發(fā)布時間：2018-05-16 19:42

  本文選題：間質(zhì)干細胞 + 宮頸癌��； 參考：《江蘇大學》2010年博士論文

【摘要】：目的：本研究旨在探討間質(zhì)干細胞(MSCs)有哪些組織來源,G-CSF是否可以對骨髓和外周血MSCs進行動員,并探討多種藥物因素對MSCs生物學特性的影響,以及MSCs與殼聚糖之間的生物相容性。 方法：①用貼壁方法分離成人骨髓、人胚胎骨髓、新生兒臍帶和臍帶血來源的間質(zhì)干細胞,觀察其形態(tài)與生長曲線,并用流式細胞儀測定其表面標志。貼壁法分離宮頸癌來源的MSCs,并通過細胞形態(tài)、流式分析表面標志、生長曲線、細胞周期、染色體分析、組織化學和RT-PCR鑒定細胞分化潛能等方法進行鑒別。②用G-CSF為動員劑,分離培養(yǎng)動員前后的大鼠骨髓和外周血來源的MSCs。③用GSH、葛根素和醋酸鉛等三種藥物分別對臍帶MSCs的進行干預,通過其測定細胞活性、流式分析檢測細胞表面標志、細胞凋亡、組織化學和RT-PCR法分析細胞誘導分化能力的改變,從而探討這幾種藥物對MSCs可能產(chǎn)生的影響。④用冷凍干燥法制備殼聚糖支架,將臍帶間質(zhì)干細胞種植到殼聚糖支架上,觀察細胞在支架上的形態(tài)特征,并檢測細胞粘附性和細胞增殖活性。 結(jié)果：①骨髓標本均可成功分離出MSCs,但是人胚胎骨髓MSCs增殖能力要明顯強于成人骨髓MSCs,臍帶組織中也可成功分離出MSCs,而利用臍血分離MSCs沒有得到成功。宮頸癌組織中同樣存在MSCs。②經(jīng)一定劑量的G-CSF動員后外周血CD44+細胞明顯增加,并與G-CSF呈劑量效應(yīng)關(guān)系,在未動員或20μg/kg G-CSF動員的情況下,外周血未能成功分離出MSCs,只在50μg/kg和80μg/kg G-CSF動員的情況下從外周血中分離培養(yǎng)出典型的MSCs樣集落。該細胞經(jīng)流式細胞儀分析結(jié)果證實符合MSCs的表面標志特征。③GSH對MSCs的影響：0.15mg/ml GSH可以縮短MSCs的群體倍增時間,并可減少MSCs凋亡,而對細胞的表面標志以及多向分化潛能無明顯改變；葛根素對MSCs的影響：葛根素可抑制細胞增殖并呈一定的劑量效應(yīng),但是0.0012 mol/L葛根素卻具有促進成骨細胞生成的作用；醋酸鉛對MSCs的影響：醋酸鉛可以引起抑制細胞增殖并呈濃度劑量效應(yīng),可以引起細胞凋亡,抑制細胞的成骨分化,并導致MSCs分泌細胞因子的能力也下降。④臍帶間質(zhì)干細胞能夠在殼聚糖支架上粘附生長,并保持良好的細胞形態(tài),與對照組相比,其粘附率和細胞增殖活性均無顯著性差異。 結(jié)論：①人胚胎骨髓、臍帶組織、骨髓都存在MSCs,但是臍帶血中是否存在MSCs還需要進一步研究。在宮頸癌組織標本中也發(fā)現(xiàn)了MSCs的存在,推測MSCs可能在腫瘤的發(fā)生發(fā)展中有著重要的作用。②在特定劑量的G-CSF的動員下,大鼠外周血MSCs可以得到動員。③GSH可促進細胞增殖速度并對MSCs的表面標志與多向分化能力無影響,可應(yīng)用于提高MSCs的體外擴增速度；葛根素可以影響抑制細胞增殖并促進MSCs向成骨分化；醋酸鉛可以影響的MSCs的增殖活性,引起MSCs凋亡和分化潛能降低,使其分泌因子能力下降,醋酸鉛破壞了MSCs即破壞了造血微環(huán)境,這可能在貧血的發(fā)生中有一定的價值。④臍帶間質(zhì)干細胞與殼聚糖支架之間具有良好的生物相容性,可應(yīng)用于復合材料的制備。
[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.
【學位授予單位】：江蘇大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R329

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