脂肪干细胞治疗小鼠卵巢早衰的初步研究

发布时间：2018-02-24 11:17

本文关键词： 卵巢早衰环磷酰胺脂肪干细胞卵巢干细胞治疗生殖脂肪干细胞移植　出处：《第四军医大学》2014年博士论文　论文类型：学位论文

【摘要】：研究背景：卵巢早衰近年来发病率日益增高，与肿瘤患者治疗方案（化疗、放疗）的不断完善密不可分，有效的治疗使得患者的长期生存率显著提高。尤其是年轻的生育期肿瘤患者，由于放化疗破坏了大多数的分裂细胞，，化疗后她们都可能会面临闭经、不孕不育以及卵巢早衰的问题。目前临床主要以雌孕激素替代疗法和辅助生殖技术来治疗卵巢早衰，免疫抑制治疗主要用于免疫因素引起的卵巢早衰。但目前这些治疗方法尚不能从根本上修复受损的卵巢功能，且长期应用还可能会引起一些副作用。所以，怎样提前预防和恢复衰退的卵巢功能，寻求新的治疗方法，从而恢复患者的生育功能是科研工作者面临的一个挑战。随着对干细胞认识的不断深入，其在医学领域的巨大潜力被逐渐发掘出来。已有研究发现，骨髓干细胞，脐带干细胞等可以减少卵巢结构及功能损伤，而脂肪干细胞对卵巢早衰的治疗作用尚不清楚。脂肪干细胞具有较低的免疫原性，来源丰富，获取方便，生长迅速，可以作为自体移植的来源。许多研究报道，脂肪干细胞移植可以改善损伤组织的结构和功能。本研究通过探讨脂肪干细胞治疗化疗性及自身免疫性卵巢早衰及其机制，以期为卵巢早衰的治疗提供一种新的思路和方法。第一部分：脂肪干细胞对小鼠化疗性卵巢早衰治疗作用的初步研究实验目的：建立小鼠化疗性卵巢早衰模型，分离小鼠脂肪干细胞并探讨其对化疗性卵巢早衰的治疗作用。研究方法： 1.应用酶消化法分离小鼠脂肪干细胞，用流式细胞术对脂肪干细胞细胞表面标记进行鉴定，并通过诱导成骨、成脂来鉴定干细胞多向分化的潜能。 2.采用腹腔注射化疗药物环磷酰胺的方法来建立小鼠卵巢早衰模型。并采用卵巢原位注射法及尾静脉注射法进行移植。 3.分别于干细胞移植后1周、1月处死各实验组小鼠，并取小鼠卵巢，通过HE染色法观察各组卵巢卵泡形态及数目变化。 4.比较各组小鼠排卵数目和质量的变化。 5. Tunel染色观察小鼠卵巢颗粒细胞凋亡的情况。实验结果： 1.经体外培养及鉴定我们获得了性质稳定的脂肪干细胞。 2.早衰组卵巢内卵泡数量明显减少；干细胞移植后各级卵泡数目明显增多，治疗后一周，原始卵泡和窦状卵泡数目明显升高，治疗一月后，成熟卵泡数目也明显升高。 3.与早衰组相比，移植组在移植一周及一月时小鼠排卵数目显著增高。 4.移植后一周后小鼠卵巢中凋亡的颗粒细胞明显减少。结论：脂肪干细胞对化疗性卵巢早衰有一定的治疗作用，在临床应用方面具有潜在价值。第二部分：脂肪干细胞治疗化疗性卵巢早衰分子机制的初步研究实验目的：探讨脂肪干细胞治疗化疗性卵巢早衰的分子机制。研究方法： 1.用绿色荧光蛋白（Green Fluorescent Protein, GFP）转基因小鼠培养的脂肪干细胞移植治疗卵巢早衰，追踪其归巢及定位情况。 2.通过基因芯片来研究脂肪干细胞移植对小鼠化疗性卵巢早衰基因表达谱的影响。从而了解脂肪干细胞治疗卵巢早衰的内在机制。 3.采用实时定量聚合酶链反应（quantitative Real-Time Polymerase Chain Reaction，qRT-PCR）以鉴定随机选取的差异表达基因。实验结果： 1.移植后一周和一月均能在卵巢中追踪到脂肪干细胞。 2.基因芯片结果显示，与早衰组相比，治疗后尾静脉组有176个基因发生变化，原位注射组有225个基因发生变化，许多基因在卵泡发育及卵子形成中起重要作用。 3. qRT-PCR随机挑选的差异基因表达变化与相应的基因芯片结果相一致。结论：脂肪干细胞对能通过调控某些基因来改善小鼠卵巢功能。脂肪干细胞分泌的细胞因子对修复卵巢功能也起到重要的作用。第三部分：脂肪干细胞对小鼠免疫性卵巢早衰治疗作用的初步研究实验目的：建立小鼠卵巢早衰的免疫模型，移植ADSCs后观察脂肪干细胞对小鼠自身免疫性卵巢早衰的初步治疗作用。研究方法： 1.依据小鼠透明带3(zonapellucida3，ZP3)的第330-342个氨基酸序列合成ZP3多肽，通过小鼠双后掌皮下多点注射ZP3来建立卵巢早衰免疫模型。 2.干细胞治疗组于免疫后14天原位注射脂肪干细胞。于干细胞移植后1周处死各实验组小鼠，观察小鼠动情周期及卵巢组织学变化，并比较各组小鼠卵巢抗透明带抗体阳性率。 3.比较各组小鼠排卵数目和质量的变化。 4.用CM-Dil标记脂肪干细胞，追踪脂肪干细胞归巢情况。 5.观察各组小鼠颗粒细胞凋亡情况。实验结果： 1.皮下注射ZP3后，免疫模型组卵巢组织学表现以大量淋巴细胞和浆细胞浸润为特征的自身免疫性卵巢炎。移植后小鼠自身免疫性卵巢炎明显好转，抗透明带抗体阳性率降低。 2.移植后排卵率显著增高。 3.移植一周及两周后可在卵巢追踪到CM-Dil阳性细胞。 4.移植一周后颗粒细胞凋亡减少。结论脂肪干细胞对自身免疫性卵巢早衰有一定的疗效，为进一步的研究及临床应用奠定了基础。
[Abstract]:Background: in recent years, the incidence of premature ovarian failure is increasing, and the tumor treatment (chemotherapy, radiotherapy) continuous improvement are inseparable, effective treatment makes the long-term survival rate of patients were significantly improved. Especially the growth period of young patients with cancer, due to chemotherapy and destroyed the most mitotic cells, they are likely to be after chemotherapy faced with amenorrhea, infertility and premature ovarian failure.
The current clinical mainly in hormone replacement therapy and assisted reproductive technology to treat premature ovarian failure and premature ovarian failure of immunosuppressive therapy is mainly used for immune factors. But these treatments cannot repair the impaired ovarian function fundamentally, and long-term use may cause some side effects. Therefore, how to prevent ovarian function the recession and recovery, to seek a new treatment method, so as to restore the patient's reproductive function is a challenge facing researchers.
With the deepening of understanding of stem cells, its great potential in the field of medicine has been discovered. It has been found that bone marrow stem cells, umbilical cord stem cells can reduce the structure and function of ovary injury, and adipose derived stem cells on treatment of premature ovarian failure is not clear. Adipose stem cells have immunogenicity, compared with low abundant sources, convenient access, rapid growth, can be used as a source of autologous transplantation. Many studies reported that adipose derived stem cell transplantation can improve the structure and function of tissues. This study was to explore the adipose derived stem cells for treatment of chemotherapy and autoimmune premature ovarian failure and its mechanism, in order to provide a new idea and method for the treatment of premature ovarian failure.
The first part: a preliminary study on the effect of adipose stem cells on the treatment of chemotherapeutic ovarian failure in mice
Objective:
To establish a model of chemotherapeutic premature ovarian failure in mice, to separate the mouse fat stem cells and to explore the therapeutic effect on the chemotherapeutic ovarian premature failure.
Research methods:
Isolation of mouse adipose derived stem cells using 1. enzyme digestion method for identification of adipose derived stem cells, cell surface markers by flow cytometry, and the osteogenic differentiation of stem cells into tallow, identification of the potential of multi-directional differentiation.
2., a mouse model of premature ovarian failure was established by intraperitoneal injection of chemotherapeutic drugs and cyclophosphamide.
3. in the 1 weeks after the stem cell transplantation, the mice were killed in January and the ovaries of the mice were taken. The changes in the follicle morphology and number of ovarian follicles were observed by HE staining.
4. the changes in the number and quality of ovulation in each group were compared.
The apoptosis of mouse ovarian granulosa cells was observed by 5. Tunel staining.
Experimental results:
1. the stable fat stem cells were obtained by culture and identification in vitro.
2., the number of ovarian follicles in the premature senescence group was significantly reduced. The number of follicles at all levels increased significantly after stem cell transplantation. The number of primordial follicles and sinusoidal follicles increased significantly after treatment, and the number of mature follicles increased significantly after one month treatment.
3. compared with the premature senility group, the number of ovulation in the transplanted group was significantly higher in one week and one month.
4. after one week of transplantation, the apoptotic granulosa cells in the mouse ovary decreased significantly.
Conclusion:
Adipose stem cells have a certain therapeutic effect on chemotherapy induced premature ovarian failure and have potential value in clinical application.
The second part: a preliminary study on the molecular mechanism of adipose stem cells in the treatment of chemotherapeutic premature ovarian failure
Objective:
To investigate the molecular mechanism of adipose stem cells in the treatment of chemotherapeutic premature ovarian failure.
Research methods:
1., transplantation of adipose derived stem cells from Green Fluorescent Protein transgenic mice was used to treat premature ovarian failure, followed by homing and localization of Protein.
2., we studied the effect of adipose derived stem cell transplantation on the gene expression profile of mice with chemo induced premature ovarian failure through gene chip, so as to understand the intrinsic mechanism of adipose derived stem cells in the treatment of premature ovarian failure.
3. the random selected differentially expressed genes were identified by real-time quantitative polymerase chain reaction (quantitative Real-Time Polymerase Chain Reaction, qRT-PCR).
Experimental results:
Fat stem cells can be traced in the ovary in the ovary one week and one month after 1. transplantation.
2. gene chip results showed that compared with premature senescence group, there were 176 genes in the caudal vein group after treatment, and 225 genes in the in situ injection group changed. Many genes played an important role in follicular development and egg formation.
3. qRT-PCR randomly selected differentially expressed gene expression changes were consistent with the corresponding gene chip results.
Conclusion:
Adipose derived stem cells can improve the ovarian function of mice by regulating some genes. The cytokines secreted by adipose derived stem cells also play an important role in the repair of ovarian function.
The third part: a preliminary study on the effect of fat stem cells on the treatment of premature ovarian failure in mice
Objective:
The immune model of premature ovarian failure in mice was established. After transplantation of ADSCs, the primary therapeutic effect of adipose stem cells on premature ovarian failure in mice was observed.
Research methods:
1. on the basis of mouse zona pellucida 3 (zonapellucida3, ZP3) of the 330-342 amino acid sequence of the synthetic peptide ZP3, to establish the immune model of POF mice by double back subcutaneous injection of ZP3.
2. the stem cell treatment group was injected with adipose derived stem cells in situ on the 14 day after immunization. The mice in each experimental group were sacrificed at 1 weeks after the stem cell transplantation. The estrous cycle and histological changes of the ovaries in mice were observed. The positive rate of zona pellucida antibody in the ovary of each group was compared.
3. the changes in the number and quality of ovulation in each group were compared.
4. the adipose stem cells were labeled with CM-Dil, and the homing of adipose stem cells was traced.
5. the apoptosis of mice granulosa cells was observed.
Experimental results:
1. after subcutaneous injection of ZP3, the immune model group showed autoimmune ovarian inflammation characterized by a large number of lymphocytes and plasma cells infiltration. After transplantation, autoimmune Otis in mice improved significantly, and the positive rate of anti zona pellucida antibody decreased.
2. after transplantation, the rate of ovulation increased significantly.
3. a week and two weeks after transplantation, the CM-Dil positive cells could be traced in the ovary.
4. a week after transplantation, the apoptosis of granulosa cells decreased.
conclusion
Adipose stem cells have a certain effect on the autoimmune ovarian premature failure, which lays a foundation for further research and clinical application.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R711.75

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