人膀胱间充质干细胞分离及其对膀胱癌细胞增殖迁移及自噬的影响

发布时间：2018-04-19 16:49

本文选题：人膀胱间充质干细胞 + 膀胱尿路上皮癌　；参考：《第二军医大学》2016年博士论文

【摘要】：一、研究背景膀胱癌是泌尿生殖系统最常见的恶性肿瘤之一。膀胱癌占中国泌尿生殖系肿瘤发病率的首位。膀胱尿路上皮癌既往被称为膀胱移行细胞癌,它是最常见的膀胱恶性肿瘤类型,占膀胱癌患者总数的80%以上。目前膀胱尿路上皮癌总体呈现出发病率升高与年轻化的特点,早期非浸润型膀胱癌外科手术治疗后复发率高,行膀胱根治性切除术后患者生活质量显著下降。膀胱尿路上皮癌对放化疗的敏感性较低,一旦肿瘤发生转移,患者预后较差。针对膀胱尿路上皮癌的治疗急需一种新的治疗靶点以提高治疗效果。肿瘤是由许多类型的细胞组成的一种器官样结构。这些细胞通过相互作用,推动和促进肿瘤的生长和转移。致癌细胞从本地和邻近组织中招募非致瘤性细胞,同时通过循环系统构建肿瘤微环境。肿瘤微环境可以交互消息,刺激肿瘤的生长。它包含癌相关成纤维细胞,内皮细胞,免疫细胞,脂肪细胞,分化成转移性上皮细胞的癌症干细胞,具有分化成为成纤维细胞和间充质细胞谱系中的代表性细胞能力的间充质干细胞/基质细胞,以及不同类型的细胞外基质蛋白。他们通过旁分泌信号及物理性作用相互影响,促进癌症的进展。这些不同的癌症间质,特别是间充质干细胞和从干细胞中提取的基质细胞,最近已被确认从肿瘤发生的早期开始影响肿瘤的生长、发展、进展,在癌症患病过程中扮演重要的角色。它通过上皮间质的转变和转移,影响微环境的构造,使得肿瘤得以维护并转移到其他组织。膀胱粘膜下层和基质层中存在间质细胞。我们推测膀胱组织中存在间充质干细胞(mesenchymal stem cell,MSC),因为MSC广泛分部于人体组织中,目前可以从骨髓、肌肉、脂肪、脐带等多种具有间质的组织中分离得到。MSC具有向多组织细胞分化的潜能,能产生和分泌多种细胞因子和生长因子,参与炎症反应和肿瘤发生等。因此,深入研究膀胱间充质干细胞与膀胱尿路上皮癌之间的关系,可能为治疗癌症、预防复发提供新策略。二、研究目的本论文探索从人膀胱组织中分离培养得到膀胱间充质干细胞(human bladder derived Mesenchymal stem cells,h BSC)的实验方法,进而分析h BSC与脂肪、骨髓等其他组织来源间充质干细胞的差别,然后通过诱导h BSC三系分化及向其它类型膀胱组织细胞的分化实验以进一步验证其干细胞潜能。然后通过与癌细胞系的共培养测量细胞的生长与迁移并研究其可能的机制。最后在营养缺乏环境下检测实验细胞凋亡率,评估h BSC对膀胱癌细胞系细胞自噬作用的影响其作用机制。据此探索膀胱间充质干细胞在膀胱尿路上皮癌疾病发展过程中所发挥的作用机制。三、主要研究内容1.从全膀胱癌切除的正常组织中分离培养人膀胱间充质干细胞(h BSC),流式细胞仪鉴定细胞表面抗原分子,诱导h BSC向成脂、成骨和成软骨三系分化,诱导h BSC向其他类型膀胱组织细胞分化;2.从细胞增殖、端粒酶活性以及细胞因子分泌几个方面比较h BSC与骨髓和脐带来源的人间充质干细胞的差别;3.将h BSC与膀胱癌细胞系5637和J82通过transwell小室共培养,或收集h BSC条件性培养基用于5637和J82培养,测定细胞增殖和细胞迁移,Western blot检测p70、AKT和STAT3蛋白质的磷酸化水平,荧光定量PCR检测EMT相关基因表达。4.收集h BSC条件性培养基用于5637和J82培养,细胞免疫荧光检测LAMP2,Western blot检测细胞自噬基因LC3B,转染EGFP-LC3B质粒观察处理后的荧光蛋白分布,Annexin V-FITC标记后采用流式细胞仪检测营养缺乏时的细胞凋亡率,评估h BSC对膀胱癌细胞系细胞自噬作用的影响。四、结果1.分离得到的人膀胱间充质干细胞(h BSC),建立合适的培养体系,流式细胞仪鉴定h BSC表达CD13、CD29、CD73、CD90和CD105分子,不表达CD14、CD19、CD31、CD34、CD45和HLA-DR,低表达HLA-ABC。h BSC经过诱导可分化成为脂肪、骨和软骨细胞,经特殊培养基诱导能够分化成尿路上皮样、内皮样及平滑肌样细胞。2.h BSC与人骨髓来源间充质干细胞(h BM-MSC)和人脐带来源间充质干细胞(h UC-MSC)比较,h BSC和h UC-MSC的细胞生长速度相同,但h BSC的端粒酶活性却与h BM-MSC相似,细胞因子分泌方面h BSC中EGF、IGF1、IL-1β和IL-6的分泌量略高于h BM-MSC和h UC-MSC。3.h BSC与膀胱癌细胞系5637和J82共培养,采用transwell共培养或条件培养基培养都能够促进5637生长及迁移,而对J82的作用不明显,机制分析认为胞内p70和STAT3蛋白质被诱导磷酸化是5637细胞生长速度增加的重要原因,而h BSC促进5637细胞发生EMT转化,从而促进5637迁移。4.低血清或无血清饥饿时,h BSC的条件培养基能够促进5637细胞自噬,诱导胞内LC3BⅡ增加,LC3B和LAMP2在5637细胞中出现点状聚集。五、结论通过本课题建立的方法,可以从人膀胱粘膜下层和肌层组织中分离得到膀胱间充质干细胞。人膀胱间充质干细胞(h BSC)具有与其他组织来源间充质干细胞相同的细胞表面抗原分子和三系分化能力。h BSC细胞增殖、端粒酶活性和细胞因子分泌与其他组织来源MSC相比较有其特点。h BSC能够促进体外共培养的膀胱癌细胞系5637生长,在此过程中,m TOR和STAT3途径可能起到重要作用;h BSC能够诱导5627细胞EMT转变,从而促进细胞迁移。在血清饥饿时h BSC促进5637细胞自噬和存活。本课题研究提高了人们对膀胱组织细胞构成的认知,认识到人膀胱间充质干细胞在膀胱尿路上皮癌细胞生长及迁移等生理过程中发挥重要作用,提出人膀胱间充质干细胞可能是膀胱尿路上皮癌治疗的新靶点。
[Abstract]:Bladder cancer is one of the most common malignant tumors in the genitourinary system. Bladder cancer accounts for the highest incidence of urogenital cancer in China. Bladder urothelial carcinoma is formerly known as bladder transitional cell carcinoma. It is the most common type of bladder cancer, accounting for more than 80% of the total number of bladder cancer. The overall incidence of cancer was higher and younger. The recurrence rate of early non invasive bladder cancer was higher after surgical treatment. The quality of life decreased significantly after radical cystectomy. The sensitivity of bladder urothelial carcinoma to radiotherapy and chemotherapy was low, and the prognosis of the patients was poor. Cancer therapy is in urgent need of a new therapeutic target to improve the therapeutic effect. A tumor is an organ like structure composed of many types of cells. These cells promote and promote tumor growth and metastasis through interaction. Carcinogenic cells recruit non tumorigenic cells from local and adjacent tissues and construct tumors through a circulatory system. Microenvironment. A tumor microenvironment can interact messages to stimulate the growth of a tumor. It contains cancer related fibroblasts, endothelial cells, immune cells, adipocytes, cancer stem cells differentiated into metastatic epithelial cells, and a mesenchymal stem cell / base that differentiates into the representative cell capacity of fibroblasts and mesenchymal cells. Stromal cells and different types of extracellular matrix proteins. They interact with each other through paracrine signals and physical effects to promote cancer progress. These different cancers, especially mesenchymal stem cells and stromal cells extracted from stem cells, have recently been confirmed to affect the growth of the tumor from the early stage of the tumorigenesis. Development, progress, plays an important role in the process of cancer disease. It affects the microstructure of the microenvironment through the transformation and transfer of epithelial stroma, which enables the tumor to be maintained and transferred to other tissues. Interstitial cells exist in the submucosa of the bladder and the matrix layer. We speculate that the mesenchymal stem cell exists in the bladder fabric. MSC), because MSC is widely used in human tissue, it is possible to separate the potential of.MSC from bone marrow, muscle, fat, umbilical cord and many other interstitial tissues, which can produce and secrete a variety of cytokines and growth factors, participate in inflammatory reactions and tumorigenesis. The relationship between stromal cells and bladder urothelial carcinoma may provide a new strategy for the treatment of cancer and prevention of recurrence. Two. The purpose of this paper is to explore the experimental methods of separating and cultivating human bladder derived Mesenchymal stem cells, H BSC from human bladder tissue, and then analyzing h BSC and fat, bone marrow The differentiation of mesenchymal stem cells from other tissue sources, and then the differentiation of H BSC three lines and differentiation to other types of bladder tissue cells to further verify their stem cell potential. Then the possible mechanism of cell growth and migration was measured by co culture with the cancer cell lines and the possible mechanisms were studied. Finally, in the nutritional deficiency environment. To detect the apoptosis rate of experimental cells and evaluate the effect of H BSC on the autophagy of bladder cancer cell line, and to explore the mechanism of action of bladder mesenchymal stem cells in the development of bladder urothelial carcinoma. Three, main content 1. isolated and cultured human bladder mesenchyme from normal tissues removed from bladder cancer. H BSC, flow cytometer identified cell surface antigen molecules, induced H BSC to differentiate into fat, osteogenic and chondrogenic three lines, induced H BSC to differentiate into other types of bladder tissue cells, and 2. compared h BSC with human mesenchymal stem cells derived from bone marrow and umbilical cord from several aspects of cell proliferation, telomerase activity and cytokine secretion. 3. h BSC and bladder cancer cell lines 5637 and J82 were co cultured in Transwell compartment, or H BSC conditioned medium was collected for 5637 and J82 culture, cell proliferation and cell migration, Western blot detection p70, AKT and STAT3 protein phosphorylation level. The culture medium was used in 5637 and J82 culture. Cell immunofluorescence was detected by LAMP2, Western blot was used to detect the autophagy gene LC3B. The distribution of fluorescent protein after transfection of EGFP-LC3B plasmid was observed. The apoptotic rate was detected by flow cytometry after Annexin V-FITC, and the autophagy of the bladder cancer cell line was evaluated by the flow cytometry. Four, four, result 1. isolated human bladder mesenchymal stem cells (H BSC), establish a suitable culture system. Flow cytometer identified h BSC expression CD13, CD29, CD73, CD90 and CD105 molecules, and did not express CD14, CD19, CD31, and differentiated into fat, bone and cartilage cells, through special medium. Induction can differentiate into urinary tract epithelioid, endothelioid and smooth muscle like cells.2.h BSC compared with human mesenchymal stem cells (H BM-MSC) and human umbilical cord derived mesenchymal stem cells (H UC-MSC). The growth rate of H BSC and H UC-MSC is the same, but the telomerase activity of H BSC is similar to that of the human mesenchymal stem cells. The secretion of F, IGF1, IL-1 beta and IL-6 is slightly higher than that of H BM-MSC and H UC-MSC.3.h BSC and bladder cancer cell line 5637 and J82 co culture. The use of Transwell co culture or conditioned medium can promote the growth and migration of 5637, but the effect on J82 is not obvious. Mechanism analysis suggests that intracellular and protein are induced to be phosphorylated in 5637 cells. The important reason for the increase of long speed, while h BSC promotes EMT transformation in 5637 cells, thus promoting the 5637 migration of.4. low serum or serum free starvation, the conditioned medium of H BSC can promote autophagy of 5637 cells, induce intracellular LC3B II increase, LC3B and LAMP2 appear in the 5637 cells. Five. Conclusion the method established through this subject can be established. Human bladder mesenchymal stem cells are isolated from the submucosa and myometrium of human bladder. Human bladder mesenchymal stem cells (H BSC) have the same cell surface antigen molecules as other tissue derived mesenchymal stem cells and the proliferation of three lineage differentiation ability.H BSC cells. Telomerase activity and cytokine secretion are compared with other tissue sources MSC .h BSC can promote the growth of bladder cancer cell line 5637 co cultured in vitro. In this process, m TOR and STAT3 pathway may play an important role. H BSC can induce EMT transformation of 5627 cells and promote cell migration. H BSC promotes autophagy and survival of 5637 cells in serum starvation. The cognition of the composition of the cell is important for the human bladder mesenchymal stem cells to play an important role in the growth and migration of bladder urothelial carcinoma cells. The proposed human bladder mesenchymal stem cells may be a new target for the treatment of bladder urothelial carcinoma.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.14

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