本文關(guān)鍵詞： 卵巢早衰 環(huán)磷酰胺 脂肪干細(xì)胞 卵巢 干細(xì)胞治療 生殖 脂肪干細(xì)胞移植　出處：《第四軍醫(yī)大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景：卵巢早衰近年來發(fā)病率日益增高，與腫瘤患者治療方案（化療、放療）的不斷完善密不可分，有效的治療使得患者的長期生存率顯著提高。尤其是年輕的生育期腫瘤患者，由于放化療破壞了大多數(shù)的分裂細(xì)胞，，化療后她們都可能會(huì)面臨閉經(jīng)、不孕不育以及卵巢早衰的問題。 目前臨床主要以雌孕激素替代療法和輔助生殖技術(shù)來治療卵巢早衰，免疫抑制治療主要用于免疫因素引起的卵巢早衰。但目前這些治療方法尚不能從根本上修復(fù)受損的卵巢功能，且長期應(yīng)用還可能會(huì)引起一些副作用。所以，怎樣提前預(yù)防和恢復(fù)衰退的卵巢功能，尋求新的治療方法，從而恢復(fù)患者的生育功能是科研工作者面臨的一個(gè)挑戰(zhàn)。 隨著對(duì)干細(xì)胞認(rèn)識(shí)的不斷深入，其在醫(yī)學(xué)領(lǐng)域的巨大潛力被逐漸發(fā)掘出來。已有研究發(fā)現(xiàn)，骨髓干細(xì)胞，臍帶干細(xì)胞等可以減少卵巢結(jié)構(gòu)及功能損傷，而脂肪干細(xì)胞對(duì)卵巢早衰的治療作用尚不清楚。脂肪干細(xì)胞具有較低的免疫原性，來源豐富，獲取方便，生長迅速，可以作為自體移植的來源。許多研究報(bào)道，脂肪干細(xì)胞移植可以改善損傷組織的結(jié)構(gòu)和功能。本研究通過探討脂肪干細(xì)胞治療化療性及自身免疫性卵巢早衰及其機(jī)制，以期為卵巢早衰的治療提供一種新的思路和方法。 第一部分：脂肪干細(xì)胞對(duì)小鼠化療性卵巢早衰治療作用的初步研究 實(shí)驗(yàn)?zāi)康模?建立小鼠化療性卵巢早衰模型，分離小鼠脂肪干細(xì)胞并探討其對(duì)化療性卵巢早衰的治療作用。 研究方法： 1.應(yīng)用酶消化法分離小鼠脂肪干細(xì)胞，用流式細(xì)胞術(shù)對(duì)脂肪干細(xì)胞細(xì)胞表面標(biāo)記進(jìn)行鑒定，并通過誘導(dǎo)成骨、成脂來鑒定干細(xì)胞多向分化的潛能。 2.采用腹腔注射化療藥物環(huán)磷酰胺的方法來建立小鼠卵巢早衰模型。并采用卵巢原位注射法及尾靜脈注射法進(jìn)行移植。 3.分別于干細(xì)胞移植后1周、1月處死各實(shí)驗(yàn)組小鼠，并取小鼠卵巢，通過HE染色法觀察各組卵巢卵泡形態(tài)及數(shù)目變化。 4.比較各組小鼠排卵數(shù)目和質(zhì)量的變化。 5. Tunel染色觀察小鼠卵巢顆粒細(xì)胞凋亡的情況。 實(shí)驗(yàn)結(jié)果： 1.經(jīng)體外培養(yǎng)及鑒定我們獲得了性質(zhì)穩(wěn)定的脂肪干細(xì)胞。 2.早衰組卵巢內(nèi)卵泡數(shù)量明顯減少；干細(xì)胞移植后各級(jí)卵泡數(shù)目明顯增多，治療后一周，原始卵泡和竇狀卵泡數(shù)目明顯升高，治療一月后，成熟卵泡數(shù)目也明顯升高。 3.與早衰組相比，移植組在移植一周及一月時(shí)小鼠排卵數(shù)目顯著增高。 4.移植后一周后小鼠卵巢中凋亡的顆粒細(xì)胞明顯減少。 結(jié)論： 脂肪干細(xì)胞對(duì)化療性卵巢早衰有一定的治療作用，在臨床應(yīng)用方面具有潛在價(jià)值。 第二部分：脂肪干細(xì)胞治療化療性卵巢早衰分子機(jī)制的初步研究 實(shí)驗(yàn)?zāi)康模?探討脂肪干細(xì)胞治療化療性卵巢早衰的分子機(jī)制。 研究方法： 1.用綠色熒光蛋白（Green Fluorescent Protein, GFP） 轉(zhuǎn)基因小鼠培養(yǎng)的脂肪干細(xì)胞移植治療卵巢早衰，追蹤其歸巢及定位情況。 2.通過基因芯片來研究脂肪干細(xì)胞移植對(duì)小鼠化療性卵巢早衰基因表達(dá)譜的影響。從而了解脂肪干細(xì)胞治療卵巢早衰的內(nèi)在機(jī)制。 3.采用實(shí)時(shí)定量聚合酶鏈反應(yīng)（quantitative Real-Time Polymerase Chain Reaction，qRT-PCR）以鑒定隨機(jī)選取的差異表達(dá)基因。 實(shí)驗(yàn)結(jié)果： 1.移植后一周和一月均能在卵巢中追蹤到脂肪干細(xì)胞。 2.基因芯片結(jié)果顯示，與早衰組相比，治療后尾靜脈組有176個(gè)基因發(fā)生變化，原位注射組有225個(gè)基因發(fā)生變化，許多基因在卵泡發(fā)育及卵子形成中起重要作用。 3. qRT-PCR隨機(jī)挑選的差異基因表達(dá)變化與相應(yīng)的基因芯片結(jié)果相一致。 結(jié)論： 脂肪干細(xì)胞對(duì)能通過調(diào)控某些基因來改善小鼠卵巢功能。脂肪干細(xì)胞分泌的細(xì)胞因子對(duì)修復(fù)卵巢功能也起到重要的作用。 第三部分：脂肪干細(xì)胞對(duì)小鼠免疫性卵巢早衰治療作用的初步研究 實(shí)驗(yàn)?zāi)康模?建立小鼠卵巢早衰的免疫模型，移植ADSCs后觀察脂肪干細(xì)胞對(duì)小鼠自身免疫性卵巢早衰的初步治療作用。 研究方法： 1.依據(jù)小鼠透明帶3(zonapellucida3，ZP3)的第330-342個(gè)氨基酸序列合成ZP3多肽，通過小鼠雙后掌皮下多點(diǎn)注射ZP3來建立卵巢早衰免疫模型。 2.干細(xì)胞治療組于免疫后14天原位注射脂肪干細(xì)胞。于干細(xì)胞移植后1周處死各實(shí)驗(yàn)組小鼠，觀察小鼠動(dòng)情周期及卵巢組織學(xué)變化，并比較各組小鼠卵巢抗透明帶抗體陽性率。 3.比較各組小鼠排卵數(shù)目和質(zhì)量的變化。 4.用CM-Dil標(biāo)記脂肪干細(xì)胞，追蹤脂肪干細(xì)胞歸巢情況。 5.觀察各組小鼠顆粒細(xì)胞凋亡情況。 實(shí)驗(yàn)結(jié)果： 1.皮下注射ZP3后，免疫模型組卵巢組織學(xué)表現(xiàn)以大量淋巴細(xì)胞和漿細(xì)胞浸潤為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顯好轉(zhuǎn)，抗透明帶抗體陽性率降低。 2.移植后排卵率顯著增高。 3.移植一周及兩周后可在卵巢追蹤到CM-Dil陽性細(xì)胞。 4.移植一周后顆粒細(xì)胞凋亡減少。 結(jié)論 脂肪干細(xì)胞對(duì)自身免疫性卵巢早衰有一定的療效，為進(jìn)一步的研究及臨床應(yīng)用奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R711.75

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