發(fā)布時間：2018-09-15 05:32

【摘要】：干細(xì)胞(stem cells，SC)是現(xiàn)代醫(yī)學(xué)中的重大發(fā)現(xiàn)，分為胚胎干細(xì)胞(embryonic stem cell，，ES)和成體干細(xì)胞(adult stem cell，ADS)，兩種細(xì)胞都能夠再生成為各種組織器官。但相對應(yīng)的各種倫理道德問題也隨之出現(xiàn)。近年來，羊水來源干細(xì)胞（amniotic fluid-derived stem cell，AFS）的發(fā)現(xiàn)及研究提供了一種既避免了倫理道德問題，又具有更強(qiáng)大的醫(yī)學(xué)應(yīng)用前景的新方法。羊水容易獲取，且獲取過程安全性高，可于產(chǎn)前診斷時經(jīng)羊膜穿刺獲得，也可于孕婦分娩時獲得，兩種獲取方法不會對胎兒造成傷害，僅對母體造成微創(chuàng)。經(jīng)實(shí)驗(yàn)測定，羊水來源干細(xì)胞同時具備胚胎干細(xì)胞和成體干細(xì)胞的部分生理特性。實(shí)驗(yàn)表明，胚胎干細(xì)胞可以在體外被誘導(dǎo)為生殖細(xì)胞，成體干細(xì)胞可以在體外被誘導(dǎo)為卵母樣細(xì)胞。本實(shí)驗(yàn)旨在對羊水來源干細(xì)胞向卵母樣細(xì)胞分化的能力進(jìn)行研究，同時探討其在細(xì)胞治療方面以及組織工程學(xué)方面治療卵巢早衰（premature ovarian failure，POF）的可能性。 目的： 1.研究某株源自孕中期羊水的羊水來源干細(xì)胞系分化成為卵母樣細(xì)胞的能力。 2.研究羊水來源干細(xì)胞對卵巢早衰的治療效果。 方法： 1.采用特定培養(yǎng)基誘導(dǎo)一株源自孕中期羊水的羊水來源干細(xì)胞系，使其向卵母樣細(xì)胞分化，然后采用Real-time RT-PCR方法檢測卵母細(xì)胞標(biāo)志基因ZP2、ZP3，采用Western blotting方法檢測透明帶蛋白ZP2和標(biāo)志性蛋白Vasa并采用免疫熒光化學(xué)的方法檢測誘導(dǎo)后羊水來源干細(xì)胞ZP2的表達(dá)。 2.將實(shí)驗(yàn)用的SD大鼠隨機(jī)分為四組：A組為正常組、B組為模型組、C組為一次移植組、D組為兩次移植組。A組采用生理鹽水灌胃，B組、C組、D組連續(xù)14天采用雷公藤多苷片懸液灌胃，D組在第1天與第7天灌胃后分別移植一次AFS，C組在第14天灌胃后移植一次AFS。最后觀察大鼠的陰道涂片、性激素水平以及卵巢的組織形態(tài)學(xué)變化。 結(jié)果： 1.羊水來源干細(xì)胞在培養(yǎng)基中生長迅速，證明了其經(jīng)加入人卵泡液的特殊培養(yǎng)基誘導(dǎo)后，可在體外誘導(dǎo)分化成為卵母樣細(xì)胞，分化的卵母樣細(xì)胞表達(dá)標(biāo)志基因ZP2、ZP3和標(biāo)志性蛋白Vasa、ZP2。 2.使用雷公藤多苷片懸液灌胃法造模后，模型組有明顯的卵巢衰竭現(xiàn)象，造模成功；經(jīng)羊水來源干細(xì)胞治療后，C組與D組卵巢衰竭的情況得到恢復(fù)，其中分階段移植比一次移植恢復(fù)的更好。D組卵巢內(nèi)均可以發(fā)現(xiàn)新生卵泡。 3.經(jīng)羊水來源干細(xì)胞治療后，四組間FSH值的差異有統(tǒng)計學(xué)意義；四組間E2值的差異也有統(tǒng)計學(xué)意義，且P均0.01。 結(jié)論： 1.羊水來源干細(xì)胞具有較強(qiáng)的增殖潛力且具有向卵母樣細(xì)胞分化的能力。 2.羊水來源干細(xì)胞會對卵巢早衰具有一定的治療作用，從促進(jìn)卵巢生成卵泡、調(diào)節(jié)性激素水平兩方面改善了卵巢功能。
[Abstract]:Stem cells (stem cells,SC) are important discoveries in modern medicine. They are divided into embryonic stem cell (embryonic stem cell,ES) and adult stem cell (adult stem cell,ADS), both of which can regenerate into various tissues and organs. However, the corresponding ethical and moral problems also appear. In recent years, the discovery and research of amniotic fluid derived stem cells (amniotic fluid-derived stem cell,AFS) have provided a new method which can avoid ethical problems and have a stronger prospect of medical application. Amniotic fluid is easy to be obtained, and the process of obtaining amniotic fluid is safe, which can be obtained by amniotic membrane puncture during prenatal diagnosis and delivery by pregnant women. The two methods of obtaining amniotic fluid are not harmful to the fetus, but only minimally invasive to the mother. Experimental results show that amniotic fluid derived stem cells have some physiological characteristics of both embryonic stem cells and adult stem cells. The results showed that embryonic stem cells could be induced into germ cells in vitro and adult stem cells could be induced into oocyte like cells in vitro. The purpose of this study was to investigate the ability of amniotic fluid derived stem cells to differentiate into oocyte like cells and to explore the possibility of the treatment of premature ovarian failure (premature ovarian failure,POF) in cell therapy and tissue engineering. Objective: 1. To study the ability of an amniotic fluid derived stem cell line derived from amniotic fluid in the second trimester to differentiate into oocyte like cells. 2. To study the therapeutic effect of amniotic fluid derived stem cells on premature ovarian failure. Methods: 1. An amniotic fluid derived stem cell line derived from amniotic fluid in the second trimester of pregnancy was induced by a specific medium to differentiate into oocyte like cells. Then Real-time RT-PCR method was used to detect the expression of the oocyte marker gene ZP2,ZP3,. The pellucida protein ZP2 and the iconic protein Vasa were detected by Western blotting method and the expression of ZP2 in amniotic fluid derived stem cells was detected by immunofluorescence method. 2. The experimental SD rats were randomly divided into four groups: group A, normal group, group B, model group, group C, group D, two times transplantation, group A, group B: normal saline, group B, group C, group D, using tripterygium wilfordii polyglycosides for 14 days. Group D received intragastric transplantation of AFS. once on day 1 and day 7 after intragastric perfusion of tablet suspension, group AFS,C was transplanted once after gastric perfusion on day 14, and AFS. was transplanted once after gastric perfusion on day 1 and day 7, respectively. Finally, the vaginal smear, sex hormone level and histological changes of ovary were observed. Results: 1. Amniotic fluid derived stem cells (amniotic fluid) grew rapidly in the medium, which proved that they could be induced to differentiate into oocyte like cells in vitro by adding the special medium to human follicular fluid. Differentiated oocyte expression marker gene ZP2,ZP3 and iconic protein Vasa,ZP2. 2. After the model was made with the suspension of Tripterygium wilfordii polyglycosides, the model group had obvious ovarian failure phenomenon, the model was successful, and the ovarian failure of group C and group D were recovered after treatment with amniotic fluid stem cells. Three follicles could be found in the ovary of group D after stage transplantation, which was better than that of single transplantation. After treatment with amniotic fluid derived stem cells, there were significant differences in FSH and E2 between the four groups (P < 0.01). Conclusion: 1. Amniotic fluid derived stem cells have strong proliferative potential and the ability to differentiate into oocyte. 2. Amniotic fluid derived stem cells have a certain therapeutic effect on premature ovarian failure, which can improve ovarian function by promoting ovarian follicle formation and regulating sex hormone level.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R329

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