌病的作用机制及临床研究
发布时间：2018-08-01 19:06
【摘要】：目的及意义:子宫腺肌病(Adenomyosis,AM)是女性的常见病、多发病,临床表现如继发性痛经进行性加重、经期延长、经量过多、性交痛甚至不孕等,严重影响患者生活质量,甚至危害着患者的健康。西药及手术治疗,均存在一定局限性。中医认为AM的病机核心是血瘀,而血瘀往往不单独致病,而与其他病理产物搏结相兼而病,痰湿即是其中之一,痰湿瘀结也成为导致AM的一种重要病机而备受关注。本研究以化痰消ve为法制定方剂"川夏宁坤汤",从宏观整体研究其对子宫腺肌病患者症状及证候的疗效。从微观分子生物学角度,本研究以子宫腺肌病ICR模型小鼠为研究对象,以Survivin、VEGF、Caveolin-1为研究靶点,观察模型小鼠经"川夏宁坤汤"治疗后的三组指标表达的差异,从不同角度探讨川夏宁坤汤治疗子宫腺肌病(痰湿瘀结型)的作用机制,并验证其疗效。研究方法1.实验研究本研究以小鼠模型为研究对象,在分子蛋白水平检测Survivin、VEGF、Caveolin-1含量。实验分为造模与动物实验两大部分,M时间持续长达6个月。1.1研究方法1.1.1造模:将72只新生2天雌性ICR小鼠随机分为9组:模型组1、2组,空白组1、2组,中成药组(散结镇痛组)、西药组(米非司酮组)、中药高剂量组、中药中剂量组、中药低剂量组,其中模型组、空白组均为2组,每组8只。除空白组以外,其他各组均以他莫昔芬与花生油、卵磷脂、炼乳制成混合液,给予滴灌处理,连续4天,喂养至3个月,使用组织病理方法观察肌层是否出现内膜浸润,以明确造模是否成功,并以免疫组化法检测模型组、空白组中Survivin、VEGF表达差异,以辅证造模成功与否。1.1.2动物实验:空白组和模型组小鼠均使用生理盐水灌胃。中药组采取"川夏宁坤汤"颗粒剂(配制成高、中、低剂量)分布灌胃,西药组、中成药组分布采用米非司酮、散结镇痛胶囊配制溶液灌胃。灌胃周期3个月,3个月后处死小鼠,通过免疫组化、RT-PCR方法检测不同组间Survivin、VEGF、Caveolin-1表达的差异。以探究:子宫腺肌病模型小鼠病灶子宫内CAV-1表达与正常小鼠子宫是否存在差异;不同剂量的川夏宁坤汤、米非司酮、散结镇痛胶囊灌胃后,不同组间Survivin、VEGF、Caveolin-1在小鼠子宫表达的差异。2.临床研究以"川夏宁坤汤"作为试验组,以临床常用治疗子宫腺肌病药物散结镇痛胶囊作为对照药物,将痰湿瘀结型AM患者随机分组进行观察,以明确"川夏宁坤汤"颗粒的临床疗效。2.1研究方法2.1.1通过试验组与对照组的比较分析,对"川夏宁坤汤"颗粒减轻痰湿瘀结型子宫腺肌病痛经程度、降低持续时间、减少月经血量疗效进行评价。2.1.2通过试验组与对照组的比较分析,对"川夏宁坤汤"颗粒改善痰湿瘀结型子宫腺肌病患者的中医证候积分、血清CA125、子宫体积等方面疗效进行评价。结果1.实验研究1.1造模结果:经他莫昔芬滴灌4天,并正常饲养至3个月后,处死空白组1组、模型组1组,经病理组织切片观察,可见异位至肌层腺体细胞增生。1.2实验结果:1.2.1子宫腺肌病小鼠病灶子宫内,Survivin呈高表达;经中药(高剂量组)治疗,其表达降低,但中、低浓度组灌胃后表达无明显下降。1.2.2子宫腺肌病小鼠病灶子宫内,VEGF呈高表达;且经中药(高、中、低剂量组)灌胃后,表达均降低,且无明显差异(P0.5)。1.2.3子宫腺肌病小鼠病灶子宫内,Caveolin-1呈高表达。经中药(高、中、低剂量)灌胃后,表达降低,且不同浓度组间存在差异(P0.5)。2.临床研究2.1经3个月治疗,试验组与对照组痛经NRS评分较治疗前下降(P0.5),试验组与对照组疗后比较,试验组NRS评分明显低于对照组(P0.5),表明"川夏宁坤汤"颗粒能明显改善痛经程度,且疗效优于散结镇痛胶囊。2.2经3个月治疗,试验组与对照组COX量表评分较治疗前下降(P0.5),试验组与对照组疗后比较,试验组痛经严重程度评分明显低于对照组(P0.5),表明"川夏宁坤汤"颗粒能明显改善痛经程度,且疗效优于散结镇痛胶囊,与NRS评分一致;试验组与对照组疗后比较,二者症状持续总时间积分无明显差异(P0.5)。表明"川夏宁坤汤"颗粒能有效地改善痛经持续时间及程度,在缓解症状程度方面疗效优于散结镇痛胶囊。2.3经3个月治疗,试验组与对照组中医证侯积分较疗前均有明显降低(P0.5),两组疗后积分比较无明显差异(P0.5)。表明"川夏宁坤汤"与散结镇痛胶囊在治疗子宫腺肌病痰湿瘀结型证候积分方面均有明显疗效,疗效相近。2.4经3个月治疗,试验组与对照组在治疗前后子宫体积大小无明显变化(P0.5),表明"川夏宁坤汤"颗粒在改善子宫体积大小方面无明显作用。2.5经3个月治疗,试验组与对照组在治疗前后血清CA125值无明显下降,表明"川夏宁坤汤"颗粒对降低子宫腺肌病患者血清CA125值方面无明显作用。2.6经3个月治疗,试验组与对照组治疗后月经量MBL评分较前均有明显下降(P0.5)。治疗后与对照组比较,试验组改善月经量、降低MBL评分方面疗效更显著。表明"川夏宁坤汤"在改善子宫腺肌病患者月经量方面疗效优于散结镇痛胶囊。2.7经3个月治疗,试验组有效率84.84%,对照组有效率62.96%,试验组有效率高于对照组。表明"川夏宁坤汤"治疗子宫腺肌病总有效率高于散结镇痛胶囊。结论1.实验研究1.1新生ICR小鼠滴灌他莫昔芬混合液造成子宫腺肌病小鼠模型方法可行。1.2在子宫腺肌病模型小鼠病灶子宫内,Survivin、VEGF均呈现高表达状态;经中药"川夏宁坤汤"不同浓度灌胃3个月后,VEGF表达明显降低,Survivin表达仅在使用高剂量中药灌胃后有所降低。表明"川夏宁坤汤"可能通过抑制新生血管生成、抑制细胞过度增殖而对子宫腺肌病起到治疗作用。1.3实验证实,在子宫腺肌病模型小鼠病灶子宫内Caveolin-1呈高表达。在中药"川夏宁坤汤"不同浓度组,Caveolin-1表达有明显差异。2.临床研究2.1 "川夏宁坤汤"颗粒具有明显改善子宫腺肌病患者痛经症状程度、痛经程度、持续时间的作用,可有效改善月经量及中医证候。2.2 "川夏宁坤汤"颗粒对子宫腺肌病患者子宫体积大小及血清CA125降低无明显疗效。
[Abstract]:Objective and significance: Adenomyosis (AM) is a common and frequently occurring disease of women. The clinical manifestations of secondary dysmenorrhea, such as secondary dysmenorrhea, prolonged menstrual period, excessive menstruation, sexual intercourse and even infertility, seriously affect the quality of life of the patients and even harm the health of the patients. There are certain limitations in western medicine and surgical treatment. Chinese medicine thinks AM The core of the pathogenesis is blood stasis, and the blood stasis often does not cause disease alone, but it is one of the other pathological products. Phlegm dampness is one of them. The phlegm and blood stasis also becomes an important pathogenesis of AM. This study takes the phlegm elimination ve as the prescription of "Sichuan Xia Ningkun soup", and studies the patients with adenomyosis from the macro overall. From the microscopic molecular biology point of view, this study took the ICR model mice of adenomyosis as the research object, with Survivin, VEGF, Caveolin-1 as the research target, observed the difference of the three groups of expression in the model mice after "Chuan Xia Ning Kun soup" treatment, and discussed the treatment of uterine adenomyosis (phlegm dampness) from different angles. The mechanism of the effect of blood stasis type), and to verify its effect. Study methods 1. experimental study in mice model, the molecular protein level of Survivin, VEGF, Caveolin-1 content. The experiment was divided into two parts of model and animal experiment, M time lasted for 6 months.1.1 research method 1.1.1 model: 72 newborn 2 days female ICR was small Rats were randomly divided into 9 groups: Group 1,2 of model group, group 1,2 of blank group, Chinese patent medicine group (dissection analgesic group), western medicine group (mifepristone group), high dose group of traditional Chinese medicine, middle dose group of traditional Chinese medicine, low dose group of traditional Chinese medicine, in which the model group and blank group were all 2 groups with 8 rats in each group. The other groups were made from tamoxifen with peanut oil, lecithin and condensed milk. The mixture was treated with drip irrigation for 4 days, feeding for 3 months, and using histopathology to observe the infiltration of the intima in the myometrium to determine whether the model was successful, and the model group was detected by immunohistochemistry. The difference of Survivin and VEGF expression in the blank group was supplemented by.1.1.2 animal experiment: blank group and model group. The traditional Chinese medicine group took "Chuan Xia Ning Kun soup" granules (prepared into high, medium, low dose) distribution of stomach, the western medicine group, the Chinese patent medicine group distributed using mifepristone, Sanjie analgesic capsule preparation solution gavage. 3 months after the gastric perfusion, 3 months after the death of mice, through immunohistochemical, RT-PCR method to detect Survivin, VEGF, Ca among different groups The difference between the expression of veolin-1 and the difference in the uterus CAV-1 expression in the uterine adenomyosis model mice and the normal mice uterus, the difference between the different doses of the Chuan Xia Ning Kun soup, mifepristone, the Shan Jie analgesic capsule, the difference between the different groups of Survivin, VEGF, Caveolin-1 in the uterus of the mice,.2. clinical study of "Chuan Xia Ning Kun soup" "As the experimental group, the clinical treatment of AM patients with phlegm dampness and stasis type was randomly divided into two groups to observe the clinical curative effect of" Chuan Xia Ning Kun Decoction "(Chuan Xia Ning Kun Decoction) on the clinical therapeutic effect of" Chuan Xia Ning Kun Decoction "and" Chuan Xia Ning Kun Decoction "to reduce the phlegm and dampness and blood stasis through the comparison and analysis between the experimental group and the control group, 2.1.1. The degree of dysmenorrhea, reducing duration and reducing the curative effect of menstrual blood volume of the patients with adenomyosis,.2.1.2 was evaluated through comparison and analysis between the experimental group and the control group. The curative effect of "Chuan Xia Ning Kun Decoction" on the improvement of TCM syndrome score, serum CA125, and the volume of uterus was evaluated. Results 1. experimental research was carried out. Study 1.1 model results: after 4 days of tamoxifen drip irrigation and normal feeding to 3 months after 3 months, 1 groups were killed in the blank group and 1 groups in the model group. The results of.1.2 test of the ectopic and myometrium gland cell proliferation were observed by pathological tissue section. The expression of Survivin was high in the uterus of 1.2.1 uterine adenomyosis of mice, and the expression decreased after the treatment of Chinese medicine (high dose group). In the low concentration group, the expression of VEGF was highly expressed in the endometriosis of.1.2.2 uterine adenomyosis, and the expression decreased after gavage, and there was no significant difference (P0.5) in the uterine adenomyosis of mice (P0.5) in the uterine adenomyosis of mice and the high expression of Caveolin-1. After 3 months, the NRS score of dysmenorrhea decreased (P0.5) in the experimental group and the control group compared with the control group (P0.5). The NRS score of the experimental group was significantly lower than that of the control group (P0.5), which showed that the "Chuan Xia Ning Kun Tang" granule could obviously improve the degree of dysmenorrhea and the curative effect in the experimental group and the control group. The results showed that the effect of "Chuan Xia Ning Kun Tang" can obviously improve the degree of dysmenorrhea, and the effect of the clinical study of the experimental group and the control group was significantly lower than that of the control group (P0.5). After 3 months of treatment, the score of.2.2 in the experimental group and the control group was lower than that before the treatment (P0.5). Compared with the control group, the score of the severity of dysmenorrhea in the experimental group was significantly lower than that of the control group (P0.5), indicating that the "Chuan Xia Ning Kun soup" granule could obviously improve the degree of dysmenorrhea, and the curative effect was better than that of the scattered analgesic capsule and NRS. The scores of the test group and the control group were compared with the control group, and the total time integral of the symptoms of the two patients had no significant difference (P0.5). The results showed that "Chuan Xia Ning Kun Tang" granule could effectively improve the duration and degree of dysmenorrhea, and the curative effect in alleviating the symptom degree was better than that of the Sanjie analgesic capsule for 3 months. The score of the TCM syndrome in the experimental group and the control group was more than that of the control group. Before treatment (P0.5), there was no significant difference between the two groups after treatment (P0.5). It showed that "Chuan Xia Ning Kun soup" and Sanjie analgesic capsule had obvious curative effect in the treatment of syndrome scores of phlegm dampness and stasis syndrome of adenomyosis. The curative effect was similar to that of.2.4 after 3 months, and the size of uterus in the test group and the control group had no obvious changes before and after the treatment. (P0.5) the results showed that "Chuan Xia Ning Kun soup" granules had no obvious effect on the size of the uterus for 3 months. The serum CA125 value of the test group and the control group did not decrease significantly before and after the treatment. It showed that the "Sichuan Xia Ningkun soup" granule had no significant effect on the CA125 value of the patients with uterine adenomyosis for 3 months, and the experimental group was not treated with.2.6. After treatment, the MBL score of the control group was significantly lower than that before the treatment (P0.5). Compared with the control group, the effect of the experimental group on improving the menstrual volume and reducing the MBL score was more significant. The effect of "Chuan Xia Ning Kun Decoction" in improving the menstrual volume of the patients with uterine adenomyosis was better than that of the Sanjie analgesic capsule.2.7 for 3 months, and the effective rate of the experimental group was 84.84. %, the effective rate of the control group was 62.96%, the effective rate of the experimental group was higher than that of the control group. It showed that the total effective rate of "Chuan Xia Ning Kun soup" in the treatment of adenomyosis was higher than that of the Sanjie analgesic capsule. Conclusion 1. experimental study on the mouse model of adenomyosis caused by the mixed solution of tamoxifen in 1.1 newborn ICR mice was feasible in the uterine adenomyosis model mice. The expression of Survivin and VEGF both showed high expression, and the expression of VEGF was obviously reduced after 3 months of gavage of Chinese traditional Chinese medicine "Chuan Xia Ning Kun soup", and the expression of Survivin was reduced only after the use of high dose of traditional Chinese medicine. It showed that "Chuan Xia Ningkun soup" may be treated by inhibiting the formation of neovascularization and inhibiting the excessive proliferation of cells. The.1.3 test in the uterine adenomyosis model mice showed a high expression of Caveolin-1 in the uterine adenomyosis model mice. The expression of Caveolin-1 in the different concentration groups of "Chuan Xia Ning Kun soup" in the Chinese medicine was significantly different from the.2. clinical study 2.1 "Chuan Xia Ning Kun Tang" granules which obviously improved the degree of dysmenorrhea, the degree of dysmenorrhea, and the duration of the duration of the uterine adenomyosis. 2.2 "Chuanxia Ningkun Decoction" Granule has no obvious effect on reducing uterine volume and serum CA125 in patients with adenomyosis.
【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R271.9
[Abstract]:Objective and significance: Adenomyosis (AM) is a common and frequently occurring disease of women. The clinical manifestations of secondary dysmenorrhea, such as secondary dysmenorrhea, prolonged menstrual period, excessive menstruation, sexual intercourse and even infertility, seriously affect the quality of life of the patients and even harm the health of the patients. There are certain limitations in western medicine and surgical treatment. Chinese medicine thinks AM The core of the pathogenesis is blood stasis, and the blood stasis often does not cause disease alone, but it is one of the other pathological products. Phlegm dampness is one of them. The phlegm and blood stasis also becomes an important pathogenesis of AM. This study takes the phlegm elimination ve as the prescription of "Sichuan Xia Ningkun soup", and studies the patients with adenomyosis from the macro overall. From the microscopic molecular biology point of view, this study took the ICR model mice of adenomyosis as the research object, with Survivin, VEGF, Caveolin-1 as the research target, observed the difference of the three groups of expression in the model mice after "Chuan Xia Ning Kun soup" treatment, and discussed the treatment of uterine adenomyosis (phlegm dampness) from different angles. The mechanism of the effect of blood stasis type), and to verify its effect. Study methods 1. experimental study in mice model, the molecular protein level of Survivin, VEGF, Caveolin-1 content. The experiment was divided into two parts of model and animal experiment, M time lasted for 6 months.1.1 research method 1.1.1 model: 72 newborn 2 days female ICR was small Rats were randomly divided into 9 groups: Group 1,2 of model group, group 1,2 of blank group, Chinese patent medicine group (dissection analgesic group), western medicine group (mifepristone group), high dose group of traditional Chinese medicine, middle dose group of traditional Chinese medicine, low dose group of traditional Chinese medicine, in which the model group and blank group were all 2 groups with 8 rats in each group. The other groups were made from tamoxifen with peanut oil, lecithin and condensed milk. The mixture was treated with drip irrigation for 4 days, feeding for 3 months, and using histopathology to observe the infiltration of the intima in the myometrium to determine whether the model was successful, and the model group was detected by immunohistochemistry. The difference of Survivin and VEGF expression in the blank group was supplemented by.1.1.2 animal experiment: blank group and model group. The traditional Chinese medicine group took "Chuan Xia Ning Kun soup" granules (prepared into high, medium, low dose) distribution of stomach, the western medicine group, the Chinese patent medicine group distributed using mifepristone, Sanjie analgesic capsule preparation solution gavage. 3 months after the gastric perfusion, 3 months after the death of mice, through immunohistochemical, RT-PCR method to detect Survivin, VEGF, Ca among different groups The difference between the expression of veolin-1 and the difference in the uterus CAV-1 expression in the uterine adenomyosis model mice and the normal mice uterus, the difference between the different doses of the Chuan Xia Ning Kun soup, mifepristone, the Shan Jie analgesic capsule, the difference between the different groups of Survivin, VEGF, Caveolin-1 in the uterus of the mice,.2. clinical study of "Chuan Xia Ning Kun soup" "As the experimental group, the clinical treatment of AM patients with phlegm dampness and stasis type was randomly divided into two groups to observe the clinical curative effect of" Chuan Xia Ning Kun Decoction "(Chuan Xia Ning Kun Decoction) on the clinical therapeutic effect of" Chuan Xia Ning Kun Decoction "and" Chuan Xia Ning Kun Decoction "to reduce the phlegm and dampness and blood stasis through the comparison and analysis between the experimental group and the control group, 2.1.1. The degree of dysmenorrhea, reducing duration and reducing the curative effect of menstrual blood volume of the patients with adenomyosis,.2.1.2 was evaluated through comparison and analysis between the experimental group and the control group. The curative effect of "Chuan Xia Ning Kun Decoction" on the improvement of TCM syndrome score, serum CA125, and the volume of uterus was evaluated. Results 1. experimental research was carried out. Study 1.1 model results: after 4 days of tamoxifen drip irrigation and normal feeding to 3 months after 3 months, 1 groups were killed in the blank group and 1 groups in the model group. The results of.1.2 test of the ectopic and myometrium gland cell proliferation were observed by pathological tissue section. The expression of Survivin was high in the uterus of 1.2.1 uterine adenomyosis of mice, and the expression decreased after the treatment of Chinese medicine (high dose group). In the low concentration group, the expression of VEGF was highly expressed in the endometriosis of.1.2.2 uterine adenomyosis, and the expression decreased after gavage, and there was no significant difference (P0.5) in the uterine adenomyosis of mice (P0.5) in the uterine adenomyosis of mice and the high expression of Caveolin-1. After 3 months, the NRS score of dysmenorrhea decreased (P0.5) in the experimental group and the control group compared with the control group (P0.5). The NRS score of the experimental group was significantly lower than that of the control group (P0.5), which showed that the "Chuan Xia Ning Kun Tang" granule could obviously improve the degree of dysmenorrhea and the curative effect in the experimental group and the control group. The results showed that the effect of "Chuan Xia Ning Kun Tang" can obviously improve the degree of dysmenorrhea, and the effect of the clinical study of the experimental group and the control group was significantly lower than that of the control group (P0.5). After 3 months of treatment, the score of.2.2 in the experimental group and the control group was lower than that before the treatment (P0.5). Compared with the control group, the score of the severity of dysmenorrhea in the experimental group was significantly lower than that of the control group (P0.5), indicating that the "Chuan Xia Ning Kun soup" granule could obviously improve the degree of dysmenorrhea, and the curative effect was better than that of the scattered analgesic capsule and NRS. The scores of the test group and the control group were compared with the control group, and the total time integral of the symptoms of the two patients had no significant difference (P0.5). The results showed that "Chuan Xia Ning Kun Tang" granule could effectively improve the duration and degree of dysmenorrhea, and the curative effect in alleviating the symptom degree was better than that of the Sanjie analgesic capsule for 3 months. The score of the TCM syndrome in the experimental group and the control group was more than that of the control group. Before treatment (P0.5), there was no significant difference between the two groups after treatment (P0.5). It showed that "Chuan Xia Ning Kun soup" and Sanjie analgesic capsule had obvious curative effect in the treatment of syndrome scores of phlegm dampness and stasis syndrome of adenomyosis. The curative effect was similar to that of.2.4 after 3 months, and the size of uterus in the test group and the control group had no obvious changes before and after the treatment. (P0.5) the results showed that "Chuan Xia Ning Kun soup" granules had no obvious effect on the size of the uterus for 3 months. The serum CA125 value of the test group and the control group did not decrease significantly before and after the treatment. It showed that the "Sichuan Xia Ningkun soup" granule had no significant effect on the CA125 value of the patients with uterine adenomyosis for 3 months, and the experimental group was not treated with.2.6. After treatment, the MBL score of the control group was significantly lower than that before the treatment (P0.5). Compared with the control group, the effect of the experimental group on improving the menstrual volume and reducing the MBL score was more significant. The effect of "Chuan Xia Ning Kun Decoction" in improving the menstrual volume of the patients with uterine adenomyosis was better than that of the Sanjie analgesic capsule.2.7 for 3 months, and the effective rate of the experimental group was 84.84. %, the effective rate of the control group was 62.96%, the effective rate of the experimental group was higher than that of the control group. It showed that the total effective rate of "Chuan Xia Ning Kun soup" in the treatment of adenomyosis was higher than that of the Sanjie analgesic capsule. Conclusion 1. experimental study on the mouse model of adenomyosis caused by the mixed solution of tamoxifen in 1.1 newborn ICR mice was feasible in the uterine adenomyosis model mice. The expression of Survivin and VEGF both showed high expression, and the expression of VEGF was obviously reduced after 3 months of gavage of Chinese traditional Chinese medicine "Chuan Xia Ning Kun soup", and the expression of Survivin was reduced only after the use of high dose of traditional Chinese medicine. It showed that "Chuan Xia Ningkun soup" may be treated by inhibiting the formation of neovascularization and inhibiting the excessive proliferation of cells. The.1.3 test in the uterine adenomyosis model mice showed a high expression of Caveolin-1 in the uterine adenomyosis model mice. The expression of Caveolin-1 in the different concentration groups of "Chuan Xia Ning Kun soup" in the Chinese medicine was significantly different from the.2. clinical study 2.1 "Chuan Xia Ning Kun Tang" granules which obviously improved the degree of dysmenorrhea, the degree of dysmenorrhea, and the duration of the duration of the uterine adenomyosis. 2.2 "Chuanxia Ningkun Decoction" Granule has no obvious effect on reducing uterine volume and serum CA125 in patients with adenomyosis.
【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R271.9
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