消症方对子宫腺肌病内膜间质细胞增殖、凋亡的影响

发布时间：2018-05-10 06:03

本文选题：消症方 + 子宫腺肌病　；参考：《广州中医药大学》2017年硕士论文

【摘要】：目的:通过采用胶原酶消化法处理子宫腺肌病在位及异位病灶子宫内膜组织,体外分离培养腺肌病在位及异位子宫内膜间质细胞,观察腺肌病内膜间质细胞的生长增殖曲线,通过将药物直接作用于细胞,采用MTT法检测药物对细胞的增殖抑制作用,显微镜下观察凋亡细胞形态,采用流式细胞术检测细胞凋亡及细胞周期,观察消症方对腺肌病在位及异位子宫内膜间质细胞增殖和凋亡的影响,阐释消症方治疗子宫腺肌病的作用机理,为临床治疗子宫腺肌病提供实验论证和理论基础,为中医采用消ve化瘀法治疗子宫腺肌病赋予新的内涵,为子宫腺肌病的保守治疗提供新的思路。方法:1.本实验研究选择2014年12月—2015年12月在广东省中医院大德路总院妇科行全子宫切除术,且术后病理诊断为子宫腺肌病的患者6例,子宫全切后,无菌操作分别取在位子宫内膜、异位病灶内膜组织,采用胶原酶消化法体外分离培养子宫腺肌病在位及异位内膜间质细胞,并采用免疫组织化学方法对细胞进行纯度鉴定。2.采用水提法制备含药培养液,并将实验分为5组:消症方高剂量组、消症方中剂量组、消症方低剂量组、孕三烯酮组以及空白对照组。通过将药物直接作用于子宫腺肌病在位及异位内膜间质细胞24h,采用MTT法观察消症方含药培养液对腺肌病细胞的增殖抑制作用,采用苏木素-伊红染色(HE染色)方法显微镜下观察消症方直接作用于细胞之后的凋亡细胞形态;同时利用流式细胞仪检测药物对细胞凋亡、细胞周期的作用。成果:1.本实验研究通过采用Ⅰ型胶原酶消化、筛网过滤等方法原代培养子宫腺肌病在位及异位子宫内膜间质细胞,取得标本共6例,最终培养成功5例。并通过选用对腺肌病间质细胞具有特异性的波形蛋白,对原代培养的腺肌病在位及异位内膜细胞进行免疫组织化学方法鉴定,由细胞鉴定可知,原代培养出的子宫腺肌病在位及异位内膜细胞中内膜间质细胞的纯度均高达99%以上。2.消症方及孕三烯酮组均可抑制子宫腺肌病在位及异位内膜间质细胞的增殖,且消症方在一定范围内能呈剂量依赖性的抑制腺肌病细胞增殖。3.消症方组及孕三烯酮组显微镜下均可观察到:部分细胞变得固缩,细胞质变得致密,细胞核染色质浓集,为凋亡细胞的形态学特征。4.消症方对子宫腺肌病在位及异位内膜间质细胞的凋亡的流式细胞术检测:药物直接作用于腺肌病细胞24h后,与空白对照组相比,消症方高、中、低剂量组及孕三烯酮组均能显著促进在位及异位内膜间质细胞凋亡(P0.05),且消症方组随着药物浓度的升高,凋亡率也随之增加。5.消症方对子宫腺肌病在位及异位内膜间质细胞的细胞周期的流式细胞术检测:消症方高、中、低剂量组及孕三烯酮组均可引起腺肌病在位及异位内膜间质细胞发生S期阻滞,且消症方组阻滞细胞周期呈一定的浓度依赖性。结论:1.消症方能有效抑制子宫腺肌病在位及异位内膜间质细胞的增殖,对子宫腺肌病在位及异位内膜间质细胞的生长有促进其凋亡的作用,并且能通过将子宫腺肌病在位及异位内膜间质细胞阻断于S期以阻断周期进程。2.消症方治疗子宫腺肌病的作用可能与其通过抑制子宫腺肌病在位及异位内膜间质细胞增殖、促进细胞凋亡、阻断周期进程有关,从细胞生物学方面解释了消症方限制子宫腺肌病发生和发展的过程,为消症方临床治疗子宫腺肌病提供了实验论证及理论基础,为临床对子宫腺肌病的治疗提供了新的思路。
[Abstract]:Objective: to treat endometriosis and ectopic endometrium of adenomyosis by collagenase digestion, in vitro and in vitro culture of adenomyosis in vitro and ectopic endometrial stromal cells, and observe the growth and proliferation of endometrial stromal cells in adenomyosis. By direct action of drugs to cells, the proliferation of cells was detected by MTT method. The morphology of apoptotic cells was observed under microscope, apoptosis and cell cycle were detected by flow cytometry. The effect of Xiaozheng prescription on the proliferation and apoptosis of endometrial stromal cells in adenomyosis and ectopic endometrium was observed. The mechanism of Xiaozheng prescription in the treatment of adenomyosis was explained, and the experimental demonstration of the clinical treatment of adenomyosis and the clinical treatment of adenomyosis were provided. The theory foundation provides new connotation for the treatment of adenomyosis by eliminating ve and removing stasis in traditional Chinese medicine. It provides a new idea for the conservative treatment of uterine adenomyosis. Methods: 1. experimental studies were selected from December 2014 to December 2015 in the Department of gynecology in the General Hospital of Guangdong Province Traditional Chinese Medical Hospital, and the pathological diagnosis of uterine adenomyosis after operation was made. In 6 patients, after total hysterectomy, the eutopic endometrium, endometrium and ectopic endometrium were isolated and cultured by collagenase digestion. The purity of the cells was identified by immunohistochemical method and.2. was prepared by the method of immunohistochemistry. The 5 groups: the high dose group of Xiaozheng Fang, the dose group of Xiaozheng Fang, the low dose group of Xiaozheng Fang, the pregnant three ketone group and the blank control group. Through the direct action of the drug on the eutopic and ectopic endometrium interstitial cells 24h of the uterine adenomyosis, the inhibitory effect of the culture solution of Xiaozheng Fang on the proliferation of adenomyosis cells was observed by the method of MTT, and the hematoxylin eosin was used. The staining (HE staining) method was used to observe the morphology of the apoptotic cells directly acting on the cells after the elimination of the disease. At the same time, the effect of drugs on cell apoptosis and cell cycle was detected by flow cytometry. The results of the 1. experimental studies were the primary culture of adenomyosis in situ and ectopia by using type I collagenase digestion and sieve filtration. A total of 6 cases of endometrial stromal cells were obtained, and 5 cases were successfully cultured. By selecting the specific vimentin for the interstitial cells of adenomyosis, the primary cultured adenomyosis was identified by immunohistochemistry. The primary and ectopic endometriosis of the primary cultured adenomyosis was identified by cell identification. The purity of endometrial stromal cells in membrane cells is more than 99%.2. and three ketones can inhibit the proliferation of endometriosis and ectopic endometrium stromal cells, and the elimination side can be observed under a certain range of dose-dependent inhibition of adenomyosis cell proliferation.3. elimination group and pregnancy three ketone group under microscope. Some cells become compact, the cytoplasm becomes dense, the nucleus chromatin is dense, and the morphological feature of the apoptotic cells is the flow cytometry of the apoptosis of the endometriosis and ectopic endometrium cells of.4.. The drug is directly acted on the 24h of the adenomyosis cells and compared with the blank control group, the high, middle, and low dose groups are compared with the blank control group. And gestation three ketone group can significantly promote the apoptosis of eutopic and ectopic endometrium stromal cells (P0.05). With the increase of drug concentration, the rate of apoptosis also increases the flow cytometry of.5. Xiao Zheng on the cell cycle of endometriosis and ectopic endometrial stromal cells: high, middle, low dose, and three ketones. The group of adenomyosis can cause S phase block in the eutopic and ectopic endometrial stromal cells, and the cell cycle of the Xiaozheng group has a certain concentration dependence. Conclusion: the 1. side can effectively inhibit the proliferation of the eutopic and ectopic endometrium stromal cells of the uterus adenomyosis, and promote the growth of the adenomyosis in the position and the ectopic endometrium cells. The effect of apoptosis, and the effect of blocking the interstitial cells of endometriosis and ectopic endometrium by blocking the interstitial cells of the endometrium in the S stage to block the periodic process of.2. elimination in the treatment of adenomyosis may be related to the inhibition of the proliferation of endometriosis and ectopic endometrium cells, the promotion of cell apoptosis and the interruption of the cycle process, from cell biology. It explains the process of restricting the occurrence and development of adenomyosis by Xiaozheng party. It provides an experimental demonstration and theoretical basis for the clinical treatment of adenomyosis by Xiaozheng party, and provides a new way of thinking for the treatment of adenomyosis in clinical.

【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R271.9

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