CD47基因缺陷对恶性肿瘤血管新生的影响及其机制研究
发布时间：2018-10-05 06:57
【摘要】：新生血管生成是恶性肿瘤发生、发展及浸润的重要条件。肿瘤细胞微环境中存在大量对血管新生具有影响作用的因子和信号。通常情况下,促血管生成因子在肿瘤中过度表达,例如VEGF,是促进肿瘤血管新生的重要因素,我们形象的称之为血管新生开关(angiogenic swich),由于血管新生在肿瘤病程发展中的重要作用,血管生成抑制剂可能为恶性肿瘤的治疗提供一个有效的治疗手段。CD47是分子量为50 kD的跨膜蛋白受体,也是免疫球蛋白家族的重要成员,具有一个类Ig的N-端,5个跨膜区和一个短的C-末端。CD47作为整合素蛋白,能够通过与其调节蛋白SIRP-?结合,发挥细胞与细胞间信号传导的作用。同时,CD47通过其配体TSP-1对内皮细胞的功能进行调节,CD47存在能够与TSP-1的C-末端结合的结构域,TSP-1多肽或CD47抗体能够高效的阻断由NO所激活、cGMP所介导的血管内皮细胞增殖。肿瘤细胞的增殖依赖于肿瘤血管的新生,同时肿瘤血管的增殖程度也标志着肿瘤侵袭能力的大小,因此,针对CD47的治疗可以作为恶性肿瘤治疗的新的策略,抑制肿瘤过快生长,争取宝贵的治疗时间。在本研究中,我们证实了CD47可以促进肿瘤血管新生,其机制是CD47缺失下调P53和P21表达,促进肿瘤细胞增殖和抑制细胞衰老,进一步的研究显示,CD47缺失同样可以通过下调肿瘤血管内皮细胞中P53和P21表达使内皮细胞获得永生,我们的研究提供了一个通过调节CD47及其下游通路抑制肿瘤血管新生的新的研究方向。近期的报道显示:CD47对内皮细胞功能的调节除了通过经典途径CD47-TSP-1之外,还可调节内皮细胞的干性,使成体内皮细胞出现永生化,重现细胞分化能力。在我们课题组前期对CD47缺失内皮细胞的研究中,我们也曾获得体外无限传代,并且干性相关基因表达上调的内皮细胞,但难以重复。通过我们对其功能深入研究发现,我们观察到CD47缺失可以使内皮细胞中细胞周期相关因子的表达,延缓细胞衰老,这一结果在肿瘤模型中得到了验证,CD47缺失的小鼠体内的肿瘤血管新生更加旺盛,且抗氧化应激能力增强。本课题共分为两部分:第一部分:体内实验:研究目的:深入研究cd47对于肿瘤血管增殖能力的影响对于研究血管新生的过程及调控机制非常重要,建立小鼠肿瘤模型,对肿瘤血管新生情况进行研究,为进一步的体外机制研究奠定基础。研究方法:将rm1肿瘤细胞复苏传代后,将细胞注入小鼠腹股沟皮下。注射后11d处死小鼠,剥离肿瘤,切片后分别采用cd31,cd144抗体进行肿瘤组织染色,镜下拍照,利用imagepro-plus6.0软件进行分析。研究结果:在同样生长周期下,cd47-/-小鼠体内的肿瘤生长更为旺盛,肿瘤体积明显大于wt小鼠。通过免疫组化研究发现肿瘤组织切片cd31+,cd144+的染色面积cd47-/-小鼠均高于wt小鼠,证实cd47-/-小鼠肿瘤组织中血管较wt小鼠丰富。结论:cd47缺失可促进肿瘤组织血管新生,从而促进肿瘤组织的快速生长。第二部分:体外实验研究目的:通过分子生物学手段,解析cd47缺失对内皮细胞在血管新生过程中功能变化的作用机制。1.内皮细胞分离纯化:研究方法:利用磁珠分选及apc分选的方法,对小鼠肿瘤细胞进行分选,获得内皮细胞。结论:经分离纯化获得内皮细胞。2.cd47对内皮细胞抗衰老能力影响的研究:研究方法:对cd47-/-和wt内皮细胞分别培养2d,4d,6d后进行β-半乳糖苷酶染色实验,检测其抗衰老能力,并对二者进行对比。结论:cd47-/-可以使内皮细胞抗衰老能力增强。3.cd47对内皮细胞成管能力影响的研究:研究方法:利用成管实验检测cd47-/-和wt内皮细胞成管能力,加样后3h,8h分别在光镜下观察内皮细胞成管的形态及长度。结论:cd47-/-可以使内皮细胞成管能力增强。4.cd47对内皮细胞周期影响的研究:研究方法:内皮细胞传代第2,3,4,5代(分别标示为P2,P3,P4,P5),分别利用PI单染色法分别检测内皮细胞中G0/G1期,S期,G2期细胞所占比例,并对CD47-/-和WT内皮细胞进行比较。结论:CD47影响内皮细胞的细胞周期,CD47-/-促进细胞有丝分裂,导致细胞增殖能力加强。5.CD47影响内皮细胞周期机制的研究:研究方法:利用PCR法分别检测CD47-/-和WT内皮细胞p53,p21,c-myc mRNA表达水平并对二者进行比较。结论:CD47对细胞周期调控蛋白具有影响,CD47-/-导致内皮细胞p53,p21表达下降,促进细胞的有丝分裂,内皮细胞c-myc表达上调,内皮细胞干性增强,增殖能力增强。
[Abstract]:Neoangiogenesis is an important condition for the occurrence, development and infiltration of malignant tumors. There is a large number of factors and signals that affect angiogenesis in tumor cell microenvironment. in general, that angiogenesis factor is excessively express in the tumor, such as VEGF, is an important factor for promoting the angiogenesis of the tumor, and the image is called a angiogenesis switch, and the angiogenesis factor plays an important role in the development of the tumor course, angiogenesis inhibitors may provide an effective means of treatment for the treatment of malignant tumors. CD47 is a transmembrane protein receptor with a molecular weight of 50 kD and an important member of the immunoglobulin family with N-terminal, 5 transmembrane regions and a short C-terminus of a class Ig. CD47, as a whole protein, can be used to regulate protein SIRP-? In combination, play a role in signal transduction between cells and cells. At the same time, CD47 regulates endothelial cell function by its ligand TSP-1. CD47 has a domain capable of binding to the C-terminal of TSP-1, and TSP-1 polypeptide or CD47 antibody can effectively block the proliferation of vascular endothelial cells mediated by NO. The proliferation of tumor cells depends on the angiogenesis of tumor angiogenesis, and the degree of proliferation of tumor vessels also marks the size of tumor invasion ability. Therefore, the treatment of CD47 can be used as a new strategy for malignant tumor treatment, so as to inhibit tumor growth and seek valuable treatment time. in this study, we confirm that CD47 can promote angiogenesis of tumor, its mechanism is CD47 deletion down-regulation of P53 and P21 expression, promote tumor cell proliferation and inhibit cell senescence, further study and display, CD47 deletion can also reduce the expression of P53 and P21 in vascular endothelial cells of tumor cells so that the endothelial cells can be immortal, and our research provides a new research direction for inhibiting tumor angiogenesis by modulating CD47 and its downstream pathway. Recent reports show that the regulation of the function of endothelial cells by CD47 can regulate the drying of endothelial cells in addition to the classical pathway of CD47-TSP-1, so as to make the endothelial cells immortal and reproduce the ability of cell differentiation. In the study of CD47-deficient endothelial cells in our previous research group, we have also obtained infinite passages in vitro, and the dry-related genes express up-regulated endothelial cells, but it is difficult to repeat. We have found that CD47 deletion can lead to the expression of cell cycle-related factors in endothelial cells and delay cell senescence. and the resistance to oxidative stress is enhanced. The objective of this study is to study the effect of cd47 on tumor angiogenesis and to establish a mouse tumor model to study the angiogenesis of tumor. laying the foundation for further in vitro mechanism research. Methods: After the rm1 tumor cells were recovered and passaged, the cells were injected into the inguinal skin of the mice. After the injection, the mice were sacrificed and the tumor was peeled off. The cd31 and cd144 antibodies were used for tumor tissue staining, and then photographed under the microscope and analyzed using the imagepro-plus6.0 software. Results: In the same growth cycle, tumor growth in cd47-/-mice was more vigorous and tumor volume was significantly larger than that of weight mice. It was found that cd31 +, cd144 + staining area cd47-/-mice were higher in cd47-/-mice than wt mice by immunohistochemistry. Conclusion: CD47 deletion can promote the angiogenesis of tumor tissue and promote the rapid growth of tumor tissue. Part two: The purpose of in vitro experimental study is to analyze the mechanism of cd47 deletion on the function of endothelial cells during angiogenesis by means of molecular biology. Isolation and purification of endothelial cells: A method for sorting and sorting tumor cells of mice by magnetic bead sorting and apc sorting was carried out to obtain endothelial cells. Conclusion: The effects of isolated and purified endothelial cells on anti-aging ability of endothelial cells were studied. The anti-aging ability of cd47-/-and wt endothelial cells were cultured for 2d, 4d and 6d, respectively, and their anti-aging abilities were tested and compared. Conclusion: cd47-/-can enhance the anti-aging ability of endothelial cells. The morphology and length of endothelial cells were observed under light microscope. Conclusion: cd47-/-can enhance the ability of endothelial cells to enhance the cycle of endothelial cells. 4. cd47 studies the effects of cd47 on the cycle of endothelial cells: the first, third, fourth and fifth generations of endothelial cells (labeled P2, P3, P4, P5, respectively), respectively detecting G0/ G1 phase and S phase in endothelial cells by using PI single staining method. The percentage of G2 phase cells was compared with that of CD47-/-and WT endothelial cells. Conclusion: CD47 affects the cell cycle of endothelial cells, CD47-/-promotes the mitosis of cells, and leads to the enhancement of cell proliferation ability. 5. CD47 studies the mechanism of endothelial cell cycle: study methods: CD47-/-and WT endothelial cell p53, p21 were detected by PCR, respectively. c-myc mRNA expression level and compare them. Conclusion: CD47 has an effect on cell cycle regulation protein. CD47-/-leads to the decrease of p53 and p21 expression in endothelial cells, promotes the mitosis of cells, the expression of c-myc in endothelial cells is upregulated, the dry enhancement and proliferation of endothelial cells are enhanced.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R730.2
[Abstract]:Neoangiogenesis is an important condition for the occurrence, development and infiltration of malignant tumors. There is a large number of factors and signals that affect angiogenesis in tumor cell microenvironment. in general, that angiogenesis factor is excessively express in the tumor, such as VEGF, is an important factor for promoting the angiogenesis of the tumor, and the image is called a angiogenesis switch, and the angiogenesis factor plays an important role in the development of the tumor course, angiogenesis inhibitors may provide an effective means of treatment for the treatment of malignant tumors. CD47 is a transmembrane protein receptor with a molecular weight of 50 kD and an important member of the immunoglobulin family with N-terminal, 5 transmembrane regions and a short C-terminus of a class Ig. CD47, as a whole protein, can be used to regulate protein SIRP-? In combination, play a role in signal transduction between cells and cells. At the same time, CD47 regulates endothelial cell function by its ligand TSP-1. CD47 has a domain capable of binding to the C-terminal of TSP-1, and TSP-1 polypeptide or CD47 antibody can effectively block the proliferation of vascular endothelial cells mediated by NO. The proliferation of tumor cells depends on the angiogenesis of tumor angiogenesis, and the degree of proliferation of tumor vessels also marks the size of tumor invasion ability. Therefore, the treatment of CD47 can be used as a new strategy for malignant tumor treatment, so as to inhibit tumor growth and seek valuable treatment time. in this study, we confirm that CD47 can promote angiogenesis of tumor, its mechanism is CD47 deletion down-regulation of P53 and P21 expression, promote tumor cell proliferation and inhibit cell senescence, further study and display, CD47 deletion can also reduce the expression of P53 and P21 in vascular endothelial cells of tumor cells so that the endothelial cells can be immortal, and our research provides a new research direction for inhibiting tumor angiogenesis by modulating CD47 and its downstream pathway. Recent reports show that the regulation of the function of endothelial cells by CD47 can regulate the drying of endothelial cells in addition to the classical pathway of CD47-TSP-1, so as to make the endothelial cells immortal and reproduce the ability of cell differentiation. In the study of CD47-deficient endothelial cells in our previous research group, we have also obtained infinite passages in vitro, and the dry-related genes express up-regulated endothelial cells, but it is difficult to repeat. We have found that CD47 deletion can lead to the expression of cell cycle-related factors in endothelial cells and delay cell senescence. and the resistance to oxidative stress is enhanced. The objective of this study is to study the effect of cd47 on tumor angiogenesis and to establish a mouse tumor model to study the angiogenesis of tumor. laying the foundation for further in vitro mechanism research. Methods: After the rm1 tumor cells were recovered and passaged, the cells were injected into the inguinal skin of the mice. After the injection, the mice were sacrificed and the tumor was peeled off. The cd31 and cd144 antibodies were used for tumor tissue staining, and then photographed under the microscope and analyzed using the imagepro-plus6.0 software. Results: In the same growth cycle, tumor growth in cd47-/-mice was more vigorous and tumor volume was significantly larger than that of weight mice. It was found that cd31 +, cd144 + staining area cd47-/-mice were higher in cd47-/-mice than wt mice by immunohistochemistry. Conclusion: CD47 deletion can promote the angiogenesis of tumor tissue and promote the rapid growth of tumor tissue. Part two: The purpose of in vitro experimental study is to analyze the mechanism of cd47 deletion on the function of endothelial cells during angiogenesis by means of molecular biology. Isolation and purification of endothelial cells: A method for sorting and sorting tumor cells of mice by magnetic bead sorting and apc sorting was carried out to obtain endothelial cells. Conclusion: The effects of isolated and purified endothelial cells on anti-aging ability of endothelial cells were studied. The anti-aging ability of cd47-/-and wt endothelial cells were cultured for 2d, 4d and 6d, respectively, and their anti-aging abilities were tested and compared. Conclusion: cd47-/-can enhance the anti-aging ability of endothelial cells. The morphology and length of endothelial cells were observed under light microscope. Conclusion: cd47-/-can enhance the ability of endothelial cells to enhance the cycle of endothelial cells. 4. cd47 studies the effects of cd47 on the cycle of endothelial cells: the first, third, fourth and fifth generations of endothelial cells (labeled P2, P3, P4, P5, respectively), respectively detecting G0/ G1 phase and S phase in endothelial cells by using PI single staining method. The percentage of G2 phase cells was compared with that of CD47-/-and WT endothelial cells. Conclusion: CD47 affects the cell cycle of endothelial cells, CD47-/-promotes the mitosis of cells, and leads to the enhancement of cell proliferation ability. 5. CD47 studies the mechanism of endothelial cell cycle: study methods: CD47-/-and WT endothelial cell p53, p21 were detected by PCR, respectively. c-myc mRNA expression level and compare them. Conclusion: CD47 has an effect on cell cycle regulation protein. CD47-/-leads to the decrease of p53 and p21 expression in endothelial cells, promotes the mitosis of cells, the expression of c-myc in endothelial cells is upregulated, the dry enhancement and proliferation of endothelial cells are enhanced.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R730.2
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