胎盘细胞的分离培养及其与母血、脐血细胞嵌合情况的研究

发布时间：2018-08-22 12:35

【摘要】：近年来,干细胞由于其自我更新及多向分化潜能的双重特性,在再生医学、细胞替代治疗及药物筛选等领域显示出了广阔的应用前景。并且随着干细胞以及组织工程相关基础及临床研究的深入,已经在一部分疾病中显示出了良好的治疗效果。但当代医学中,仍然存在着许多以目前医疗水平所不能彻底治愈的疾病,其中有一部分有关细胞、组织甚至器官的坏死如糖尿病、免疫功能障碍、阿尔兹海默病及帕金森等。干细胞的出现及逐渐深入研究,也为这些疾病的治愈带来的崭新的希望。目前干细胞的主要来源及潜在来源包括胚泡,胎儿组织,脐带血及成人组织。在诸多种类干细胞的临床试验及应用中,造血干细胞移植的理论基础和技术方法相对较成熟。造血干细胞移植目前对于大多数恶性血液系统肿瘤及一些免疫缺陷病是很有潜力的一种根治性治疗方法,在临床上已有广泛的应用。造血干细胞移植主要包括自体移植和异体移植两种方式。在造血干细胞的来源方面,过去比较成熟理想的来源为骨髓及外周血,但其来自于成体因而干性较弱。研究者们一直在坚持不懈的寻找更为理想并容易获得的细胞来源。20世纪80年代起,脐血因其丰富的造血干细胞含量吸引了研究者们的注意。脐血来源干细胞在临床上的应用迅速发展,在造血重建及免疫系统重建方面有较理想的效果,扩展了造血干细胞的来源。脐血干细胞来源丰富且免疫原性较低,但单份脐血含的细胞数量较少限制了其临床应用范畴。因此,找到更加广泛,更加适合的细胞来源成为造血干细胞移植临床应用的突破口。胎盘为圆盘形,嵌于子宫壁中,是孕期支持胎儿和母体之间进行各种物质交换并兼具内分泌等功能的器官,由底蜕膜、叶状绒毛膜和羊膜三部分组成。通常,胎盘属于分娩过程中所产生的废弃物,然而近年来由于许多研究发现多种干、祖细胞存在于胎盘中,即胎盘本身便是一个含量丰富的干、祖细胞库,因而受到越来越多的关注和探索。大量细胞组群的存在显示出其在未来再生医学临床应用中的巨大潜能。研究显示胎盘中含有许多不同种类的干细胞,近些年来,诸多研究的主要方向及重点针对于其所含间充质干细胞方面,对于造血干细胞相关的发掘研究较少。基于已有的实验研究及相关结果,我们可以推断,对胎盘来源的细胞加以更为深入和细致的研究,充分了解其生物学特性,对于选择和促进其尽早应用于临床有着至关重要的作用,可以解决临床多种相关疾病干细胞来源短缺或不合的问题。并且胎盘来源的细胞较体细胞而言,更为原始干性更强,可能获得更优的临床疗效。本研究的主要目的即为检测胎盘中单个核细胞总数以及CD34+细胞数量并与单份脐血中细胞数量做比较,观察胎盘及脐血来源细胞的集落形成情况,分别检测胎盘来源细胞、母血来源细胞和脐血来源细胞的HLA型别,应用STR-PCR技术检测和探索胎盘来源细胞与母血、脐血细胞的嵌合程度,为今后应用于临床提供相关理论基础。本研究主要分以下两部分内容:1.胎盘和脐血中细胞总数及其中CD34+细胞所占比例研究。胎盘及脐血来源细胞在体外接种培养的集落形成情况。在产妇及其家属知情同意并签署知情同意书的前提下,在研究获得解放军307医院伦理委员会审批的情况下,我们收集了五位健康足月剖宫产产妇的脐血及其对应胎盘。分娩过程中将脐血抽出置入一次性塑料血袋暂时保存,取回后离心并取下层细胞计数。胎盘用生理盐水反复冲洗以去除其表面残存血液及杂质,用注射器推注并收集胎盘脉管中的血液,应用含有10g/L AMD3100的生理盐水灌注胎盘脉管系统,夹闭脐动脉并孵育30分钟,收集灌注液,胎盘脉管中血液及灌注液离心并取下层细胞计数。胎盘剩余组织部分用剪切加酶消化法、研磨法处理,由100目钢网过滤,反复冲洗离心后计细胞数。将胎盘脉管血液、灌注液及组织来源的细胞计总数。用流式细胞仪检测脐血来源细胞及胎盘来源细胞中CD34+细胞的数量及所占比例。以5×104将脐血来源及胎盘来源细胞分别添加进1.1m L培养基中并接种于培养皿,于含5%CO2的培养箱中以37℃培养并定期观察。实验结果显示,胎盘中所获取的细胞总数、CD34+细胞数及其所占比例均明显高于从脐血所获取的细胞数。胎盘和脐血的集落培养皿均在接种后第六天可以观察到集落的出现,第十四天可以观察到集落形成良好。但与脐血细胞相同的接种密度下,胎盘来源细胞所形成的集落数量较少。胎盘中所含细胞数量上明显的优势提示我们,胎盘可能是未来临床干细胞移植治疗各类疾病很有潜力的来源。集落形成实验部分显示胎盘来源细胞具有多种集落形成的能力,而集落形成能力是造血干祖细胞分化以及增殖潜能的一项重要评估内容。2.胎盘来源细胞的HLA检测及其与母血、脐血来源细胞嵌合的检测。于产妇进入产房前自贵要静脉抽取外周血5m L,离心后取下层细胞。分别检测胎盘来源细胞、母血细胞及脐血细胞的HLA-A、HLA-B、HLA-C、HLA-DRB1、HLA-DQB1位点。HLA检测结果显示本研究五例样本中有两例胎盘细胞HLA位点检测提示其含有母体成分,其余三例结果显示胎盘来源细胞与脐血细胞位点完全相合。STR-PCR方法检测嵌合率的结果显示五例胎盘样本中均有母体成分嵌合,而HLA检测提示有母体成分的两例母体成分嵌合率尤其高。因此我们推断,HLA检测结果不同可能与其检测灵敏度及胎盘组织根据解剖结构肉眼分离有偏差相关,但与STR检测结果综合考虑提示,就目前的分离方法,胎盘中确实混有母体来源的成分,而其是否对临床移植的效果产生影响,还需要进一步的研究。本研究的创新性在于系统并完整的将脐血、胎盘和母血来源细胞以同样的检测方法检测,并得出可以对比并有一定意义的检测结果。同时,将HLA检测和STR-PCR检测方法共同应用于检测胎盘细胞来源及其与母体成分和胎儿成分嵌合的探索研究中,更加精准的检测出微小的嵌合比率,为潜在的临床应用可行性及方法的选择及奠定了基础。
[Abstract]:In recent years, stem cells have shown broad application prospects in regenerative medicine, cell replacement therapy and drug screening due to their dual characteristics of self-renewal and multi-directional differentiation potential. But in modern medicine, there are still many diseases which can not be cured completely by the current medical level. Some of them are related to the necrosis of cells, tissues and even organs, such as diabetes, immune dysfunction, Alzheimer's disease and Parkinson's disease. The emergence and gradual in-depth study of stem cells also bring about the cure of these diseases. The main sources and potential sources of stem cells include blastocysts, fetal tissues, umbilical cord blood and adult tissues. In clinical trials and applications of many types of stem cells, the theoretical basis and technical methods of hematopoietic stem cell transplantation are relatively mature. Hematopoietic stem cell transplantation is currently used in most malignant hematological malignancies. Hematopoietic stem cell transplantation mainly includes autologous transplantation and allogeneic transplantation. In the past, the ideal source of hematopoietic stem cells was bone marrow and peripheral blood, but it came from adult and therefore dry. Since the 1980s, umbilical cord blood has attracted the attention of researchers because of its abundant hematopoietic stem cell content. The clinical application of umbilical cord blood-derived stem cells has developed rapidly, and they are ideal for hematopoietic reconstruction and immune system reconstruction. Umbilical cord blood stem cells have abundant sources and low immunogenicity, but the small number of cells contained in a single umbilical cord blood limits the scope of its clinical application. Therefore, to find a wider and more suitable cell source becomes a breakthrough in the clinical application of hematopoietic stem cell transplantation. The placenta is usually a waste produced during childbirth. However, in recent years, many studies have found that many kinds of stem, progenitor cells exist in the placenta, that is, the placenta. As a rich stem-progenitor cell bank, it has attracted more and more attention and exploration. The existence of a large number of cell groups shows its great potential in the future clinical application of regenerative medicine. Based on the existing experimental studies and related results, we can infer that more in-depth and detailed study of placental-derived cells and a full understanding of their biological characteristics are crucial to the selection and promotion of their early clinical application. The main purpose of this study is to detect the total number of mononuclear cells in the placenta and the number of CD34 + cells in the placenta and to compare them with a single umbilical cord. The number of cells in blood was compared, the colony formation of cells from placenta and umbilical cord blood was observed, the HLA types of placenta-derived cells, maternal blood-derived cells and umbilical cord blood-derived cells were detected respectively, and the degree of chimerism between placenta-derived cells and maternal blood and umbilical cord blood cells was detected by STR-PCR, which provided relevant theory for clinical application in the future. This study is divided into the following two parts: 1. The total number of cells in placenta and umbilical cord blood and the proportion of CD34 + cells in them. With the approval of the committee, we collected the umbilical cord blood and its placenta from five healthy full-term cesarean mothers. During delivery, the umbilical cord blood was extracted and placed in a disposable plastic blood bag for temporary preservation, centrifuged and counted the cells in the lower layer. Push and collect the blood in the placental vein, perfuse the placental vein system with saline containing 10g/L AMD3100, clamp the umbilical artery and incubate for 30 minutes, collect the perfusion fluid, centrifuge the blood and perfusion fluid in the placental vein and count the cells in the lower layer. The number and proportion of CD34 + cells in cord blood and placenta derived cells were measured by flow cytometry. The cord blood and placenta derived cells were added into 1.1mL medium by 5 *104 and inoculated into 1.1mL medium respectively. The results showed that the total number of cells, the number of CD34 + cells and their proportion in placenta were significantly higher than those in umbilical cord blood. Colony culture dishes of placenta and umbilical cord blood were observed on day 6 and day 14 after inoculation. Good colony formation can be observed. However, at the same inoculation density as umbilical cord blood cells, the number of colonies formed by placental-derived cells is relatively small. Placenta-derived cells have a variety of colony-forming abilities, and colony-forming ability is an important assessment of the differentiation and proliferation potential of hematopoietic stem-progenitor cells. 2. HLA detection of placenta-derived cells and detection of their chimerism with maternal and umbilical cord blood-derived cells. The HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci of placenta-derived cells, maternal blood cells and umbilical cord blood cells were detected respectively. The results of HLA detection showed that two of the five samples in this study contained maternal components, and the other three showed that placenta-derived cells and umbilical cord blood cell loci were identical. The results of TR-PCR showed that all five placenta samples had maternal component chimerism, and HLA detection suggested that the maternal component chimerism rate was especially high in two cases with maternal component. The results suggest that the current isolation methods do mix maternal-derived components in the placenta, and further research is needed to determine whether the maternal-derived components have an impact on clinical transplantation outcomes. At the same time, HLA and STR-PCR were used to detect the origin of placental cells and their chimerism with maternal and fetal components, and the small chimerism ratio was detected more accurately, which laid a foundation for the potential clinical application feasibility and method selection. Foundation.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R457.7

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