TNFSF15抑制小鼠颅内血管母细胞瘤的生长及出血

发布时间：2019-06-26 10:34

【摘要】：目的：手术切除是治疗人的颅内血管母细胞瘤的最佳方式,但是生长在关键部位(例如颅底或者脑干)及颅内多发的血管母细胞瘤阻碍的手术的实施,并且当前没有一种有效的药物治疗方式。通过一种有效的化学药物,来抑制血管母细胞瘤的异常过多增生的血管,也许能能够成为一种有效的治疗方式。方法：通过免疫组织化学染色来检测人的血管母细胞瘤的VEGF/TNFSF15及小胶质/巨噬细胞的表达。利用bEnd.3细胞立体定向注射到小鼠的基底节,建立小鼠的血管母细胞瘤模型。通过HE染色来评价血管瘤及出血的体积,ELISA检测VEGF/TNFSF15的表达水平,CD68+,7/4+等免疫荧光染色分析炎性细胞对血管瘤的浸润情况。通过慢病毒转染bEnd.3细胞,致使该细胞高表达TNFSF15,通过RT-PCR, Western blotting及ELISA,分析VEGF/TNFSF15的表达水平。细胞增殖实验和管腔形成实验来评价bEnd.3细胞的体外血管新生能力。结果：肿瘤坏死因子超家族成员-15(TNFSF15,又名血管内皮生长抑制因子)被认为是一种抗血管新生的因子,TNFSF15能够抑制血管内皮细胞的生长,诱导分裂期血管内皮细胞的凋亡,抑制血管内皮祖细胞向血管内皮细胞的转化。通过免疫组织化学染色发现,TNFSF15在正常人的微血管结构中高度表达,但是在人的血管母细胞瘤内极低的表达,上调血管母细胞瘤的TNFSF15也许能成为治疗该病的有效靶点。来源于小鼠内皮瘤的bEnd.3细胞系能够分泌大量的血管内皮生长因子(VEGF), VEGF不但能够促进血管的生长也能促进血管的渗漏,但是b.End3细胞表达极少量的TNFSF15,研究发现bEnd.3细胞能在小鼠的皮下组织形成稳定的血管瘤。我们把b. End3细胞移植到C57BL/6小鼠的颅内,能形成与人的血管母细胞瘤病理结构极其相似的小鼠的颅内细胞瘤。与对照组相比,当把bEnd.3细胞移植到高TNFSF15表达的转基因小鼠颅内,能够形成较小体积的血管瘤和能减少血管瘤的出血,并且伴有较少的炎性细胞浸润和增多的周细胞募集。更重要的是,在TNFSF15高表达的转基因小鼠中,能够下调血管瘤中VEGF的表达水平。通过慢病毒转染bEnd.3细胞,促使bEnd.3细胞高表达TNFSF15,亦能发现抑制血管瘤的生长和出血,并也能下调VEGF的表达。体外研究亦证实TNFSF15能够抑制bEnd.3细胞的生长和下调VEGF的分泌。进一步的研究发现,通过使用死亡受体-3(DR-3)的拮抗剂来封闭DR-3的功能,发现TNFSF15不能发挥抑制bEnd.3细胞增殖和不能下调VEGF的表达,为此我们推论TNSF15能够通过DR3受体下调VEGF的表达水平,进而抑制血管瘤的生长及出血的发生。结论：与正常的脑的微血管相比,我们发现在人的血管母细胞瘤标本中,存在高VEGF表达和极低TNFSF15表达的模式,这种异常的表达模式可能诱导了或者加重了血管母细胞瘤疾病的发生发展。在小鼠的颅内血管瘤的模型中,TNFSF15能够抑制血管瘤的生长和减少出血的发生,抑制血管瘤周围炎性细胞的浸润和促进周细胞的募集,并能下调血管瘤中VEGF的表达水平。机制研究发现,TNFSF15是通过或者是部分通过DR-3受体来下调VEGF的表达。
[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.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.41

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