上皮间质细胞转化及其介导的纤维化与子宫腺肌症的相关性研究

发布时间：2018-05-18 15:20

本文选题：子宫腺肌症 + TAM　；参考：《复旦大学》2014年博士论文

【摘要】：子宫腺肌病(Adenomyosis, ADM)作为子宫内膜异位症(Endometriosis, EMs)的“内在性”表现,是子宫内膜侵入子宫肌层的一种良性病变。’ADM最常发生在子宫后壁,临床主要表现为经量增多和经期延长、进行性痛经和性交痛、性欲减退等,约35%的患者无任何临床症状或仅感到轻微的不适。核磁共振(MRI)近年来作为确诊腺肌病的检查手段开始逐渐被应用于临床,而在MRI之前,确诊ADM的唯一办法是子宫切除后的病理检查。这样往往延误了患者的求诊,导致就诊时大多病情或症状已进展较重,最终只能以全子宫切除为结局,因此对育龄期妇女的伤害尤为巨大。ADM的病因至今不清楚。目前多数研究者认为ADM是基底层内膜细胞增生、侵入到肌层间质的结果。而关于引起内膜基底层细胞侵入肌层的因素尚不清楚。上皮-间质细胞转化(epithelial-mesenchymal transition, EMT)是指上皮细胞在特定的生理和病理情况下向间质细胞转分化的现象。在EMT过程中,上皮细胞失去细胞极性,丧失细胞间紧密连接和粘附连接,获得了浸润性和游走迁移能力,变成了具有间质细胞功能和特性的细胞。EMT在肿瘤中赋予细胞迁移、浸润的能力。而ADM的内膜细胞的侵入过程与这一过程非常相似,因此本课题通过研究ADM发生发展中相关因子,探讨EMT与ADM的关系。第一部分他莫昔芬诱导的ICR小鼠腺肌症模型目的利用他莫昔芬的诱导作用,对ICR小鼠进行ADM的建模。方法购入10只ICR孕鼠(孕周17-19周),将新生小鼠的雌性小鼠随机平均分为建模组和对照组。出生当天算第0天第1天至第5天给予TAM(浓度200μg/mL)诱导,按5μL/g的剂量直接口服给予建模组新生小鼠,对照组予相同剂量的空白溶剂。建模组和对照组再分别随机分为5组,分别在第5天、第10天、第15天、第42天和第60天以10%水合氯醛麻醉,心脏灌注处死,取小鼠Y型子宫,保存在4%多聚甲醛里。石蜡包埋后,横截面切片保存。同时,小鼠断奶后(21天时)与母鼠分隔开,在第28天、第42天和第56天分别行热板实验。将热板仪设定温度为55℃,待温度达到后将小鼠置于热板仪的热板上面,开始计时,以小鼠开始舔舐后脚掌或从热板上跳起为结束。这段时间视为小鼠的热板实验的时间。结果HE染色观察,建模组5天时未见异位内膜侵入小鼠子宫肌层；10天时,有2/5例侵入小鼠子宫肌层；15天时,有3/5例侵入肌层；42天时,全部建模组小鼠子宫均可见腺体侵入肌层；60天时,侵入肌层的腺体较大,个数较多。对照组未见异位内膜。对照组热板实验有轻微的下降,建模组每组时间均低于对照组,且呈现明显的下降趋势,与对照组有明显差别。结论TAM口服给药诱导了ICR小鼠的ADM形成。42天时,建模成功率为100%。建模过程中ICR小鼠建模组的痛觉耐受力也有下降。第二部分上皮-间质细胞转化在腺肌症发生发展过程中的作用目的探讨EMT相关因子在ADM形成过程中的表达,以及EMT在此过程中的作用。方法利用免疫组化方法对ADM小鼠子宫及ADM患者病灶标本进行检测。主要检测EMT的相关因子E-cadherin、Vimentin,以及EMT的TGF-β/Smad3通路中的TGF-β、p-Snad3、Six1的表达,通过对比表达量的变化,探讨ADM发生发展过程中的EMT的作用。结果ADM建模组小鼠的E-cadherin在建模过程中表达降低。相同天数的建模组与对照组有显著差异(p0.05),建模组的不同时间的5组间也有显著差异(p0.05)。在ADM患者的免疫组化染色中观察到,在子宫肌层中可见异位腺上皮细胞,与对照组的在位腺上皮的E-cadherin免疫组化染色相比明显变浅。两组间有显著差异(p0.05)。ADM建模组小鼠的Vimentin在建模过程中在异位内膜的表达升高。相同天数的建模组与对照组有显著差异(p0.05),建模组的不同时间的5组间也有显著差异(p0.05)。而ADM患者的Vimentin表达于异位内膜,与对照组的在位腺上皮相比有显著差异(p0.05)。ADM建模组小鼠的TGF-β在建模过程中在异位内膜的表达升高。相同天数的建模组与对照组有显著差异(p0.05),建模组的不同时间的5组间也有显著差异(p0.05)。而ADM患者的TGF-p表达于异位内膜,与对照组的在位腺上皮相比有显著差异(p0.05)。ADM建模组小鼠的Six1在建模过程中在异位内膜胞核内的表达升高。相同天数的建模组与对照组有显著差异(p0.05),建模组的不同时间的5组间也有显著差异(p0.05)。而ADM患者的Sixl表达于异位内膜胞核,与对照组的在位腺上皮相比有显著差异(p0.05)。ADM建模组小鼠的p-Smad3在建模过程中在异位内膜胞核内的表达升高。相同天数的建模组与对照组有显著差异(p0.05),建模组的不同时间的5组间也有显著差异(p0.05)。而ADM患者的p-Smad3表达于异位内膜胞核,与对照组的在位腺上皮相比有显著差异(p0.05)。结论ADM形成过程中涉及EMT的发生,并与Six1调控的TGF-β/Smad3通路相关。第三部分EMT导致的纤维化在腺肌症发生发展中的作用目的通过特殊染色及纤维化相关因子的检测,观察在EMT导致的ADM中是否存在纤维化的发生。方法利用Masson染色及苦味酸-天狼星红染色对ADM建模成功的小鼠子宫及ADM异位病灶进行染色,利用纤维对染料的特殊感应,检测ADM中是否有胶原纤维的生成。同时用免疫组化的方法对ADM小鼠子宫及ADM异位病灶进行a-SMA 及 Collagen I表达量的检测,探讨ADM中纤维化的可能。结果Masson染色：在ADM建模组小鼠中,42和60天组小鼠子宫整个间质部分可见蓝色纤维染色弥漫性分布,腺体及肌层呈现红色；ADM患者切片病灶处可见大片红色肌肉纤维,之间夹杂红色腺体及蓝色纤维。苦味酸-天狼星红染色：普通光学显微镜下观察,ADM小鼠子宫腺上皮及肌层呈黄色,42天及60天的建模组小鼠间质内弥漫分布红色纤维状染色；ADM患者腺体与肌纤维均为黄色,肌纤维之间及腺体周围有红色染色,400倍镜下观察可见纤维状。偏振光显微镜下观察,光镜下红色纤维部分可见红黄绿三色胶原纤维交织分布,红黄两色光较强,纤维较粗,绿色光弱,纤维较细。ADM小鼠近病灶处折光较强,红黄光较多；ADM患者异位内膜周围折光强,红黄光多于绿光。ADM建模组小鼠的Collagen I在建模过程中在异位内膜的表达升高。相同天数的建模组与对照组有显著差异(p0.05),建模组的不同时间的5组间也有显著差异(p0.05)。而ADM患者的Collagen I表达于异位内膜及其周围间质内,与对照组的在位腺上皮相比有显著差异(p0.05)。ADM建模组小鼠的α-SMA除了子宫肌层的表达,建模过程中在异位内膜的表达升高。相同天数的建模组与对照组有显著差异(p0.05),建模组的不同时间的5组间也有显著差异(p0.05)。而ADM患者除了肌层外,其a-SMA表达于异位内膜及其周围间质内,与对照组的在位腺上皮相比有显著差异(p0.05)。结论在ADM的发生发展过程中产生了胶原纤维,可能与EMT相关,对ADM病程的进展有着重要作用。
[Abstract]:Adenomyosis (ADM), as an "intrinsic" manifestation of Endometriosis (EMs), is a benign lesion of the endometrium intruding into the myometrium of the uterus. 'ADM most often occurs in the posterior wall of the uterus. The main clinical manifestation is the increase of the uterus and the prolonged menstruation, progressive dysmenorrhea, sexual pain, loss of sexuality, and so on, about 35% of the patients. There is no clinical symptoms or slight discomfort. MRI has been gradually applied to the clinic as an examination of adenomyosis in recent years, and before MRI, the only way to diagnose ADM is the pathological examination after hysterectomy. This often delays the patient's diagnosis and causes most of the illness or symptoms to come in. However, the cause of.ADM is not clear. Most researchers believe that ADM is the result of endometrial hyperplasia in the basal layer and intruding into the intermuscular layer. The factors causing the invasion of the intima to the basal layer of the intima are not clear. Epithelial-mesenchymal transition (EMT) refers to the transdifferentiation of epithelial cells to interstitial cells in a specific physiological and pathological condition. In the process of EMT, the epithelial cells lose the polarity of cells, lose the close connection and adhesion of cells, and obtain the ability of impregnation and migration, and become interstitial cells. Function and characteristic cell.EMT endow cells with the ability to migrate and infiltrate in the tumor. And the invasion process of ADM endometrium cells is very similar to this process. Therefore, the relationship between EMT and ADM is explored by studying the related factors in the development of ADM. The first part of tamoxifen induced ICR mouse adenopathy model was used to make use of him. The inducement of celecoxifene was used to model the ADM in ICR mice. Methods 10 ICR pregnant mice were purchased (17-19 weeks of pregnancy). The female mice were randomly divided into modeling group and control group. The birth day was given from first days to fifth days from zeroth days to fifth days (200 mu g/mL), and the model group was given a direct oral administration of 5 mu L/g to the new mice. The control group was given the same dose of blank solvent. The model group and the control group were randomly divided into 5 groups, respectively at fifth days, tenth days, fifteenth days, forty-second and sixtieth days with chloral anaesthesia with 10% hydrate and heart perfusion, the mice Y uterus was taken and preserved in the paraformaldehyde. After paraffin embedded, the cross section was preserved. At the same time, the mice were weaned (21 days). At twenty-eighth days, forty-second days and fifty-sixth days, the hot plate was set at 55 degrees, and the mice were placed on the hot plate of the hot plate after the temperature was reached, and the time was started. The mice began licking the feet or jumping from the hot plate to the end. This period was considered as the time of the hot plate experiment of the mice. Results HE No ectopic endometrium intruded into the myometrium of the mouse uterus at 5 days, and at the 10 day, 2/5 cases intruded into the myometrium of the mice; on the 15 day, 3/5 intruded into the myometrium; at the time of 42 days, all the mice in the modeling group showed the glands of the glands into the myometrium; at the 60 day, the glands of the intruded layers were larger and more. The control group did not see the ectopic endometrium. There was a slight decrease in the heat plate test in the group, and the modeling group was lower than the control group, and showed a significant downward trend, and there was a significant difference with the control group. Conclusion TAM oral administration induced the ADM formation of ICR mice for.42 days, the success rate of modeling was also decreased in the 100%. modeling module of ICR mice in the process of 100%. modeling. The role of the transformation of skin mesenchymal cells in the development of adenopathy and to explore the expression of EMT related factors in the process of ADM formation and the role of EMT in this process. Methods the immunohistochemical method was used to detect the lesions of the ADM mice uterus and ADM patients. The main detection of EMT related factors E-cadherin, Vimentin, and EM. The expression of TGF- beta, p-Snad3, Six1 in T's TGF- beta /Smad3 pathway, through contrast expression changes, to explore the role of EMT in the developing process of ADM. The expression of E-cadherin in the ADM modeling group was reduced during the modeling process. The modeling group of the same days had significant difference between the control group and the control group (P0.05), and the 5 groups at different time of the modeling group were also between the same days. There were significant differences (P0.05). Ectopic epithelial cells were observed in the myometrium of the uterine myometrium in the immunohistochemical staining of the ADM patients, which was significantly lighter than the E-cadherin immunohistochemical staining in the control group. There was a significant difference between the two groups (P0.05) the expression of Vimentin in the ectopic endometrium in the modeling process of the.ADM model group. There was significant difference between the modeling group and the control group (P0.05), and there was significant difference between the 5 groups at different time in the modeling group (P0.05). The Vimentin of the ADM patients was expressed in the ectopic endometrium, which was significantly different from the control group. (P0.05) the expression of TGF- beta in the ectopic endometrium in the modeling group of.ADM modeling group in the modeling process. There was a significant difference between the modeling group and the control group (P0.05), and the 5 groups in the modeling group had significant differences (P0.05). The TGF-p of the ADM patients was expressed in the ectopic endometrium and was significantly different from that of the control group (P0.05) the Six1 in the.ADM modeling group was in the ectopic endocardium during the modeling process. There was significant difference between the model group and the control group (P0.05), and there was a significant difference between the 5 groups at different time in the modeling group (P0.05). The Sixl of the ADM patients was expressed in the ectopic endometrium, and there was a significant difference from the control group in the eutopic gland epithelium (P0.05) the p-Smad3 in the.ADM modeling group was in the ectopic endometrium during the modeling process. There was a significant difference in the expression of the nucleus in the nucleus of the same day (P0.05), and there was a significant difference between the 5 groups at different time in the modeling group (P0.05). The p-Smad3 of the ADM patients was expressed in the ectopic endometrium, and compared with the control group, there was a significant difference (P0.05). Conclusion the formation of ADM involved the occurrence of EMT. Related to the TGF- beta /Smad3 pathway regulated by Six1. Third part of the role of EMT induced fibrosis in the development of adenopathy. Objective To observe the presence of fibrosis in ADM induced by EMT by specific staining and fibrosis related factors. Methods Masson staining and picric acid and Sirius red staining were used to model ADM. The mouse uterus and ADM ectopic focus were stained, and the formation of collagen fibers in ADM was detected by the special induction of fiber on the dye. At the same time, the expression of a-SMA and Collagen I in ADM mice uterus and ADM ectopic focus was detected by immunohistochemical method, and the possibility of fibrosis in ADM was discussed. Results Masson staining: ADM. In the mice of the model group, the whole interstitial part of the uterus in the 42 and 60 days group showed a diffuse distribution of blue fiber, and the glands and muscularis were red. The lesions of the ADM patients showed large red muscle fibers, with red glands and blue fibers. Picric acid - Sirius red staining: common optical microscope observation, ADM mice The epithelium of the uterus and the myometrium were yellow. The interstitial tissue of the mice in the modeling group of 42 days and 60 days was diffuse red fibrous staining. The glands and the muscle fibers of the ADM patients were all yellow, red between the muscle fibers and around the glands, and the fiber was observed under the 400 times of the microscope. Under the polarizing microscope, the red fiber was partly red yellow under the light microscope. The green tricolor collagen fiber is interwoven, the red and yellow two color light is stronger, the fiber is thicker and the green light is weak. The near focus of the.ADM mice is stronger, and the red and yellow light is more. The ADM patients have strong refraction around the ectopic endometrium, and the red and yellow light is more than the green light.ADM modeling group. The expression of Collagen I in the ectopic endometrium is elevated in the process of building the model. The same days are the same. There was significant difference between the modeling group and the control group (P0.05), and there was a significant difference between the 5 groups at different time in the modeling group (P0.05). The Collagen I of the ADM patients was expressed in the ectopic endometrium and its surrounding interstitium, and was significantly different from that of the control group (P0.05) the alpha -SMA in the.ADM modeling group was modeled after the expression of the myometrium of the uterus. The expression of the ectopic endometrium increased in the process. The modeling group with the same days was significantly different from the control group (P0.05), and there were significant differences between the 5 groups at different time in the modeling group (P0.05). But the a-SMA expression in the ectopic endometrium and the surrounding interstitial tissue except the myometrium of the ADM patients was significantly different from that of the reigned gland epithelium (P0.05). On the occurrence and development of ADM, collagen fibers are produced, which may be related to EMT and play an important role in the progression of ADM.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R711.74

【相似文献】