發(fā)布時間：2018-10-05 06:57

【摘要】：新生血管生成是惡性腫瘤發(fā)生、發(fā)展及浸潤的重要條件。腫瘤細(xì)胞微環(huán)境中存在大量對血管新生具有影響作用的因子和信號。通常情況下,促血管生成因子在腫瘤中過度表達(dá),例如VEGF,是促進(jìn)腫瘤血管新生的重要因素,我們形象的稱之為血管新生開關(guān)(angiogenic swich),由于血管新生在腫瘤病程發(fā)展中的重要作用,血管生成抑制劑可能為惡性腫瘤的治療提供一個有效的治療手段。CD47是分子量為50 kD的跨膜蛋白受體,也是免疫球蛋白家族的重要成員,具有一個類Ig的N-端,5個跨膜區(qū)和一個短的C-末端。CD47作為整合素蛋白,能夠通過與其調(diào)節(jié)蛋白SIRP-?結(jié)合,發(fā)揮細(xì)胞與細(xì)胞間信號傳導(dǎo)的作用。同時,CD47通過其配體TSP-1對內(nèi)皮細(xì)胞的功能進(jìn)行調(diào)節(jié),CD47存在能夠與TSP-1的C-末端結(jié)合的結(jié)構(gòu)域,TSP-1多肽或CD47抗體能夠高效的阻斷由NO所激活、cGMP所介導(dǎo)的血管內(nèi)皮細(xì)胞增殖。腫瘤細(xì)胞的增殖依賴于腫瘤血管的新生,同時腫瘤血管的增殖程度也標(biāo)志著腫瘤侵襲能力的大小,因此,針對CD47的治療可以作為惡性腫瘤治療的新的策略,抑制腫瘤過快生長,爭取寶貴的治療時間。在本研究中,我們證實了CD47可以促進(jìn)腫瘤血管新生,其機(jī)制是CD47缺失下調(diào)P53和P21表達(dá),促進(jìn)腫瘤細(xì)胞增殖和抑制細(xì)胞衰老,進(jìn)一步的研究顯示,CD47缺失同樣可以通過下調(diào)腫瘤血管內(nèi)皮細(xì)胞中P53和P21表達(dá)使內(nèi)皮細(xì)胞獲得永生,我們的研究提供了一個通過調(diào)節(jié)CD47及其下游通路抑制腫瘤血管新生的新的研究方向。近期的報道顯示:CD47對內(nèi)皮細(xì)胞功能的調(diào)節(jié)除了通過經(jīng)典途徑CD47-TSP-1之外,還可調(diào)節(jié)內(nèi)皮細(xì)胞的干性,使成體內(nèi)皮細(xì)胞出現(xiàn)永生化,重現(xiàn)細(xì)胞分化能力。在我們課題組前期對CD47缺失內(nèi)皮細(xì)胞的研究中,我們也曾獲得體外無限傳代,并且干性相關(guān)基因表達(dá)上調(diào)的內(nèi)皮細(xì)胞,但難以重復(fù)。通過我們對其功能深入研究發(fā)現(xiàn),我們觀察到CD47缺失可以使內(nèi)皮細(xì)胞中細(xì)胞周期相關(guān)因子的表達(dá),延緩細(xì)胞衰老,這一結(jié)果在腫瘤模型中得到了驗證,CD47缺失的小鼠體內(nèi)的腫瘤血管新生更加旺盛,且抗氧化應(yīng)激能力增強(qiáng)。本課題共分為兩部分:第一部分:體內(nèi)實驗:研究目的:深入研究cd47對于腫瘤血管增殖能力的影響對于研究血管新生的過程及調(diào)控機(jī)制非常重要,建立小鼠腫瘤模型,對腫瘤血管新生情況進(jìn)行研究,為進(jìn)一步的體外機(jī)制研究奠定基礎(chǔ)。研究方法:將rm1腫瘤細(xì)胞復(fù)蘇傳代后,將細(xì)胞注入小鼠腹股溝皮下。注射后11d處死小鼠,剝離腫瘤,切片后分別采用cd31,cd144抗體進(jìn)行腫瘤組織染色,鏡下拍照,利用imagepro-plus6.0軟件進(jìn)行分析。研究結(jié)果:在同樣生長周期下,cd47-/-小鼠體內(nèi)的腫瘤生長更為旺盛,腫瘤體積明顯大于wt小鼠。通過免疫組化研究發(fā)現(xiàn)腫瘤組織切片cd31+,cd144+的染色面積cd47-/-小鼠均高于wt小鼠,證實cd47-/-小鼠腫瘤組織中血管較wt小鼠豐富。結(jié)論:cd47缺失可促進(jìn)腫瘤組織血管新生,從而促進(jìn)腫瘤組織的快速生長。第二部分:體外實驗研究目的:通過分子生物學(xué)手段,解析cd47缺失對內(nèi)皮細(xì)胞在血管新生過程中功能變化的作用機(jī)制。1.內(nèi)皮細(xì)胞分離純化:研究方法:利用磁珠分選及apc分選的方法,對小鼠腫瘤細(xì)胞進(jìn)行分選,獲得內(nèi)皮細(xì)胞。結(jié)論:經(jīng)分離純化獲得內(nèi)皮細(xì)胞。2.cd47對內(nèi)皮細(xì)胞抗衰老能力影響的研究:研究方法:對cd47-/-和wt內(nèi)皮細(xì)胞分別培養(yǎng)2d,4d,6d后進(jìn)行β-半乳糖苷酶染色實驗,檢測其抗衰老能力,并對二者進(jìn)行對比。結(jié)論:cd47-/-可以使內(nèi)皮細(xì)胞抗衰老能力增強(qiáng)。3.cd47對內(nèi)皮細(xì)胞成管能力影響的研究:研究方法:利用成管實驗檢測cd47-/-和wt內(nèi)皮細(xì)胞成管能力,加樣后3h,8h分別在光鏡下觀察內(nèi)皮細(xì)胞成管的形態(tài)及長度。結(jié)論:cd47-/-可以使內(nèi)皮細(xì)胞成管能力增強(qiáng)。4.cd47對內(nèi)皮細(xì)胞周期影響的研究:研究方法:內(nèi)皮細(xì)胞傳代第2,3,4,5代(分別標(biāo)示為P2,P3,P4,P5),分別利用PI單染色法分別檢測內(nèi)皮細(xì)胞中G0/G1期,S期,G2期細(xì)胞所占比例,并對CD47-/-和WT內(nèi)皮細(xì)胞進(jìn)行比較。結(jié)論:CD47影響內(nèi)皮細(xì)胞的細(xì)胞周期,CD47-/-促進(jìn)細(xì)胞有絲分裂,導(dǎo)致細(xì)胞增殖能力加強(qiáng)。5.CD47影響內(nèi)皮細(xì)胞周期機(jī)制的研究:研究方法:利用PCR法分別檢測CD47-/-和WT內(nèi)皮細(xì)胞p53,p21,c-myc mRNA表達(dá)水平并對二者進(jìn)行比較。結(jié)論:CD47對細(xì)胞周期調(diào)控蛋白具有影響,CD47-/-導(dǎo)致內(nèi)皮細(xì)胞p53,p21表達(dá)下降,促進(jìn)細(xì)胞的有絲分裂,內(nèi)皮細(xì)胞c-myc表達(dá)上調(diào),內(nèi)皮細(xì)胞干性增強(qiáng),增殖能力增強(qiáng)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R730.2

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