本文選題：子宮腺肌癥 + TAM��； 參考：《復旦大學》2014年博士論文

【摘要】：子宮腺肌病(Adenomyosis, ADM)作為子宮內(nèi)膜異位癥(Endometriosis, EMs)的“內(nèi)在性”表現(xiàn),是子宮內(nèi)膜侵入子宮肌層的一種良性病變�！疉DM最常發(fā)生在子宮后壁,臨床主要表現(xiàn)為經(jīng)量增多和經(jīng)期延長、進行性痛經(jīng)和性交痛、性欲減退等,約35%的患者無任何臨床癥狀或僅感到輕微的不適。核磁共振(MRI)近年來作為確診腺肌病的檢查手段開始逐漸被應用于臨床,而在MRI之前,確診ADM的唯一辦法是子宮切除后的病理檢查。這樣往往延誤了患者的求診,導致就診時大多病情或癥狀已進展較重,最終只能以全子宮切除為結局,因此對育齡期婦女的傷害尤為巨大。ADM的病因至今不清楚。目前多數(shù)研究者認為ADM是基底層內(nèi)膜細胞增生、侵入到肌層間質的結果。而關于引起內(nèi)膜基底層細胞侵入肌層的因素尚不清楚。上皮-間質細胞轉化(epithelial-mesenchymal transition, EMT)是指上皮細胞在特定的生理和病理情況下向間質細胞轉分化的現(xiàn)象。在EMT過程中,上皮細胞失去細胞極性,喪失細胞間緊密連接和粘附連接,獲得了浸潤性和游走遷移能力,變成了具有間質細胞功能和特性的細胞。EMT在腫瘤中賦予細胞遷移、浸潤的能力。而ADM的內(nèi)膜細胞的侵入過程與這一過程非常相似,因此本課題通過研究ADM發(fā)生發(fā)展中相關因子,探討EMT與ADM的關系。第一部分他莫昔芬誘導的ICR小鼠腺肌癥模型目的利用他莫昔芬的誘導作用,對ICR小鼠進行ADM的建模。方法 購入10只ICR孕鼠(孕周17-19周),將新生小鼠的雌性小鼠隨機平均分為建模組和對照組。出生當天算第0天第1天至第5天給予TAM(濃度200μg/mL)誘導,按5μL/g的劑量直接口服給予建模組新生小鼠,對照組予相同劑量的空白溶劑。建模組和對照組再分別隨機分為5組,分別在第5天、第10天、第15天、第42天和第60天以10%水合氯醛麻醉,心臟灌注處死,取小鼠Y型子宮,保存在4%多聚甲醛里。石蠟包埋后,橫截面切片保存。同時,小鼠斷奶后(21天時)與母鼠分隔開,在第28天、第42天和第56天分別行熱板實驗。將熱板儀設定溫度為55℃,待溫度達到后將小鼠置于熱板儀的熱板上面,開始計時,以小鼠開始舔舐后腳掌或從熱板上跳起為結束。這段時間視為小鼠的熱板實驗的時間。結果HE染色觀察,建模組5天時未見異位內(nèi)膜侵入小鼠子宮肌層；10天時,有2/5例侵入小鼠子宮肌層；15天時,有3/5例侵入肌層；42天時,全部建模組小鼠子宮均可見腺體侵入肌層；60天時,侵入肌層的腺體較大,個數(shù)較多。對照組未見異位內(nèi)膜。對照組熱板實驗有輕微的下降,建模組每組時間均低于對照組,且呈現(xiàn)明顯的下降趨勢,與對照組有明顯差別。結論TAM口服給藥誘導了ICR小鼠的ADM形成。42天時,建模成功率為100%。建模過程中ICR小鼠建模組的痛覺耐受力也有下降。第二部分上皮-間質細胞轉化在腺肌癥發(fā)生發(fā)展過程中的作用目的探討EMT相關因子在ADM形成過程中的表達,以及EMT在此過程中的作用。方法利用免疫組化方法對ADM小鼠子宮及ADM患者病灶標本進行檢測。主要檢測EMT的相關因子E-cadherin、Vimentin,以及EMT的TGF-β/Smad3通路中的TGF-β、p-Snad3、Six1的表達,通過對比表達量的變化,探討ADM發(fā)生發(fā)展過程中的EMT的作用。結果ADM建模組小鼠的E-cadherin在建模過程中表達降低。相同天數(shù)的建模組與對照組有顯著差異(p0.05),建模組的不同時間的5組間也有顯著差異(p0.05)。在ADM患者的免疫組化染色中觀察到,在子宮肌層中可見異位腺上皮細胞,與對照組的在位腺上皮的E-cadherin免疫組化染色相比明顯變淺。兩組間有顯著差異(p0.05)。ADM建模組小鼠的Vimentin在建模過程中在異位內(nèi)膜的表達升高。相同天數(shù)的建模組與對照組有顯著差異(p0.05),建模組的不同時間的5組間也有顯著差異(p0.05)。而ADM患者的Vimentin表達于異位內(nèi)膜,與對照組的在位腺上皮相比有顯著差異(p0.05)。ADM建模組小鼠的TGF-β在建模過程中在異位內(nèi)膜的表達升高。相同天數(shù)的建模組與對照組有顯著差異(p0.05),建模組的不同時間的5組間也有顯著差異(p0.05)。而ADM患者的TGF-p表達于異位內(nèi)膜,與對照組的在位腺上皮相比有顯著差異(p0.05)。ADM建模組小鼠的Six1在建模過程中在異位內(nèi)膜胞核內(nèi)的表達升高。相同天數(shù)的建模組與對照組有顯著差異(p0.05),建模組的不同時間的5組間也有顯著差異(p0.05)。而ADM患者的Sixl表達于異位內(nèi)膜胞核,與對照組的在位腺上皮相比有顯著差異(p0.05)。ADM建模組小鼠的p-Smad3在建模過程中在異位內(nèi)膜胞核內(nèi)的表達升高。相同天數(shù)的建模組與對照組有顯著差異(p0.05),建模組的不同時間的5組間也有顯著差異(p0.05)。而ADM患者的p-Smad3表達于異位內(nèi)膜胞核,與對照組的在位腺上皮相比有顯著差異(p0.05)。結論ADM形成過程中涉及EMT的發(fā)生,并與Six1調(diào)控的TGF-β/Smad3通路相關。第三部分EMT導致的纖維化在腺肌癥發(fā)生發(fā)展中的作用目的通過特殊染色及纖維化相關因子的檢測,觀察在EMT導致的ADM中是否存在纖維化的發(fā)生。方法 利用Masson染色及苦味酸-天狼星紅染色對ADM建模成功的小鼠子宮及ADM異位病灶進行染色,利用纖維對染料的特殊感應,檢測ADM中是否有膠原纖維的生成。同時用免疫組化的方法對ADM小鼠子宮及ADM異位病灶進行a-SMA 及 Collagen I表達量的檢測,探討ADM中纖維化的可能。結果Masson染色：在ADM建模組小鼠中,42和60天組小鼠子宮整個間質部分可見藍色纖維染色彌漫性分布,腺體及肌層呈現(xiàn)紅色；ADM患者切片病灶處可見大片紅色肌肉纖維,之間夾雜紅色腺體及藍色纖維�？辔端�-天狼星紅染色：普通光學顯微鏡下觀察,ADM小鼠子宮腺上皮及肌層呈黃色,42天及60天的建模組小鼠間質內(nèi)彌漫分布紅色纖維狀染色；ADM患者腺體與肌纖維均為黃色,肌纖維之間及腺體周圍有紅色染色,400倍鏡下觀察可見纖維狀。偏振光顯微鏡下觀察,光鏡下紅色纖維部分可見紅黃綠三色膠原纖維交織分布,紅黃兩色光較強,纖維較粗,綠色光弱,纖維較細。ADM小鼠近病灶處折光較強,紅黃光較多；ADM患者異位內(nèi)膜周圍折光強,紅黃光多于綠光。ADM建模組小鼠的Collagen I在建模過程中在異位內(nèi)膜的表達升高。相同天數(shù)的建模組與對照組有顯著差異(p0.05),建模組的不同時間的5組間也有顯著差異(p0.05)。而ADM患者的Collagen I表達于異位內(nèi)膜及其周圍間質內(nèi),與對照組的在位腺上皮相比有顯著差異(p0.05)。ADM建模組小鼠的α-SMA除了子宮肌層的表達,建模過程中在異位內(nèi)膜的表達升高。相同天數(shù)的建模組與對照組有顯著差異(p0.05),建模組的不同時間的5組間也有顯著差異(p0.05)。而ADM患者除了肌層外,其a-SMA表達于異位內(nèi)膜及其周圍間質內(nèi),與對照組的在位腺上皮相比有顯著差異(p0.05)。結論在ADM的發(fā)生發(fā)展過程中產(chǎn)生了膠原纖維,可能與EMT相關,對ADM病程的進展有著重要作用。
[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.
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R711.74

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