【摘要】：目的：手術(shù)切除是治療人的顱內(nèi)血管母細(xì)胞瘤的最佳方式,但是生長(zhǎng)在關(guān)鍵部位(例如顱底或者腦干)及顱內(nèi)多發(fā)的血管母細(xì)胞瘤阻礙的手術(shù)的實(shí)施,并且當(dāng)前沒(méi)有一種有效的藥物治療方式。通過(guò)一種有效的化學(xué)藥物,來(lái)抑制血管母細(xì)胞瘤的異常過(guò)多增生的血管,也許能能夠成為一種有效的治療方式。方法：通過(guò)免疫組織化學(xué)染色來(lái)檢測(cè)人的血管母細(xì)胞瘤的VEGF/TNFSF15及小膠質(zhì)/巨噬細(xì)胞的表達(dá)。利用bEnd.3細(xì)胞立體定向注射到小鼠的基底節(jié),建立小鼠的血管母細(xì)胞瘤模型。通過(guò)HE染色來(lái)評(píng)價(jià)血管瘤及出血的體積,ELISA檢測(cè)VEGF/TNFSF15的表達(dá)水平,CD68+,7/4+等免疫熒光染色分析炎性細(xì)胞對(duì)血管瘤的浸潤(rùn)情況。通過(guò)慢病毒轉(zhuǎn)染bEnd.3細(xì)胞,致使該細(xì)胞高表達(dá)TNFSF15,通過(guò)RT-PCR, Western blotting及ELISA,分析VEGF/TNFSF15的表達(dá)水平。細(xì)胞增殖實(shí)驗(yàn)和管腔形成實(shí)驗(yàn)來(lái)評(píng)價(jià)bEnd.3細(xì)胞的體外血管新生能力。結(jié)果：腫瘤壞死因子超家族成員-15(TNFSF15,又名血管內(nèi)皮生長(zhǎng)抑制因子)被認(rèn)為是一種抗血管新生的因子,TNFSF15能夠抑制血管內(nèi)皮細(xì)胞的生長(zhǎng),誘導(dǎo)分裂期血管內(nèi)皮細(xì)胞的凋亡,抑制血管內(nèi)皮祖細(xì)胞向血管內(nèi)皮細(xì)胞的轉(zhuǎn)化。通過(guò)免疫組織化學(xué)染色發(fā)現(xiàn),TNFSF15在正常人的微血管結(jié)構(gòu)中高度表達(dá),但是在人的血管母細(xì)胞瘤內(nèi)極低的表達(dá),上調(diào)血管母細(xì)胞瘤的TNFSF15也許能成為治療該病的有效靶點(diǎn)。來(lái)源于小鼠內(nèi)皮瘤的bEnd.3細(xì)胞系能夠分泌大量的血管內(nèi)皮生長(zhǎng)因子(VEGF), VEGF不但能夠促進(jìn)血管的生長(zhǎng)也能促進(jìn)血管的滲漏,但是b.End3細(xì)胞表達(dá)極少量的TNFSF15,研究發(fā)現(xiàn)bEnd.3細(xì)胞能在小鼠的皮下組織形成穩(wěn)定的血管瘤。我們把b. End3細(xì)胞移植到C57BL/6小鼠的顱內(nèi),能形成與人的血管母細(xì)胞瘤病理結(jié)構(gòu)極其相似的小鼠的顱內(nèi)細(xì)胞瘤。與對(duì)照組相比,當(dāng)把bEnd.3細(xì)胞移植到高TNFSF15表達(dá)的轉(zhuǎn)基因小鼠顱內(nèi),能夠形成較小體積的血管瘤和能減少血管瘤的出血,并且伴有較少的炎性細(xì)胞浸潤(rùn)和增多的周細(xì)胞募集。更重要的是,在TNFSF15高表達(dá)的轉(zhuǎn)基因小鼠中,能夠下調(diào)血管瘤中VEGF的表達(dá)水平。通過(guò)慢病毒轉(zhuǎn)染bEnd.3細(xì)胞,促使bEnd.3細(xì)胞高表達(dá)TNFSF15,亦能發(fā)現(xiàn)抑制血管瘤的生長(zhǎng)和出血,并也能下調(diào)VEGF的表達(dá)。體外研究亦證實(shí)TNFSF15能夠抑制bEnd.3細(xì)胞的生長(zhǎng)和下調(diào)VEGF的分泌。進(jìn)一步的研究發(fā)現(xiàn),通過(guò)使用死亡受體-3(DR-3)的拮抗劑來(lái)封閉DR-3的功能,發(fā)現(xiàn)TNFSF15不能發(fā)揮抑制bEnd.3細(xì)胞增殖和不能下調(diào)VEGF的表達(dá),為此我們推論TNSF15能夠通過(guò)DR3受體下調(diào)VEGF的表達(dá)水平,進(jìn)而抑制血管瘤的生長(zhǎng)及出血的發(fā)生。結(jié)論：與正常的腦的微血管相比,我們發(fā)現(xiàn)在人的血管母細(xì)胞瘤標(biāo)本中,存在高VEGF表達(dá)和極低TNFSF15表達(dá)的模式,這種異常的表達(dá)模式可能誘導(dǎo)了或者加重了血管母細(xì)胞瘤疾病的發(fā)生發(fā)展。在小鼠的顱內(nèi)血管瘤的模型中,TNFSF15能夠抑制血管瘤的生長(zhǎng)和減少出血的發(fā)生,抑制血管瘤周?chē)仔约?xì)胞的浸潤(rùn)和促進(jìn)周細(xì)胞的募集,并能下調(diào)血管瘤中VEGF的表達(dá)水平。機(jī)制研究發(fā)現(xiàn),TNFSF15是通過(guò)或者是部分通過(guò)DR-3受體來(lái)下調(diào)VEGF的表達(dá)。
[Abstract]:Objective: The surgical removal is the best way to treat the intracranial hemangioblastoma, but it is not effective in the treatment of the key parts (for example, the skull base or the brain stem) and the intracranial multiple angioblastoma. It may be an effective way to treat an abnormal hyperplastic vessel of a hemangioblastoma by an effective chemical agent. Methods: The expression of VEGF/ TNFSF15 and microglia/ macrophage in human hemangioblastoma was detected by immunohistochemical staining. The mouse's hemangioblastoma model was established by stereospecific injection of bEnd.3 cells into the basal ganglia of the mice. The volume of hemangiomas and hemorrhage was evaluated by HE staining, and the expression level of VEGF/ TNFSF15, CD68 +,7/4 + and other immunofluorescent staining were used to analyze the infiltration of hemangiomas. The expression level of VEGF/ TNF15 was analyzed by RT-PCR, Western blotting and ELISA. The cell proliferation assay and the lumen formation assay were used to evaluate the in vitro angiogenesis of bEnd.3 cells. Results: The tumor necrosis factor superfamily member-15 (TNFSF15, also known as the vascular endothelial growth inhibitory factor) was considered an anti-angiogenic factor, and TNFSF15 was able to inhibit the growth of vascular endothelial cells and induce the apoptosis of vascular endothelial cells during the division. To inhibit the transformation of the vascular endothelial progenitor cells to the vascular endothelial cells. TNFSF15 is highly expressed in the microvessel structure of normal persons by immunohistochemical staining, but the expression of TNFSF15 in the human hemangioblastoma is very low, and the up-regulation of the TNFSF15 of the hemangioblastoma may be an effective target for the treatment of the disease. The bEnd.3 cell line derived from mouse endothelioma can secrete a large amount of vascular endothelial growth factor (VEGF), which not only can promote the growth of blood vessels, but also promote the leakage of blood vessels, but the b. End3 cells express a very small amount of TNFSF15, The study found that bEnd.3 cells could form stable hemangiomas in the subcutaneous tissue of the mice. We transplanted b. End3 cells into the intracranial of C57BL/6 mice, and can form the intracranial cell tumor of mice with extremely similar pathological structure to human hemangioblastoma. Compared with the control group, when bEnd.3 cells were transplanted to the high TNFSF15-expressed transgenic mouse, the smaller volume of the hemangioma and the bleeding of the hemangioma can be reduced, with less inflammatory cell infiltration and increased peripheral cell recruitment. More importantly, in the highly expressed transgenic mice of TNFSF15, the level of VEGF expression in the hemangioma can be reduced. By transfecting bEnd.3 cells with lentivirus, the high-expression TNFSF15 of bEnd.3 cells can be induced, and the growth and bleeding of the hemangioma can be inhibited, and the expression of VEGF can also be reduced. The in vitro study also confirmed that TNFSF15 was able to inhibit the growth of bEnd.3 cells and down-regulate the secretion of VEGF. Further studies have found that by using an antagonist of the death receptor-3 (DR-3) to close the function of the DR-3, it is found that TNFSF15 is not capable of exerting the inhibition of bEnd.3 cell proliferation and the inability to downregulate the expression of VEGF, and for this reason we conclude that the TNSF15 is able to down-regulate the expression level of VEGF by the DR3 receptor, Thereby inhibiting the growth of the hemangioma and the occurrence of the hemorrhage. Conclusion: Compared with the normal brain microvessel, we find that there is a pattern of high VEGF expression and very low TNFSF15 expression in human hemangioblastoma, which may induce or aggravate the development of angioblastoma disease. In the model of the intracranial hemangioma of the mouse, the TNFSF15 can inhibit the growth of the hemangioma and reduce the occurrence of the hemorrhage, inhibit the infiltration of the inflammatory cells around the hemangioma and promote the recruitment of the peripheral cells, and can reduce the expression level of VEGF in the hemangioma. The mechanism study found that TNFSF15 is the expression of VEGF by or in part through the DR-3 receptor.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.41

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