不同来源间质干细胞的分离及多种药物因素对其生物学特性的影响

发布时间：2018-05-16 19:42

  本文选题：间质干细胞 + 宫颈癌　； 参考：《江苏大学》2010年博士论文

【摘要】：目的：本研究旨在探讨间质干细胞(MSCs)有哪些组织来源,G-CSF是否可以对骨髓和外周血MSCs进行动员,并探讨多种药物因素对MSCs生物学特性的影响,以及MSCs与壳聚糖之间的生物相容性。 方法：①用贴壁方法分离成人骨髓、人胚胎骨髓、新生儿脐带和脐带血来源的间质干细胞,观察其形态与生长曲线,并用流式细胞仪测定其表面标志。贴壁法分离宫颈癌来源的MSCs,并通过细胞形态、流式分析表面标志、生长曲线、细胞周期、染色体分析、组织化学和RT-PCR鉴定细胞分化潜能等方法进行鉴别。②用G-CSF为动员剂,分离培养动员前后的大鼠骨髓和外周血来源的MSCs。③用GSH、葛根素和醋酸铅等三种药物分别对脐带MSCs的进行干预,通过其测定细胞活性、流式分析检测细胞表面标志、细胞凋亡、组织化学和RT-PCR法分析细胞诱导分化能力的改变,从而探讨这几种药物对MSCs可能产生的影响。④用冷冻干燥法制备壳聚糖支架,将脐带间质干细胞种植到壳聚糖支架上,观察细胞在支架上的形态特征,并检测细胞粘附性和细胞增殖活性。 结果：①骨髓标本均可成功分离出MSCs,但是人胚胎骨髓MSCs增殖能力要明显强于成人骨髓MSCs,脐带组织中也可成功分离出MSCs,而利用脐血分离MSCs没有得到成功。宫颈癌组织中同样存在MSCs。②经一定剂量的G-CSF动员后外周血CD44+细胞明显增加,并与G-CSF呈剂量效应关系,在未动员或20μg/kg G-CSF动员的情况下,外周血未能成功分离出MSCs,只在50μg/kg和80μg/kg G-CSF动员的情况下从外周血中分离培养出典型的MSCs样集落。该细胞经流式细胞仪分析结果证实符合MSCs的表面标志特征。③GSH对MSCs的影响：0.15mg/ml GSH可以缩短MSCs的群体倍增时间,并可减少MSCs凋亡,而对细胞的表面标志以及多向分化潜能无明显改变；葛根素对MSCs的影响：葛根素可抑制细胞增殖并呈一定的剂量效应,但是0.0012 mol/L葛根素却具有促进成骨细胞生成的作用；醋酸铅对MSCs的影响：醋酸铅可以引起抑制细胞增殖并呈浓度剂量效应,可以引起细胞凋亡,抑制细胞的成骨分化,并导致MSCs分泌细胞因子的能力也下降。④脐带间质干细胞能够在壳聚糖支架上粘附生长,并保持良好的细胞形态,与对照组相比,其粘附率和细胞增殖活性均无显著性差异。 结论：①人胚胎骨髓、脐带组织、骨髓都存在MSCs,但是脐带血中是否存在MSCs还需要进一步研究。在宫颈癌组织标本中也发现了MSCs的存在,推测MSCs可能在肿瘤的发生发展中有着重要的作用。②在特定剂量的G-CSF的动员下,大鼠外周血MSCs可以得到动员。③GSH可促进细胞增殖速度并对MSCs的表面标志与多向分化能力无影响,可应用于提高MSCs的体外扩增速度；葛根素可以影响抑制细胞增殖并促进MSCs向成骨分化；醋酸铅可以影响的MSCs的增殖活性,引起MSCs凋亡和分化潜能降低,使其分泌因子能力下降,醋酸铅破坏了MSCs即破坏了造血微环境,这可能在贫血的发生中有一定的价值。④脐带间质干细胞与壳聚糖支架之间具有良好的生物相容性,可应用于复合材料的制备。
[Abstract]:Objective: the purpose of this study was to explore the tissue sources of mesenchymal stem cells (MSCs), whether G-CSF could mobilize bone marrow and peripheral blood MSCs, and explore the effects of various drug factors on the biological characteristics of MSCs, and the biocompatibility between MSCs and chitosan.
Methods: (1) the bone marrow, bone marrow of human embryo, umbilical cord and umbilical cord blood stem cells were separated by the method of adherence. The morphology and growth curve of the umbilical cord and umbilical cord were observed and the surface markers were measured by flow cytometry. The MSCs was separated by the flow cytometry, and the cell morphology, flow analysis of surface markers, growth curve, cell cycle, Chromosomal analysis, histochemistry and RT-PCR identification of cell differentiation potential were identified. (2) G-CSF was used as a mobilization agent to isolate and culture the bone marrow and peripheral blood MSCs. of the rats before and after mobilization. Three drugs, such as GSH, puerarin and lead acetate, were used to dry the umbilical cord MSCs, and the cell activity was measured by flow analysis. Cell surface markers, apoptosis, histochemistry and RT-PCR method were used to analyze the changes in the ability of cell differentiation, and to explore the possible effects of these drugs on MSCs. (4) the chitosan scaffold was prepared by freeze drying method, and the umbilical cord mesenchymal stem cells were planted on the chitosan scaffold to observe the morphological characteristics of the cells on the scaffold and to detect the details. Cell adhesion and cell proliferation activity.
Results: (1) MSCs can be successfully isolated from bone marrow specimens, but the proliferation ability of MSCs in human fetal bone marrow is stronger than that of adult bone marrow MSCs, and MSCs can be successfully separated from umbilical cord tissue, and MSCs is not successfully used in the isolation of MSCs from umbilical cord blood. In cervical cancer tissues, there are also CD44+ cells in peripheral blood after G-CSF mobilization after a fixed dose of G-CSF. There was a significant increase in the dose effect relationship with G-CSF. In the absence of mobilization or 20 mu g/kg G-CSF mobilization, the peripheral blood failed to isolate MSCs successfully. The typical MSCs like colony was isolated and cultured in the peripheral blood only in the case of 50 mu g/kg and 80 mu g/kg G-CSF. The cells were confirmed to be in line with the surface markers of MSCs by flow cytometry. Characteristics. (3) the effect of GSH on MSCs: 0.15mg/ml GSH can shorten the group doubling time of MSCs, and reduce the apoptosis of MSCs, but there is no obvious change in the cell surface markers and multidirectional differentiation potential; the effect of Puerarin on MSCs: Puerarin can inhibit cell proliferation and have a certain dose effect, but 0.0012 mol/L puerarin has The effect of lead acetate on the formation of osteoblast; lead acetate on MSCs: lead acetate can inhibit cell proliferation and have a concentration dose effect, which can induce apoptosis, inhibit the osteogenic differentiation of cells, and lead to the decrease in the ability of MSCs to secrete cytokines. There was no significant difference in adherence rate and cell proliferation activity between the two groups.
Conclusion: (1) there is MSCs in human fetal bone marrow, umbilical cord tissue and bone marrow, but the existence of MSCs in umbilical cord blood needs further study. The existence of MSCs is also found in the specimens of cervical cancer. It is presumed that MSCs may play an important role in the development of the tumor. (2) in the mobilization of the G-CSF of the specific dose of G-CSF, the peripheral blood of the rat can be MSCs. To get mobilization. (3) GSH can promote cell proliferation and have no effect on the surface markers of MSCs and multidirectional differentiation, and can be applied to increase the expansion rate of MSCs in vitro; puerarin can inhibit the proliferation of cells and promote the differentiation of MSCs into osteogenic differentiation; lead acetate can affect the proliferation of MSCs, causing the apoptosis and differentiation potential of MSCs. Low, the decrease of its secretory capacity, lead acetate destruction by destroying MSCs, which destroys the hematopoietic microenvironment, which may have a certain value in the occurrence of anemia. (4) there is a good biocompatibility between the umbilical cord mesenchymal stem cells and chitosan scaffold, which can be used in the preparation of composite materials.
【学位授予单位】：江苏大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

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