VEGF促进脐带组织来源的人内皮祖细胞增殖作用机制的研究

发布时间：2018-07-09 12:11

本文选题：内皮祖细胞 + 脐带组织　；参考：《第二军医大学》2012年硕士论文

【摘要】：内皮祖细胞(endothelial progenitor cells, EPCs)是指出生后机体中存在能特异性归巢至血管新生组织并分化成内皮细胞的一群干细胞，包括从成血一血管干细胞到完全分化的内皮细胞之间一定阶段的细胞群体，它不仅参与胚胎时期血管生成，还与损伤的血管内皮的愈合和血管新生密切相关。内皮祖细胞是血管内皮细胞的前体细胞，在骨髓中与周围基质细胞紧密接触，在生理或病理因素刺激下，可从骨髓中动员而出，此后循环内皮祖细胞经过进一步的增殖和分化归巢至外周组织进行损伤的修复。近来的多项研究显示，EPC是参与出生后生理性及病理性血管形成最主要的细胞。因此，内皮祖细胞在心脑血管疾病、肿瘤血管形成及创伤愈合等方面均发挥重要作用，具有广阔的应用前景。内皮祖细胞应用于临床治疗主要包括三个领域，包括损伤血管壁的修复、缺血组织新血管形成与再生以及人造血管的包被。限制其临床应用的一个最主要的原因是从骨髓和外周血中分离培养出EPC数量都非常有限，不能满足实验研究及临床应用的需要。解决这个问题的途径包括应用脐带血或脐带组织培养人内皮祖细胞，或者用细胞因子、生长因子、药物来促进人内皮祖细胞动员。因此，对人内皮祖细胞培养方法的研究仍然很有必要。为了培养扩增出大量稳定的内皮祖细胞，本研究参照国内外多家实验室的培养及鉴定方案，在实验中反复摸索，对影响EPC培养扩增的部分重要因素进行比较，通过对人骨髓、脐带血、脐带组织培养内皮祖细胞的比较，以及分别用不同浓度VEGF与EPC共培养研究VEGF对人内皮祖细胞动员的促进作用，进而选择培养获得率高、祖细胞特征稳定以及增殖能力、成血管能力都较强的人内皮祖细胞培养方案，为下一步移植内皮祖细胞用于治疗心血管疾病及多器官功能障碍综合症奠定良好的基础。本实验研究共分三部分：第一部分：不同组织来源的人内皮祖细胞的分离、培养目的：通过对人骨髓、脐带血、脐带组织培养内皮祖细胞的比较，建立起人EPC的标准化分离、培养和扩增的方法，为内皮祖细胞的移植奠定基础。方法：采用密度梯度差速贴壁培养法及组织块植块法，分别从骨髓、脐带血及脐带组织中分离培养内皮祖细胞，按照不同接种密度、换液时间、基础培养液、血清浓度等进行分组，比较原代EPC细胞集落个数及EPC获得率，成熟后EPC通过比较细胞的生长曲线及增殖倍数，利用SPSS统计软件，对相关数据进行总结、分析、比较。结果：通过各种不同培养条件及方法得到的EPC细胞形态基本相似。骨髓来源的人内皮祖细胞在连续传代3代以后，细胞形态即发生明显改变，而脐带血及脐带组织来源的人内皮祖细胞可稳定传代10代以上，细胞形态不发生明显改变。脐带组织及脐带血来源的人内皮祖细胞较骨髓来源的人内皮祖细胞祖细胞形态特征稳定且增殖能力较强。结论：从骨髓、脐带血及脐带组织中能成功分离培养出内皮祖细胞，并能传代扩增。脐带组织及脐带血来源的人内皮祖细胞较骨髓来源的人内皮祖细胞细胞形态特征稳定且增殖能力较强。脐带组织来源的人内皮祖细胞获得率较高且培养方法简便、可靠，可为最终临床应用于防治多器官功能障碍做准备。第二部分内皮祖细胞鉴定与功能检测目的：鉴定培养扩增出的人内皮祖细胞并测定其功能，保证所分离及培养扩增的细胞的性质及纯度。方法：通过采用细胞形态和细胞超微结构观察，用摄取Dil标记的人乙酰化低密度脂蛋白（Dil-ac-LDL）与FITC标记的特异性荆豆凝集素（FITC-UEA-1）的双色荧光染色法和用CD133、CD34、CD31、KDR等表型表达行免疫组化鉴定EPC及流式细胞仪(FACS)检测EPC纯度以及体外血管生成功能试验进行鉴定，对人骨髓、脐带血、脐带组织培养的人内皮祖细胞进行比较。结果：细胞形态及细胞超微结构符合内皮祖细胞特征，Dil-ac-LDL、FITC-UEA-1双染超过80%，免疫组化、流式细胞仪检测CD133、CD34、CD31、KDR等的阳性率均符合EPC表型特征；体外具有成血管能力。结论：本研究采用综合的鉴定体系（形态特征、流式细胞仪、双色荧光、体外血管生成功能试验）所得结果完全可证明我们培养扩增的细胞为人内皮祖细胞。脐带组织来源的人内皮祖细胞及脐带血来源的人内皮祖细胞与骨髓来源的人内皮祖细胞相比祖细胞特征稳定且成血管能力较强。第三部分VEGF促进脐带组织来源的人内皮祖细胞增殖作用机制的研究目的：研究VEGF促进人脐带组织来源的内皮祖细胞增殖作用机制。方法：比较不同浓度VEGF与人脐带组织来源的内皮祖细胞共培养后内皮祖细胞数量变化及细胞培养液一氧化氮(NO)含量。用PI3K的特异性抑制剂LY294002处理脐带EPCs，同时设空白组和溶剂（DMSO）对照组和10ng/mlVEGF+抑制剂LY294002组，用western blot检测磷酸化Akt蛋白的表达，硝酸还原酶法测定细胞培养液一氧化氮(NO)含量，，检测加入抑制剂后细胞增殖能力及成血管能力。结果：10ng/ml浓度的VEGF与人脐带组织来源的内皮祖细胞共培养后内皮祖细胞数量明显增加，细胞培养液一氧化氮(NO)含量明显提高，而20ng/ml浓度的VEGF与人脐带组织来源的内皮祖细胞共培养后内皮祖细胞数量及细胞培养液一氧化氮(NO)含量与10ng/ml浓度相比变化不明显。加入PI3K的特异性抑制剂LY294002抑制PI3K后，磷酸化Akt蛋白表达也明显减少；细胞培养液NO含量明显减少；细胞增殖能力及成血管能力明显下降。结论：10ng/ml的VEGF浓度对人内皮祖细胞的增殖具有明显促进作用，同时培养液中NO浓度明显增高。而20ng/ml浓度的VEGF与10ng/ml浓度的VEGF相比对人内皮祖细胞增殖的促进作用不明显，可能与受体饱和有关。加入PI3K的特异性抑制剂LY294002抑制PI3K后，磷酸化Akt蛋白表达也明显减少；细胞培养液NO含量明显减少；细胞增殖能力及成血管能力明显下降，提示VEGF通过PI3K/Akt/eNOS通路促进人内皮祖细胞的增殖。总之，通过对人骨髓、脐带血、脐带组织培养人内皮祖细胞的比较发现脐带组织及脐带血来源的人内皮祖细胞较骨髓来源的人内皮祖细胞细胞形态及祖细胞特征稳定且脐带组织及脐带血来源的人内皮祖细胞增殖能力及成血管能力均强于骨髓来源的人内皮祖细胞，且脐带组织来源的人内皮祖细胞获得率较高且培养方法简便、可靠。通过分别用不同浓度VEGF与人脐带组织来源的EPC共培养研究VEGF对人内皮祖细胞增殖的促进作用发现10ng/ml的VEGF浓度对人内皮祖细胞的增殖具有明显促进作用，同时培养液中NO浓度明显增高，加入PI3K的特异性抑制剂LY294002抑制PI3K后，磷酸化Akt蛋白表达也明显减少；细胞培养液NO含量明显减少；细胞增殖能力及成血管能力明显下降，提示VEGF通过PI3K/Akt/eNOS通路促进人内皮祖细胞的增殖。这一结论为为下一步研究临床应用移植人内皮祖细胞防治MODS提供确实的细胞学基础并且为下一步研究VEGF促进脐带组织来源的人内皮祖细胞增殖的作用机制打下良好基础。
[Abstract]:Endothelial progenitor cells (EPCs) is a group of stem cells in the postnatal body that can be specifically homed to neovascularization and differentiate into endothelial cells, including a group of cells from a blood - derived stem cell to a fully differentiated endothelial cell. It is not only involved in embryonic angiogenesis. It is also closely related to the healing of vascular endothelial cells and angiogenesis. Endothelial progenitor cells are the precursor cells of vascular endothelial cells, which are closely contacted with the surrounding matrix cells in the bone marrow, and can be mobilized from the bone marrow under the stimulation of physiological or pathological factors. After that, the circulating endothelial progenitor cells are returned to the outside by further proliferation and differentiation. A number of recent studies have shown that EPC is the most important cell to participate in the physiological and pathological angiogenesis after birth. Therefore, endothelial progenitor cells play an important role in cardiovascular and cerebrovascular diseases, tumor angiogenesis and wound healing, which have broad prospects for application.
The application of endothelial progenitor cells to clinical treatment mainly consists of three fields, including repair of damaged vascular walls, new vascular formation and regeneration of ischemic tissue, and inclusion of artificial blood vessels. One of the main reasons for limiting its clinical application is that the number of EPC isolated from bone marrow and peripheral blood is very limited and cannot satisfy experimental research and The needs of clinical applications include the use of umbilical cord blood or umbilical cord tissue to cultivate human endothelial progenitor cells, or the use of cytokines, growth factors, and drugs to promote the mobilization of human endothelial progenitor cells.
Therefore, it is still necessary to study the culture method of human endothelial progenitor cells. In order to develop a large number of stable endothelial progenitor cells, this study, referring to the training and identification schemes of many laboratories at home and abroad, has been repeatedly explored in the experiment, and compared some important factors affecting the amplification of EPC culture, through the human bone marrow, umbilical cord blood, and umbilical cord. The comparison of tissue culture endothelial progenitor cells and the effects of VEGF on the mobilization of human endothelial progenitor cells were studied with different concentrations of VEGF and EPC, and the culture of human endothelial progenitor cells with high acquisition rate, stable progenitor cell characteristics and proliferation ability and strong vascular ability were selected for the next transplantation of endothelial progenitor cells. The cells laid a good foundation for the treatment of cardiovascular diseases and multiple organ dysfunction syndrome.
The study is divided into three parts:
Part one: isolation and culture of human endothelial progenitor cells from different tissue sources
Objective: to establish a standardized method of isolation, culture and amplification of human EPC by comparison of human bone marrow, umbilical cord blood and umbilical cord tissue culture endothelial progenitor cells, and lay a foundation for the transplantation of endothelial progenitor cells.
Methods: the endothelial progenitor cells were isolated and cultured from bone marrow, umbilical cord blood and umbilical cord tissue by density gradient differential adhesion culture method and tissue block method. According to different inoculation density, change time, basic culture fluid, serum concentration and so on, the number of primary EPC cell colonies and the EPC acquisition rate were compared. After the maturation, the EPC was comparatively fine. Cell growth curve and proliferation multiplier, using SPSS statistical software, the relevant data were summarized, analyzed and compared.
Results: the morphology of EPC cells obtained by various culture conditions and methods was basically similar. The cell morphology of human endothelial progenitor cells derived from bone marrow changed obviously after 3 generations of continuous passage, while human endothelial progenitor cells from umbilical cord blood and umbilical cord tissue were stable over 10 generations, and the cell morphology did not change obviously. Human endothelial progenitor cells derived from tissue and umbilical cord blood are more stable and proliferative than human bone marrow derived progenitor progenitor cells.
Conclusion: endothelial progenitor cells can be successfully isolated and cultured from bone marrow, umbilical cord blood and umbilical cord tissue. Human endothelial progenitor cells from umbilical cord tissue and umbilical cord blood are more stable and more proliferating than human endothelial progenitor cells derived from bone marrow. Human endothelial progenitor cells from umbilical cord tissue derived from human umbilical cord tissue have higher rate and culture. The method is simple, reliable, and can be used for the final clinical application in the prevention and treatment of multiple organ dysfunction syndrome.
Identification and functional detection of endothelial progenitor cells in second parts
Objective: to identify cultured and expanded human endothelial progenitor cells and to determine their functions, and to ensure the properties and purity of the cells isolated and cultured.
Methods: through the observation of cell morphology and cell ultrastructure, Dil labeled human acetylated LDL (Dil-ac-LDL) and FITC labeled specific bingbean agglutinin (FITC-UEA-1) were used as a double color fluorescence staining method and CD133, CD34, CD31, KDR and other phenotypic tables with CD133, CD34, CD31 and KDR to identify EPC and flow cytometry (FACS). Purity and in vitro vascular function tests were carried out to compare human bone marrow, umbilical cord blood and umbilical cord tissue cultured human endothelial progenitor cells.
Results: the cell morphology and cell ultrastructure conformed to the characteristics of endothelial progenitor cells. Dil-ac-LDL, FITC-UEA-1 double staining was more than 80%. The positive rates of CD133, CD34, CD31 and KDR were all conformed to the EPC phenotypic characteristics, and the ability of angiogenesis in vitro.
Conclusion: the results of the comprehensive identification system (morphological characteristics, flow cytometry, double color fluorescence, and in vitro angiogenesis test) can fully demonstrate that the cultured cells are human endothelial progenitor cells, human endothelial progenitor cells from umbilical cord tissue and human umbilical cord blood derived human endothelial progenitor cells and human bone marrow sources Compared with progenitor cells, the progenitor cells are stable and have strong blood vessel capability.
The third part is about the mechanism of VEGF promoting the proliferation of human umbilical cord tissue derived endothelial progenitor cells.
Objective: To study the mechanism of VEGF promoting proliferation of human umbilical cord derived endothelial progenitor cells.
Methods: To compare the number of endothelial progenitor cells and the content of nitric oxide (NO) of endothelial progenitor cells after co culture of endothelial progenitor cells from different concentrations of VEGF and human umbilical cord tissue. The umbilical cord EPCs was treated with the specific inhibitor LY294002 of PI3K, and the blank group and solvent (DMSO) control group and the 10ng/mlVEGF+ inhibitor LY294002 group were set up, and Western blot was used. The expression of phosphorylated Akt protein was detected, the content of nitric oxide (NO) in cell culture liquid was measured by nitrate reductase method, and the cell proliferation ability and vascular ability after the addition of the inhibitor were detected.
Results: the number of VEGF in 10ng/ml concentration and the endothelial progenitor cells of human umbilical cord tissue derived from endothelial progenitor cells increased significantly, and the content of nitric oxide (NO) in cell culture fluid increased significantly. The number of VEGF in 20ng/ml concentration and the number of inner progenitor cells of endothelial progenitor cells from human umbilical cord tissue derived endothelial progenitor cells and the nitric oxide (NO) in cell culture fluid. There was no obvious change in content compared with 10ng/ml concentration. After the addition of PI3K specific inhibitor LY294002 to PI3K, the expression of phosphorylated Akt protein decreased obviously, the content of NO in cell culture fluid decreased obviously, and the proliferation ability and vascular ability of cells decreased significantly.
Conclusion: the concentration of VEGF in 10ng/ml has a significant effect on the proliferation of human endothelial progenitor cells, and the concentration of NO in the culture medium is significantly higher, while the VEGF of the 20ng/ml concentration is not obvious to the 10ng/ml concentration of VEGF and is not obvious to the proliferation of human endothelial progenitor cells, and may be related to the receptor saturation. After PI3K, the expression of phosphorylated Akt protein decreased obviously, the content of NO in cell culture fluid decreased obviously, and the proliferation ability and vascular ability of cell decreased obviously, suggesting that VEGF promoted the proliferation of human endothelial progenitor cells through PI3K/Akt/eNOS pathway.
In summary, a comparison of human endothelial progenitor cells from human umbilical cord tissue and umbilical cord blood from human bone marrow, umbilical cord blood and umbilical cord tissue shows that human endothelial progenitor cells from umbilical cord and umbilical cord blood are more stable than those of bone marrow derived human endothelial progenitor cells and the proliferation and vascular ability of human endothelial progenitor cells from umbilical cord tissue and umbilical cord blood from human umbilical cord tissue and umbilical cord blood. Human endothelial progenitor cells, which are stronger than bone marrow, have high rate of acquisition of human endothelial progenitor cells from umbilical cord tissue, and the culture method is simple and reliable. EPC co culture of different concentrations of VEGF and human umbilical cord tissue was used to study the effect of VEGF on the proliferation of human endothelial progenitor cells and to present the VEGF concentration of 10ng/ml to human endothelial progenitor cells The proliferation has an obvious promoting effect, while the concentration of NO in the culture medium is obviously increased, and the expression of phosphorylated Akt protein is obviously reduced after the addition of PI3K specific inhibitor LY294002 to PI3K, the content of NO in the cell culture solution decreases obviously, and the cell proliferation ability and the capacity of blood vessel descend obviously, suggesting that VEGF promotes human within the PI3K/Akt/eNOS pathway. This conclusion provides a good basis for the next step in the study of the clinical application of transplanted human endothelial progenitor cells to the prevention and treatment of MODS and to further study the mechanism of VEGF to promote the proliferation of human endothelial progenitor cells from the umbilical cord tissue.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

【参考文献】