羊水来源干细胞治疗大鼠卵巢早衰的初步研究

发布时间：2018-09-15 05:32

【摘要】：干细胞(stem cells，SC)是现代医学中的重大发现，分为胚胎干细胞(embryonic stem cell，，ES)和成体干细胞(adult stem cell，ADS)，两种细胞都能够再生成为各种组织器官。但相对应的各种伦理道德问题也随之出现。近年来，羊水来源干细胞（amniotic fluid-derived stem cell，AFS）的发现及研究提供了一种既避免了伦理道德问题，又具有更强大的医学应用前景的新方法。羊水容易获取，且获取过程安全性高，可于产前诊断时经羊膜穿刺获得，也可于孕妇分娩时获得，两种获取方法不会对胎儿造成伤害，仅对母体造成微创。经实验测定，羊水来源干细胞同时具备胚胎干细胞和成体干细胞的部分生理特性。实验表明，胚胎干细胞可以在体外被诱导为生殖细胞，成体干细胞可以在体外被诱导为卵母样细胞。本实验旨在对羊水来源干细胞向卵母样细胞分化的能力进行研究，同时探讨其在细胞治疗方面以及组织工程学方面治疗卵巢早衰（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来源干细胞系分化成为卵母样细胞的能力。 2.研究羊水来源干细胞对卵巢早衰的治疗效果。方法： 1.采用特定培养基诱导一株源自孕中期羊水的羊水来源干细胞系，使其向卵母样细胞分化，然后采用Real-time RT-PCR方法检测卵母细胞标志基因ZP2、ZP3，采用Western blotting方法检测透明带蛋白ZP2和标志性蛋白Vasa并采用免疫荧光化学的方法检测诱导后羊水来源干细胞ZP2的表达。 2.将实验用的SD大鼠随机分为四组：A组为正常组、B组为模型组、C组为一次移植组、D组为两次移植组。A组采用生理盐水灌胃，B组、C组、D组连续14天采用雷公藤多苷片悬液灌胃，D组在第1天与第7天灌胃后分别移植一次AFS，C组在第14天灌胃后移植一次AFS。最后观察大鼠的阴道涂片、性激素水平以及卵巢的组织形态学变化。结果： 1.羊水来源干细胞在培养基中生长迅速，证明了其经加入人卵泡液的特殊培养基诱导后，可在体外诱导分化成为卵母样细胞，分化的卵母样细胞表达标志基因ZP2、ZP3和标志性蛋白Vasa、ZP2。 2.使用雷公藤多苷片悬液灌胃法造模后，模型组有明显的卵巢衰竭现象，造模成功；经羊水来源干细胞治疗后，C组与D组卵巢衰竭的情况得到恢复，其中分阶段移植比一次移植恢复的更好。D组卵巢内均可以发现新生卵泡。 3.经羊水来源干细胞治疗后，四组间FSH值的差异有统计学意义；四组间E2值的差异也有统计学意义，且P均0.01。结论： 1.羊水来源干细胞具有较强的增殖潜力且具有向卵母样细胞分化的能力。 2.羊水来源干细胞会对卵巢早衰具有一定的治疗作用，从促进卵巢生成卵泡、调节性激素水平两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.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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