大鼠骨髓间充质干细胞的分离培养及向内皮样细胞诱导分化的实验研究

发布时间：2018-03-31 21:55

本文选题：大鼠骨髓间充质干细胞　切入点：分离　出处：《重庆大学》2007年硕士论文

【摘要】： 血管疾病是危及人类健康的常见疾病之一,其治疗手段之一是血管移植术。因此血管移植物的来源就成为人们关注的重要课题。目前临床上应用的血管移植物主要有自体血管、异体血管和人工材料血管三种,但不能满足临床需求。组织工程学的兴起为血管移植物的来源提供了一条崭新的思路。组织工程化的血管研究中种子细胞制取、培养和种植是其关键性的步骤。血管发生的主体是内皮细胞(endothelial cells,ECs)。但内皮细胞属于成熟细胞,其分裂增殖能力不强,获得的细胞数量有限,且细胞还存在去分化的倾向,因此必须寻找新的种子细胞。间充质干细胞(mesenchymal stem cells,MSCs)是起源于中胚层的多能干细胞,具有高度增殖、自我更新的能力及多向分化潜能。MSCs因取材方便,损伤轻微,对健康无害;取自自体,和无伦理、移植免疫排斥问题;另外它还可以体外大量培养扩增、易定向诱导分化以及外源基因易植入表达等优势,有望成为组织工程化血管种子细胞的主要来源。然而,目前MSCs的分离、纯化、鉴定及体外培养尚无统一方案,体外培养生长较慢,长期培养还有自动分化的可能。另外,MSCs虽然具有内皮细胞的某些表型,但有关MSCs在血管组织中的作用及其与血管内皮细胞之间的关系还需作进一步的研究。因此,如何在体外得到大量纯化的MSCs,以及在体外条件下诱导分化为内皮样细胞(endothelial-like cells,ELCs)并探讨其发生的机制,就成为组织工程血管种子细胞研究的基础。为此,本实验通过建立大鼠骨髓MSCs(rat bone marrow MSCs, rBMMSCs)体外培养体系,探讨在体外条件下,血管内皮生长因子(vascular endothelial growth factor,VEGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor,b-FGF)对MSCs分化为ELCs的可行性,期望为血管组织工程基础研究提供资料。方法本实验采用1.073g/mL Percoll密度梯度离心与贴壁培养相结合的方法,体外培养rBMMSCs。采用24小时后贴壁的细胞进行培养,以后每隔3天换液一次。待细胞80%融合后,消化传代,使之逐渐纯化。研究在体外培养条件下,rBMMSCs细胞形态、超微结构、生长曲线、细胞周期、表型等生物学特征。免疫组化法和流式细胞仪检测CD34, CD44、CD29和CD31的表达。用细胞诱导液(10ng/mL VEGF、2ng/mL b-FGF)诱导其向ELCs分化,倒置显微镜观察细胞形态,激光扫描共聚焦显微镜、流式细胞仪、免疫组化检测诱导细胞CD34、CD31、VE-cadherin的表达以及ELCs的功能,即对乙酰化低密度脂蛋白(Dil-ac-LDL)摄取和与荆豆凝集素(UEA-1)的结合。结果 1、采用Percoll密度梯度离心与贴壁培养相结合的方法所培养的rBMMSCs呈长梭形,胞核圆形或椭圆形,位于细胞中央;免疫组化检测的结果为CD29、CD44阳性,CD34阴性;流式细胞检测rBMMSCs不表达ELCs的CD31标记。 2、从传代细胞生长曲线可见:第1~2d为细胞生长潜伏期,第3~7d为对数增长期,8d以后细胞的生长进入平台期;符合干细胞的生长规律。 3、透射电镜结果显示:rBMMSCs有两种不同的形态结构,其中体积较小、核质比大、胞质内细胞器稀少者,为幼稚的rBMMSCs,表明它是处于未分化或分化较低状态的干细胞。 4、细胞周期分析显示:在一定范围内,随着传代的进行,处于G0/G1期的细胞数量增多,说明所培养的rBMMSCs绝大部分处于非增殖状态。 5、rBMMSCs在VEGF和b-FGF的刺激下,细胞形态逐渐缩短,部分细胞呈扁平形或圆形,可见串珠样结构和管状结构。纤连蛋白能显著促进管状结构的形成。6、分化细胞免疫表型为:CD31、VE-cadherin、CD34阳性,具有ELCs的功能特点:摄取Dil-ac-LDL并能结合UEA-1。结论: 本论文在前人工作的基础上,通过体外分离、纯化和鉴定rBMMSCs,建立了一套较合理的间充质干细胞体外培养的技术方案,用此方案可获得rBMMSCs。进一步,用VEGF生长因子对rBMMSCs进行诱导培养,从形态、表型和功能等三个方面对其表征,rBMMSCs的确可以分化为ELCs,并初步判定这种ELCs具有向血管生成的潜力。因此本研究工作可为下一步进行的人工血管构建,尤其是所需种子细胞的来源提供重要的实验参考依据。
[Abstract]:Vascular disease is one of the most common diseases threatening human health . One of the treatments is vascular transplantation . Therefore , the source of vascular graft has become an important subject of concern . At present , there are three kinds of vascular grafts in clinical application , such as autologous blood vessels , allograft vessels and artificial blood vessels , but do not meet the clinical needs .

The rise of tissue engineering provides a brand - new idea for the origin of vascular grafts . The preparation , cultivation and planting of seed cells in tissue engineering are critical steps .

The main body of angiogenesis is endothelial cells ( ECs ) . However , the endothelial cells belong to mature cells , and their cell division and proliferation ability are not strong , the number of cells obtained is limited , and the cells also have the tendency of dedifferentiation , so it is necessary to find new seed cells .

Mesenchymal stem cells ( MSCs ) are multipotent stem cells derived from mesoderm , and have the abilities of high proliferation and self - renewal and multi - directional differentiation potential . MSCs can be taken as the main source of tissue engineering vascular seed cells because of their convenience , mild injury and no harm to health .

However , MSCs can be isolated , purified , identified and cultured in vitro . In addition , MSCs have some phenotypes of endothelial cells , but the relationship between MSCs in vascular tissue and their relationship with vascular endothelial cells is further studied .

Therefore , how to obtain a large amount of purified MSCs in vitro , and to induce differentiation into endothelial - like cells ( ELCs ) in vitro and to explore the mechanism of their occurrence is the basis for the study of vascular seed cells in tissue engineering . To this end , the feasibility of differentiation into ELCs by vascular endothelial growth factor ( VEGF ) and basic fibroblast growth factor ( b - FGF ) in vitro is discussed .

method

The expression of CD34 , CD44 , CD29 and CD31 was detected by immunohistochemistry and flow cytometry . The expression of CD34 , CD44 , CD29 and CD31 was detected by immunohistochemistry and flow cytometry . The expression of CD34 , CD44 , CD29 and CD31 was detected by immunohistochemistry and flow cytometry . The expression of CD34 , CD31 , VE - cadherin and the function of ELCs were detected by immunohistochemistry and flow cytometry .

Results

1 . The rBMMSCs cultured by the method combined with Perfluorodensity gradient centrifugation and adherent culture were spindle - shaped , round or oval in nucleus , and located in the center of the cell ; the results of immunohistochemistry were CD29 , CD44 - positive and CD34 - negative ; and the flow cytometry showed that the rBMMSCs did not express the CD31 markers of ELCs .

2 . From the growth curve of the passaging cells , the growth latency of the cells was observed in the 1st to 2nd days , and the growth of the cells in the 3rd to 7th days was logarithmic , and the growth of the cells into the stage after 8d . The growth of the stem cells was met .

3 . Transmission electron microscopy showed that rBMMSCs had two different morphological structures , of which the volume was small , the nuclear mass ratio was large , the organelle in the cytoplasm was sparse , and the immature rBMMSCs showed that it was a stem cell which was in the undifferentiated or poorly differentiated state .

4 . Cell cycle analysis showed that in a certain range , the number of cells in G0 / G1 phase increased with the generation of passages , indicating that the majority of cultured rBMMSCs were in a non - proliferative state .

5 . Under the stimulation of VEGF and b - FGF , the morphology of the cells was gradually shortened , and some cells were flattened or round , visible tandem - like structure and tubular structure .

Conclusion :

rBMMSCs were isolated , purified and identified on the basis of the previous work , and a set of more reasonable techniques for the in vitro culture of mesenchymal stem cells was established .

【学位授予单位】：重庆大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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