基于建立临床用细胞库的脐带间充质干细胞相关生物学特性研究

发布时间：2018-08-23 14:05

【摘要】：背景：干细胞(stem cells)是指一类具有多向分化潜能和自我更新能力的细胞,可分为分为胚胎干细胞和成体干细胞。胚胎干细胞(ES)是全能干细胞,理论上能分化为各种成体细胞,由于免疫表型发育不完全,可用于异体移植,但其研究受到伦理学和法理的限制,并且因胚胎干细胞的原始特性,其存在潜在的致瘤性危险,所以成体干细胞成为目前研究的热点。传统的成体干细胞获取如骨髓采集及利用细胞因子动员外周血获取干细胞或者脂肪干细胞等都会给捐献者或患者本人带来一定的风险,而脐带中的干/祖细胞较成人骨髓中的干/祖细胞更原始,有更强的增殖分化能力；脐带中的免疫细胞较为幼稚,功能活性低,免疫功能不够成熟,处于一种缄默状态,不会触发免疫反应及引起移植物抗宿主病；脐带中不会有肿瘤细胞；脐带中的干细胞易于分离；脐带潜伏性病毒和病原微生物的感染及传播几率均相对比较低；费用低；伦理学争议少；易于保存和运输。人脐带间充质干细胞(hUCMSCs)在细胞移植、基因治疗、组织工程中有广阔的应用前景,使之在众多成体干细胞中脱颖而出。hUCMSCs主要存在于脐带内部中来自中胚层的胶样结缔组织。结缔组织内有闭锁的卵黄蒂、尿囊、2条脐动脉和1条脐静脉,无毛细血管,脐带血管外富含基质成分,其营养来自于脐带血管的渗透。血管周围被胶样结缔组织所包裹,为胶质样物质,被称为华尔氏胶或沃顿胶(、Wharton's jelly,WJ),它富含胶质和葡萄糖胺聚糖(主要成分为透明质酸)。hUCMSCs勺鉴定需要符合三个条件：1.细胞在特定的培养条件下具有可塑性；2.细胞具有特殊的免疫细胞表面标志物(hUCMSCs:CD34和CD45表达阴性；CD29和CD105表达阳性)；3.细胞具有向成软骨细胞、成骨细胞、成脂细胞和成神经细胞等方向分化的能力。结合目前国内外的干细胞相关科研进展情况,hUCMSCs的规范化和大规模的临床应用和科学研究势在必行,所以干细胞建库的问题就被提上日程。GMP (Good Manufacturing Practice)级的干细胞建库有利于使其应用规范化和大量应用,这更有利于科学研究和临床创伤修复组织工程的大规模应用,广大的患者就是未来的受益者。然而干细胞建库就面临着冻存复苏后细胞活性的改变、各种成体干细胞的比较、选择何种干细胞等一系列的问题。冻存细胞储存在-196℃液氮中,理论上储存时间是无限的,但冷冻对各种细胞均会起到破坏作用。目前认为冻存细胞的损伤主要为“溶质反应”所致,后者引起渗透压和pH值变化导致细胞膜渗漏,复苏时水分进入细胞内,造成细胞水肿变性而死亡。另外hUCMSCs供体来源的不同,hUCMSCs也会有所不同。孕妇的年龄也是其中应考虑的因素之一。蜕皮甾酮(ecdysterone, EDS)又称β-蜕皮激素,最初在昆虫中发现,但它在植物界的分布更为广泛,并且含量高。现在所应用的蜕皮甾酮主要由植物中提取。目前所知其作用主要有：1)促进核酸及蛋白质的合成；2)调节糖代谢；3)调节脂代谢；4)调节基因表达；5)改善免疫功能；6)抗氧化作用；7)促进缺血区血管的生成和侧支循环的建立；8)促进多种细胞增殖的作用等。在本课题组的前期对EDS的研究中发现,蜕皮甾酮有刺激人脐静脉内皮细胞的增殖和分裂,降低肺血管通透性；促进兔皮肤缺损创伤愈合过程,并认为其机制与蜕皮激素诱导成纤维细胞、上皮细胞以及血管内皮细胞等增殖分化有关；促进人表皮干细胞增值,促进人骨髓间充质干细胞增值,促进脐带间充质干细胞增值等作用。蜕皮甾酮的生物多效性提示其对促进细胞的增殖以及干细胞诱导分化等方面有一定的影响,进行此项研究具有广泛的应用前景。本实验就其中冻存复苏后细胞的活性及不同年龄母体来源的hUCMSCs的活性有无差异进行研究,希望为hUCMSCs建库提供理论基础。目的：研究体外条件下人脐带间充质干细胞(hUCMSCs)的分离培养鉴定、不同浓度蜕皮甾酮(EDS)对hUCMSCs冻存复苏后的生物学活性的影响及不同年龄组孕妇来源的hUCMSCs生物学活性的比较。方法：1、hUCMSCs用酶消化法进行分离、培养和扩增,用向成脂成骨方向诱导分化、流式细胞术检测hUCMSCs相关表面特异标i(?)(CD34、CD45、CD29、 CD105)的表达和RT-PCR扩增hUCMSCs中提取出的产物于1%琼脂糖凝胶电泳检测hUCMSCs相关基因表达鉴定其为间充质来源干细胞。2、随后hUCMSCs采用梯度冷冻技术冻存,6个月后复苏,扩增、传代细胞,分3组培养,空白对照组用普通培养基培养；低剂量EDS组在普通培养基中加入100μg/mL EDS;高剂量EDS组在普通培养基中加入200μg/mL EDS。显微镜下观察细胞形态,10d时计算克隆形成能力并进行统计比较,绘制细胞生长曲线,油红O染色及茜素红染色观察hUCMSCs向脂肪细胞、成骨细胞分化的能力。Oil Red O是一种油溶性偶氮染料,易溶于苯,溶于乙醇(呈浅黄色红色)和丙酮。显微技术中用作脂肪染色剂。作为脂肪细胞的染色剂的原理是使用Oil Red O的油溶性,对其它的细胞结构着色性差；茜素红与细胞中钙化成分发生显色反应,产生深红色的带色化合物,这样成骨诱导的细胞外面沉积的钙结节就被染成了深红色。3、在第三部分实验中hUCMSCs分为A、B两组。A组产妇年龄为23岁-30岁,B组产妇年龄为31-38岁,每组各抽取病例6例。流式细胞仪检测两组hUCMSCs的细胞表面特异标记(CD34、CD45、CD29、CD105)表达的不同。核型分析实验中,用秋水仙素固定处于细胞分裂期的细胞,后用Giemsa染色10分钟,在显微镜下观察染色体标本分裂相的多少及分散情况。油红O染色及茜素红染色观察A、B两组hUCMSCs向脂肪细胞、成骨细胞分化能力的不同。结果：1、hUCMSCs分离、培养鉴定方面：细胞经贴壁培养4-5h,倒置显微镜下可见原代细胞接种,24h后有少量细胞贴壁,外观呈纺锤形和短棒形,培养至约5d细胞大部为双突起的长梭形、扁平形生长。培养至10d左右,大部分贴壁细胞呈长梭形成纤维状。hUCMSCs生长曲线呈S形,接种后0-1d为潜伏适应期,从第2天起细胞开始增殖并进入对数生长期,第6天达到高峰,以后进入平台期。倒置光学显微镜下观察向脂肪细胞分化的油红O染色显示,胞浆中充满红色的油滴。观察向成骨细胞分化的茜素红染色显示,细胞大量重叠成团处被染成深红色的“钙结节”。hUCMSCs流式细胞仪检测细胞表面抗原CD29、CD105表达阳性,CD34和CD45表达阴性。RT-PCR所测hUCMSCs在20db和45db均持续表达Oct-4, Nanog, Rex-1和SCF基因。2、EDS用于冻存后的保护效应研究方面：倒置光学显微镜hUCMSCs两EDS组在细胞形态、存活率及克隆形成率等细胞活性增殖方面均优于空白对照组；成骨、成脂分化能力检测发现,各组细胞均能够在相应的诱导培养条件下分别向脂肪细胞、成骨细胞分化,而高剂量EDS组细胞在未加成骨诱导培养基的情况下仍出现向成骨细胞分化的趋势。3、不同年龄组孕妇来源的hUCMSCs细胞在相应的诱导培养条件下,均能向脂肪细胞和成骨细胞分化。观察向脂肪细胞分化的油红O染色显示,胞浆中充满红色的油滴,A组细胞油滴较B组多。观察向成骨细胞分化的茜素红染色显示,A组细胞深红色“钙结节”较B组多。流式细胞仪检测第4代细胞免疫表型显示,CD29、CD105表达阳性,且A组阳性表达率明显高于B组(P0.05)；CD34和CD45表达阴性,A组表达率均小于B组(P0.05)。核型分析检测,A、B两组细胞在染色体分析中并未有明显差异。结论：在一定浓度范围内,EDS对细胞复苏后恢复细胞活性以及提高存活率方面具有积极作用,但是较高浓度的EDS有诱导细胞向成骨细胞方向分化的趋势。年龄较小的孕妇来源脐带间充质干细胞(hUCMSCs)在细胞活性、干细胞相关细胞表面标志物表达及成骨成脂等方面较年纪较大孕妇来源hUCMSCs强。
[Abstract]:BACKGROUND: Stem cells refer to a class of cells with multiple differentiation potential and self-renewal ability, which can be divided into embryonic stem cells and adult stem cells. Due to the limitation of ethics and jurisprudence, and because of the primitive characteristics of embryonic stem cells, there is a potential oncogenic risk, adult stem cells have become the focus of current research. The stem/progenitor cells in the umbilical cord are more primitive and have stronger ability of proliferation and differentiation than those in the adult bone marrow. Human umbilical cord mesenchymal stem cells (hUCMSCs) have wide applications in cell transplantation, gene therapy and tissue engineering. HUCMSCs are found mainly in mesodermal connective tissue within the umbilical cord. There are atresia of the yolk pedicle, allantoic sac, 2 umbilical arteries and 1 umbilical vein in the connective tissue. There are no capillaries. The umbilical cord is rich in extravascular matrix components. Its nutrition comes from the permeation of umbilical cord blood vessels. The tube is surrounded by adhesive-like connective tissue, a glial-like substance called Wharton's jelly or Wharton's jelly (WJ), which is rich in glia and Glucosaminoglycan (mainly hyaluronic acid). The identification of hUCMSCs requires three conditions: 1. Cells have plasticity under specific culture conditions; 2. Cells have special characteristics. Immunocyte surface markers (hUCMSCs: CD34 and CD45 expression negative; CD29 and CD105 expression positive); 3. cells have the ability to differentiate into chondroblasts, osteoblasts, adipocytes and neuroblasts. Combined with the current research progress of stem cells at home and abroad, the standardization and large-scale clinical application of hUCMSCs and Scientific research is imperative, so the issue of stem cell bank building has been put on the agenda. GMP (Good Manufacturing Practice) level of stem cell bank building is conducive to its application standardization and a large number of applications, which is more conducive to scientific research and clinical wound repair tissue engineering large-scale application, the vast number of patients are the future beneficiaries. The establishment of stem cell bank is confronted with a series of problems, such as the changes of cell activity after cryopreservation and resuscitation, the comparison of various adult stem cells, and the selection of stem cells. It is caused by "solute reaction", which causes osmotic pressure and pH changes leading to cell membrane leakage and water entering the cell during resuscitation, resulting in cell edema and degeneration and death.
Ecdysterone (EDS), also known as beta-ecdysterone (beta-ecdysterone), was first found in insects, but it is more widely distributed in the plant kingdom and has a high content. 4) Regulating gene expression; 5) Improving immune function; 6) Antioxidant effect; 7) Promoting angiogenesis and collateral circulation in ischemic region; 8) Promoting proliferation of a variety of cells and so on. Ecdysterone can promote the proliferation and differentiation of fibroblasts, epithelial cells and vascular endothelial cells, promote the proliferation of human epidermal stem cells, promote the proliferation of human bone marrow mesenchymal stem cells, and promote the proliferation of umbilical cord mesenchymal stem cells. The multipotency suggests that it can promote cell proliferation and induce differentiation of stem cells.
In this study, we studied the cell viability after cryopreservation and resuscitation and whether there was any difference in the activity of hUCMSCs derived from different age mothers.
AIM: To study the isolation, culture and identification of human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro, the effects of different concentrations of ecdysterone (EDS) on the biological activities of hUCMSCs after cryopreservation and resuscitation, and the comparison of biological activities of hUCMSCs from pregnant women of different ages.
Methods: 1. hUCMSCs were isolated, cultured and amplified by enzymatic digestion, induced to differentiate into adipogenic osteoblasts. The expression of hUCMSCs-related surface specific marker I (?) (CD34, CD45, CD29, CD105) was detected by flow cytometry, and the products extracted from hUCMSCs by RT-PCR were identified by 1% agarose gel electrophoresis. Mesenchymal-derived stem cells. 2. HUCMSCs were cryopreserved by gradient cryopreservation. After 6 months, the cells were resuscitated, amplified and subcultured. The cells were cultured in three groups. The blank control group was cultured in normal medium; the low-dose EDS group was added with 100 ug/mL EDS; the high-dose EDS group was added with 200 ug/mL EDS. Morphology, 10 days after cloning and statistical comparison, draw cell growth curve, oil red O staining and alizarin red staining to observe the ability of hUCMSCs to differentiate into adipocytes, osteoblasts. Oil Red O is an oil-soluble azo dye, soluble in benzene, soluble in ethanol (light yellow red) and acetone. As a stain for adipocytes, the principle is that the oil-soluble oil Red O is used to stain other cellular structures poorly; alizarin red reacts with calcified components in the cells to produce dark red coloring compounds, so that calcium nodules deposited outside the cells induced by osteogenesis are stained dark red.3, solid in the third part HUCMSCs were divided into group A and group B. Maternal age in group A was 23-30 years old, and that in group B was 31-38 years old. Six cases were selected from each group. The expression of cell surface specific markers (CD34, CD45, CD29, CD105) was detected by flow cytometry. After staining for 10 minutes, the number and dispersion of the mitotic phase were observed under microscope. The differentiation ability of hUCMSCs to adipocytes and osteoblasts was observed by oil red O staining and alizarin red staining.
Results: 1. hUCMSCs were isolated and cultured for 4-5 hours. Primary cells were inoculated under inverted microscope. After 24 hours, a small number of cells adhered to the wall. The cells were spindle-shaped and short rod-shaped in appearance. Most of the cells cultured for about 5 days were long spindle-shaped and flat-shaped with double processes. The growth curve of hUCMSCs was S-shaped. The cells began to proliferate from the 2nd day to the logarithmic growth period, reached the peak on the 6th day, and then entered the plateau stage. The oil red O staining of adipocyte differentiation showed that the cytoplasm was filled with red oil droplets. Alizarin red staining showed that a large number of cells were stained into dark red "calcium nodules" at the overlapping sites. hUCMSCs were positive for CD29, CD105 and negative for CD34 and CD45 by flow cytometry. Oct-4, Nanog, Rex-1 and SCF genes were continuously expressed in hUCMSCs at 20 dB and 45 dB by RT-PCR. EDS was used to study the protective effect of cryopreservation. Inverted optical microscopy showed that hUCMSCs and EDS groups were superior to the blank control group in cell morphology, survival rate and clone formation rate; osteogenic and adipogenic differentiation ability test showed that the cells in each group were able to differentiate into adipocytes and osteoblasts under the corresponding induction culture conditions, while the high-dose EDS group was able to differentiate into adipocytes and osteoblasts respectively. 3. hUCMSCs from pregnant women of different ages could differentiate into adipocytes and osteoblasts under the corresponding induction culture conditions. Oil red O staining showed that the cytoplasm was full of red oil droplets and group A was fine. Alizarin red staining showed that there were more dark red "calcium nodules" in group A than in group B. Flow cytometry showed that CD29 and CD105 were positive in the fourth generation of cells, and the positive expression rate of CD34 and CD45 in group A was significantly higher than that in group B (P 0.05). Karyotype analysis showed that there was no significant difference in chromosome analysis between the two groups of A and B.
CONCLUSION: EDS has a positive effect on the recovery of cell viability and the improvement of survival rate after resuscitation in a certain concentration range, but higher concentration of EDS has a tendency to induce cells to differentiate into osteoblasts. The expression of markers and osteogenesis and lipid formation were stronger than those of older pregnant women, hUCMSCs.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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