自体脂肪间充质干细胞线粒体移植在辅助生殖技术中应用的初步研究

发布时间：2018-02-25 03:02

本文关键词： 脂肪间充质干细胞线粒体辅助生殖胚胎　出处：《中国人民解放军医学院》2017年博士论文　论文类型：学位论文

【摘要】：研究背景及目的:女性的生殖能力在25岁左右达高峰,35岁以后生殖能力明显下降。在辅助生殖技术工作中,37岁以上不孕患者的妊娠率仅在20%左右。多项研究表明,年龄相关的妊娠率下降以及IVF反复种植失败是由于卵子线粒体功能障碍,生成ATP能力减低,影响染色体正常分离、受精及胚胎发育潜能等。因此,为了提高高龄女性辅助生殖成功率,本实验研究,通过人自体脂肪间充质干细胞中线粒体进行卵细胞内移植的初步研究,改善卵母细胞质量及胚胎发育潜能,进而提高IVF成功率。方法:第一部分:整形外科吸脂术获得腹部皮下脂肪,0.1% I型胶原酶消化,贴壁培养获得人脂肪间充质干细胞,绘制细胞增殖曲线,进行流式细胞检测细胞表型,并通过人脂肪间充质干细胞成脂、成骨诱导验证其是否具有多功能分化潜能。第二部分:通过流式细胞术检测线粒体膜电位及活性氧(ROS)含量,化学发光检测仪(luminometer)测定RLU值,进而反映ATP浓度。比较脂肪间充质干细胞与脂肪细胞中线粒体功能。数据分析采用配对t检验,P0.05具有统计学意义。第三部分:进行临床上首例自体脂肪间充质干细胞线粒体移植。通过差速离心法提取线粒体。于取卵当日,线粒体与制动的精子一同注入卵细胞胞浆内,作为实验组,单纯ICSI的胚胎作为对照组。之后观察卵子的受精情况及胚胎发育情况,并对胚胎进行植入前遗传学筛查以及线粒体DN A拷贝数的测定。结果:第一部分:获得形态均一、长梭形、贴壁生长的人脂肪间充质干细胞,细胞增殖良好,有明显的指数生长期,体外能长期培养存活,并保持不分化状态;流式检测细胞高表达CD105、CD90,低表达CD34、CD45。能向成脂、成骨诱导分化。第二部分:随着年龄的增长,脂肪细胞、脂肪干细胞线粒体功能均下降。但来源于同一脂肪组织中,脂肪间充质干细胞的线粒体ATP水平、膜电位高于脂肪组织,活性氧水平低于脂肪细胞。第三部分:临床上首例行线粒体移植的胚胎受精与胚胎发育情况无明显异常,PGS显示胚胎染色体均异常。实验组中胚胎线粒体DNA拷贝数高于对照组中未行线粒体移植的胚胎。结论:稳定分离培养脂肪间充质干细胞,研究证实脂肪间充质干细胞中线粒体功能更优。开展临床首例自体脂肪间充质干细胞线粒体移植,并且胚胎线粒体DNA拷贝数增加,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.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R714.8

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