【作者】 郑伟红；

【导师】 易继林；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【摘要】 第一部分从PBMCs及CD133免疫磁珠分选获得内皮祖细胞的体外研究目的：比较从外周血单个核细胞(PBMCs)和CD133免疫磁珠分选体外培养人外周血内皮祖细胞(EPCs)的表型、分化、扩增、功能特点,为EPCs细胞／基因治疗提供临床参考。方法：健康成年人外周血,经Ficoll密度梯度离心法得PBMCs,经直接接种法或CD133免疫磁珠分选后置于M199培养基中培养,于第7、14天比较两组细胞表型变化及分泌、扩增、体外血管形成及趋化能力。结果：相同量标本PBMCs组早期集落数高于CD133组(P<0.01),随着培养时间的推移,流式细胞检测两组细胞均显示造血干细胞标志的表达下调和内皮细胞标志表达上升,但CD133分选组CD144表达率低于PBMCs组(P<0.01)。ELISA法检测到在PBMCs组早期EPCs分泌VEGF的水平高于CD133分选组(P＜0.01)。MTT法显示PBMCs组有较强的增殖能力。基质胶及transwell实验表明PBMCs组细胞参与血管形成的能力较强。结论：CD133分选EPCs所得细胞分化、分泌、增殖及血管形成能力较低,分化为内皮表型不完全,不宜作为EPCs细胞治疗载体。第二部分氯化钴预处理对内皮祖细胞相关功能的影响目的：研究氯化钴(cobalt chloride, CoCl2)预处理对培养的内皮祖细胞(endothelial progenitor cell, EPC)体外相关功能的影响。方法：用不同浓度的CoCl2预处理培养的EPC,用MTT和ELISA检测细胞增殖、分泌能力,用细胞迁移和基质胶管型实验检测细胞趋化和体外管样形成的功能,Western蛋白印迹检测CoCl2处理后EPC相关蛋白的变化。结果：100μM浓度CoCl2预处理6小时可增加EPC分泌,趋化及体外管样结构形成；CoCl2浓度大于400μM或预处理超过24h显著降低EPC功能活性；随着CoCl2浓度的增高,BCL-2/BAX比值逐渐降低。结论：CoCl2低浓度短时间预处理可以增强EPC体外分泌,趋化及管样形成能力,而高浓度CoCl2降低EPC的生存及增殖能力,可能与促凋亡蛋白上调有关。第三部分携带自杀基因的内皮祖细胞靶向治疗人肝癌裸鼠皮下移植瘤的初步研究目的：观察转染自杀基因的EPC (EPC-CD/UPRT)对肝癌细胞HepG2和SMMC-7721的体外杀伤作用,建立人肝癌裸鼠皮下移植瘤模型,观察EPC-CD/UPRT直接和经氯化钴预处理后在裸鼠体内的杀伤效果。方法：扩增pORF5-Fcy::Fur质粒并酶切鉴定,用fugene HD将pORF5-Fcy::Fur质粒导入EPC,免疫组化检测目的基因表达,将EPC与肝癌细胞HepG2和SMMC-7721混合培养,用MTT检测其活性；在裸鼠颈部皮下接种肝癌细胞,成瘤后观察EPC-CD/UPRT在5-FC作用下对其杀伤作用,比较EPC-CD/UPRT直接注射或经氯化钴预处理后注射对肿瘤的抑制效果,比较两组肿瘤体积大小,计算抑瘤率；最后用TUNEL法检测肿瘤凋亡指数。结果：酶切电泳图显示质粒成功扩增,纯度达99%；免疫组化结果显示,EPC经基因改造后可表达目的蛋白CD;MTT分析表明EPC-CD/UPRT能发挥强大的旁观者效应,对肿瘤细胞造成明显的杀伤；EPC-CD/UPRT联合5-FC能有效的抑制裸鼠移植瘤的生长,氯化钴预处理后可增强抑制肿瘤效果；CD/UPRT/5-FC系统对肿瘤有促凋亡作用。结论：携带CD/UPRT自杀基因的EPC能有效抑制肝癌移植瘤的生长,经氯化钴预处理后将增强肿瘤抑制效果。更多还原

【Abstract】 Part-1 Isolation of Cultured Endothelial Progenitor Cells in vitro from PBMCs and CD133+Enriched CellsObjective:Two isolation methods for sorting of endothelial progenitor cells (EPCs): from peripheral blood mononuclear cells (PBMCs) and CD133+enriched cells were compared, by defining the cell morphology, phenotype, reproductive activities and function in vitro, to provide a reference for clinical application of EPCs.Methods:PBMCs from healthy subjects were used either directly for cell culture or for CD133+sorting. The two groups of cells were cultured in complete medium M199 for 7 to 14 days and the phenotypes of EPCs were analyzed by FACS. The proliferation of differentiated EPCs was studied by MTT assay, and the VEGF concentration was measured using an ELISA kit. ECM gel experiment and migration assay were performed in vivo.Results:The results showed that PBMCs produced more colony-forming units (CFU) than CD133+enriched cells from the same volume of blood (P<0.01). From day 7 to 14, the two groups show decreased expression of hematopoietic stem cell markers and increased level of endothelial markers, but CD144+cells in CD133+group were less than in PBMCs group (P＜0.01). PBMCs group secreted more VEGF than CD133+group on the day 7 (P＜0.01). As compared with CD133+group, PBMCs group had more potent potential of proliferation and vascularization in vitro.Conclusion:It was concluded that CD133+sorted cells showed a lower capacity of differentiation, secretion, proliferation and vascularization in vitro, speculating that CD133-negative cells may be a preferential way to get EPCs for clinical therapy.Part-2 Influence of Cobalt Chloride Preconditioning on The Proagiogenic Function of Endothelial Progenitor CellsObjective:To exploit the effect of cobalt chloride (CoCl2) pretreatment on angiogenic function in vitro of cultured endothelial progenitor cells(EPC).Methods:Cultured endothelial progenitor cells from patients were pretreated with CoCl2 under a range of concentrations. Then, Cell proliferation and VEGF secretion were examined by MTT and ELISA. Matrigel experiment and migration assay were imitate vascularization in vivo.the expressions of HIF-la and its target genes were examed by Western blot analysis.Results:CoCl2 pretreatment with 100μM at 6 hours enhanced the angiogenic function of EPC such as secretion, chemotaxis and tube formation in vitro, CoCl2 pretreatment with more than 400μM or 24h hinderd the functional properties of EPC, the ratio of BCL-2/BAX decreased along with an increasing concentration of CoCl2.Conclusions:CoCl2 preconditioning with low concentration and short time will enhance neovascularization of EPC in vitro, high concentration of CoCl2 reduce cell viability and proliferation, which may attributable to increased expression of proapoptotic gene. Part-3 Pilot Research on Targeted Therapy for Transplanted Human Hepatocarcinoma under Nude Mice Skin Based on Endothelial Progenitor Cells Armed with A Suicide GeneObjective:To explore the killing effect of gene modified EPC (EPC-CD/UPRT) on hepatocarcinoma HepG2 and SMMC-7721, established a transplanted tumor model of human hepatocarcinoma in nude mouse and study the killing effects of direct EPC-CD/UPRT treatment and pretreatment with cobalt chloride.Methods:pORF5-Fcy::Fur plasmid with its correctness evaluated by the means of restriction enzyme and destination protein detected by immunohistochemistry after transduction to EPC with fugene HD, HepG2/SMMC-7721 cells were mixed with modified EPC by different proportion and the total cell survival rate was analyzed with MTT. Hepatocarcinoma cells then be inoculated into the neck skin of nude mice. When the tumor is formed, the damage effects of EPC-CD/UPRT under the 5-fluorocytosine can be observed. The depressor effects in tumor with direct injection of EPC-CD/UPRT or pretreatment with cobalt chloride before application was compared, and then the tumor sizes and tumor inhibitory rate was calculated; finally, tumor Apoptosis Index was tested with TUNEL assay.Results:Enzymes electrophorogram shows that plasmids are extensively increased successfully with the purity of 99%; IHC results shows that after gene transformation, EPC can express the target protein CD; MTT analysis indicate that EPC-CD/UPRT can play a significant role of bystander effects which cause obvious damage to HepG2 and SMMC-7721; EPC-CD/UPRT mixed with 5-FC can effectively restrain the growth of transplanted tumor of nude mice; the pretreatment of cobalt chloride can enhance the inhibitory effects of tumor; CD/UPRT/5-FC system may play a proapoptosis role in tumors.Conclusions:EPC armed with CD/UPRT can effectively inhibit the growth of hepatocarcinoma transplanted tumor. The pretreatment of cobalt chloride may enhance the restraining effects by recruitment more EPC-CD/UPRTs更多还原