本文關(guān)鍵詞： 脂肪間充質(zhì)干細(xì)胞 線粒體 輔助生殖 胚胎　出處：《中國(guó)人民解放軍醫(yī)學(xué)院》2017年博士論文　論文類型：學(xué)位論文

【摘要】：研究背景及目的:女性的生殖能力在25歲左右達(dá)高峰,35歲以后生殖能力明顯下降。在輔助生殖技術(shù)工作中,37歲以上不孕患者的妊娠率僅在20%左右。多項(xiàng)研究表明,年齡相關(guān)的妊娠率下降以及IVF反復(fù)種植失敗是由于卵子線粒體功能障礙,生成ATP能力減低,影響染色體正常分離、受精及胚胎發(fā)育潛能等。因此,為了提高高齡女性輔助生殖成功率,本實(shí)驗(yàn)研究,通過人自體脂肪間充質(zhì)干細(xì)胞中線粒體進(jìn)行卵細(xì)胞內(nèi)移植的初步研究,改善卵母細(xì)胞質(zhì)量及胚胎發(fā)育潛能,進(jìn)而提高IVF成功率。方法:第一部分:整形外科吸脂術(shù)獲得腹部皮下脂肪,0.1% I型膠原酶消化,貼壁培養(yǎng)獲得人脂肪間充質(zhì)干細(xì)胞,繪制細(xì)胞增殖曲線,進(jìn)行流式細(xì)胞檢測(cè)細(xì)胞表型,并通過人脂肪間充質(zhì)干細(xì)胞成脂、成骨誘導(dǎo)驗(yàn)證其是否具有多功能分化潛能。第二部分:通過流式細(xì)胞術(shù)檢測(cè)線粒體膜電位及活性氧(ROS)含量,化學(xué)發(fā)光檢測(cè)儀(luminometer)測(cè)定RLU值,進(jìn)而反映ATP濃度。比較脂肪間充質(zhì)干細(xì)胞與脂肪細(xì)胞中線粒體功能。數(shù)據(jù)分析采用配對(duì)t檢驗(yàn),P0.05具有統(tǒng)計(jì)學(xué)意義。第三部分:進(jìn)行臨床上首例自體脂肪間充質(zhì)干細(xì)胞線粒體移植。通過差速離心法提取線粒體。于取卵當(dāng)日,線粒體與制動(dòng)的精子一同注入卵細(xì)胞胞漿內(nèi),作為實(shí)驗(yàn)組,單純ICSI的胚胎作為對(duì)照組。之后觀察卵子的受精情況及胚胎發(fā)育情況,并對(duì)胚胎進(jìn)行植入前遺傳學(xué)篩查以及線粒體DN A拷貝數(shù)的測(cè)定。結(jié)果:第一部分:獲得形態(tài)均一、長(zhǎng)梭形、貼壁生長(zhǎng)的人脂肪間充質(zhì)干細(xì)胞,細(xì)胞增殖良好,有明顯的指數(shù)生長(zhǎng)期,體外能長(zhǎng)期培養(yǎng)存活,并保持不分化狀態(tài);流式檢測(cè)細(xì)胞高表達(dá)CD105、CD90,低表達(dá)CD34、CD45。能向成脂、成骨誘導(dǎo)分化。第二部分:隨著年齡的增長(zhǎng),脂肪細(xì)胞、脂肪干細(xì)胞線粒體功能均下降。但來源于同一脂肪組織中,脂肪間充質(zhì)干細(xì)胞的線粒體ATP水平、膜電位高于脂肪組織,活性氧水平低于脂肪細(xì)胞。第三部分:臨床上首例行線粒體移植的胚胎受精與胚胎發(fā)育情況無明顯異常,PGS顯示胚胎染色體均異常。實(shí)驗(yàn)組中胚胎線粒體DNA拷貝數(shù)高于對(duì)照組中未行線粒體移植的胚胎。結(jié)論:穩(wěn)定分離培養(yǎng)脂肪間充質(zhì)干細(xì)胞,研究證實(shí)脂肪間充質(zhì)干細(xì)胞中線粒體功能更優(yōu)。開展臨床首例自體脂肪間充質(zhì)干細(xì)胞線粒體移植,并且胚胎線粒體DNA拷貝數(shù)增加,具有一定的可行性。
[Abstract]:Background and objective: the reproductive capacity of women decreased significantly after reaching the peak at the age of 25 and reached the age of 35. The pregnancy rate of infertile women over 37 years of age was only about 20% in the assisted reproductive technology work. The decline of age-related pregnancy rate and the failure of repeated implantation of IVF are due to the dysfunction of egg mitochondria, the reduced ability to produce ATP, the influence of normal chromosome separation, fertilization and embryo development potential, etc. In order to improve the success rate of assisted reproduction in elderly women, the primary study was carried out to improve the quality of oocytes and the potential of embryo development by using mitochondria in human autologous adipose mesenchymal stem cells. Methods: the first part: the abdominal subcutaneous adipose tissue was digested with 0.1% type I collagenase by liposuction, and human adipose mesenchymal stem cells were obtained by adherent culture, and the cell proliferation curve was drawn. The phenotypes of cells were detected by flow cytometry, and the differentiation potential of human adipose mesenchymal stem cells was confirmed by osteogenic induction. Part two: mitochondrial membrane potential and reactive oxygen species (Ros) content were measured by flow cytometry. The RLU value was measured by chemiluminescence detector. And then reflect the concentration of ATP. Compare the function of mitochondria between adipose mesenchymal stem cells and adipocytes. The data analysis using paired t test (P0.05) has statistical significance. Part three: the first case of autologous adipose mesenchymal stem cells was performed in clinic. Mitochondria transplantation. Mitochondria are extracted by differential centrifugation. Mitochondria and immobilized sperm were injected into the cytoplasm of egg cells as experimental group and ICSI embryos as control group. Then the fertilization and embryo development of eggs were observed. Results: part 1: homogenous, fusiform, adherent human adipose mesenchymal stem cells were obtained, and the cells proliferated well. There is a significant exponential growth period, in vitro can be cultured for a long time, and maintain an undifferentiated state; flow cytometry high expression of CD105 CD90, low expression of CD34 CD45. Can be adipose, osteogenic differentiation. Part two: with age, adipocytes, adipocytes, The mitochondrial function of adipose stem cells all decreased, but the mitochondrial ATP level of adipose mesenchymal stem cells was higher than that of adipose tissue, and the membrane potential of adipose mesenchymal stem cells was higher than that of adipose tissue. The level of reactive oxygen species was lower than that of adipocytes. Part 3: there was no obvious abnormality in embryo fertilization and embryo development in the first routine mitochondrial transfer. The DNA copy number of embryo mitochondria in the experimental group was abnormal. Conclusion: stable isolation and culture of adipose mesenchymal stem cells, It is proved that mitochondrial function of adipose mesenchymal stem cells is better. It is feasible to carry out mitochondrial transplantation of autologous adipose mesenchymal stem cells and increase the copy number of embryonic mitochondrial DNA.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R714.8

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本文編號(hào)：1532734