Neuropilin-2对胰腺神经内分泌肿瘤血管生成作用的初步研究
发布时间：2018-09-06 13:49
【摘要】：背景与目的:胰腺神经内分泌瘤(Pancreatic Neuroendocrine Tumors,PNETs)是我国最常见的神经内分泌瘤类型。虽然PNETs发病率并不高,但因其前期症状隐匿,能及时发现并接受早期手术治疗的患者很少,而后期常伴有肝脏及其他部位转移,外科治疗效果不佳,而PNETs对放、化疗治疗不敏感。PNETs是一种富血管的肿瘤,血管生成在其发生发展中起到了非常重要的作用,抗血管生成逐渐成为其重要的治疗策略之一。分子靶向药物舒尼替尼已被批准用于PNETs的治疗。但临床观察显示,患者使用舒尼替尼,在获得短暂受益后,往往出现耐药,肿瘤侵袭能力反而增强,更易发生转移,提示有必要寻找新的抗血管生成靶点。神经纤毛蛋白2(neuropilin2,NRP2)可表达于黑色素瘤、神经母细胞瘤和胶质母细胞瘤等肿瘤细胞。目前有研究报道,NRP2在损伤缺血部位的表达上调,可能参与生理性血管的修复;在如风湿性关节炎等一些以血管新生活动增强为特点的疾病中也发现NRP2高表达,提示其参与病理性血管生成的可能。然而,在富血管的胰腺神经内分泌肿瘤中NRP2的表达情况及其对血管生成的影响尚未见报道。本研究拟对PNETs中NRP2的表达水平及促血管生成作用行初步探索,希望为PNETs的抗血管治疗提供新的思路。方法:1.免疫组化检测人胰腺神经内分泌瘤组织标本中NRP2和血管标志物CD31的表达。2.建立胰腺神经内分泌肿瘤BON-1细胞NRP2缺失的细胞系,Western blot检测BON-1CON(对照组)和BON-1 NRP2KD(干扰组)细胞中NRP2的表达情况。3.细胞增殖实验检测对照组和干扰组条件上清对内皮细胞增殖的影响。4.细胞迁移实验检测对照组和干扰组条件上清对内皮细胞迁移的影响。5.小管形成实验检测对照组和干扰组条件上清对内皮细胞成管能力的影响。结果:1.免疫组化染色结果显示,NRP2在人胰腺神经内分泌肿瘤组织中高表达,在癌旁组织中低表达。NRP2的表达与微血管标志物CD31呈正相关。2.CCK-8检测显示对照组和干扰组培养上清处理的HUVEC细胞组间增殖无统计学差异(p0.05):对照组吸光度为0.35±0.04,干扰组为0.32±0.04。3.细胞迁移实验结果显示干扰组细胞培养上清处理的HUVEC细胞迁移能力较对照组减弱。(1)Transwell实验结果:对照组(203±13)个/孔,干扰组(100±10)个/孔(P0.01)。(2)划痕实验结果:对照组愈合距离为654±29,干扰组为367±24(P0.01)。4.小管形成实验结果显示干扰组细胞培养上清处理的HUVEC细胞小管形成减少,对照组为40±5,干扰组为24±3(P0.01)。结论:1.研究结果显示,NRP2在胰腺神经内分泌肿瘤组织中的表达明显高于癌旁胰腺组织,其表达与血管密度呈正相关,提示PNETs的发展与血管生成密切相关;2.进一步研究显示,PNETs中NRP2的缺失对肿瘤相关血管内皮细胞的增殖无影响,但能抑制其迁移、成管能力,抑制PNETs的血管生成。其作用机制有待更多实验证据证实。综上,我们认为,NRP2可作为新的诊疗靶点。通过抑制PNETs中NRP2的表达,抑制血管生成,进而抑制肿瘤生长。
[Abstract]:BACKGROUND & OBJECTIVE: Pancreatic Neuroendocrine Tumors (PNETs) are the most common type of neuroendocrine tumors in China. Although the incidence of PNETs is not high, few patients can be found and treated promptly because of their concealed symptoms in the early stage, and liver and other metastases often occur in the late stage. PNETs is a kind of tumor rich in blood vessels. Angiogenesis plays a very important role in its development. Antiangiogenesis has gradually become one of its important therapeutic strategies. Molecular targeted drug sunitinib has been approved for the treatment of PNETs. However, clinical observation shows that patients with PNETs are not sensitive to radiotherapy and chemotherapy. Neurocillin 2 (NRP2) can be expressed in tumor cells such as melanoma, neuroblastoma and glioblastoma. The expression of NRP2 is up-regulated in the injured ischemic site and may be involved in the repair of physiological blood vessels. High expression of NRP2 is also found in some diseases characterized by increased angiogenesis, such as rheumatoid arthritis, suggesting its involvement in pathological angiogenesis. The purpose of this study is to explore the expression of NRP 2 and its effect on angiogenesis in PNETs, and to provide new ideas for anti-angiogenesis therapy of PNETs. Methods: 1. Immunohistochemistry was used to detect the expression of NRP 2 and CD31 in human pancreatic neuroendocrine tumor tissues. Western blot was used to detect the expression of NRP2 in BON-1CON (control group) and BON-1 NRP2KD (interference group) cells. 3. Cell proliferation assay was used to detect the effect of conditional supernatant of control group and interference group on endothelial cell proliferation. 4. Cell migration assay was used to detect the conditions of control group and interference group. Results: 1. Immunohistochemical staining showed that NRP2 was highly expressed in human pancreatic neuroendocrine neoplasms and low in paracancerous tissues. The expression of NRP2 was positively correlated with the microvascular marker CD31. CCK-8 assay showed no significant difference in proliferation between the control group and the interfering group (p0.05). The absorbance of the control group was 0.35.04, and that of the interfering group was 0.32.04.3. The migration ability of the interfering group was weaker than that of the control group. (2) Scratch test results: The healing distance of control group was 654 + 29, and that of interference group was 367 + 24 (P 0.01). The tubule formation test showed that the tubule formation of HUVEC cells treated with supernatant of cell culture in interference group was reduced, that of control group was 40 + 5, and that of interference group was 24 + 3 (P 0.01). Conclusion: 1. The results showed that the expression of NRP2 in pancreatic neuroendocrine tumors was significantly higher than that in adjacent pancreatic tissues, and its expression was positively correlated with vascular density, suggesting that the development of PNETs was closely related to angiogenesis. 2. Further studies showed that the absence of NRP2 in PNETs had no effect on the proliferation of tumor-related vascular endothelial cells, but could inhibit the proliferation of tumor-related vascular endothelial cells. In conclusion, we believe that NRP2 can be used as a new target for diagnosis and treatment. It can inhibit angiogenesis and tumor growth by inhibiting the expression of NRP2 in PNETs.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.9
本文编号：2226537
[Abstract]:BACKGROUND & OBJECTIVE: Pancreatic Neuroendocrine Tumors (PNETs) are the most common type of neuroendocrine tumors in China. Although the incidence of PNETs is not high, few patients can be found and treated promptly because of their concealed symptoms in the early stage, and liver and other metastases often occur in the late stage. PNETs is a kind of tumor rich in blood vessels. Angiogenesis plays a very important role in its development. Antiangiogenesis has gradually become one of its important therapeutic strategies. Molecular targeted drug sunitinib has been approved for the treatment of PNETs. However, clinical observation shows that patients with PNETs are not sensitive to radiotherapy and chemotherapy. Neurocillin 2 (NRP2) can be expressed in tumor cells such as melanoma, neuroblastoma and glioblastoma. The expression of NRP2 is up-regulated in the injured ischemic site and may be involved in the repair of physiological blood vessels. High expression of NRP2 is also found in some diseases characterized by increased angiogenesis, such as rheumatoid arthritis, suggesting its involvement in pathological angiogenesis. The purpose of this study is to explore the expression of NRP 2 and its effect on angiogenesis in PNETs, and to provide new ideas for anti-angiogenesis therapy of PNETs. Methods: 1. Immunohistochemistry was used to detect the expression of NRP 2 and CD31 in human pancreatic neuroendocrine tumor tissues. Western blot was used to detect the expression of NRP2 in BON-1CON (control group) and BON-1 NRP2KD (interference group) cells. 3. Cell proliferation assay was used to detect the effect of conditional supernatant of control group and interference group on endothelial cell proliferation. 4. Cell migration assay was used to detect the conditions of control group and interference group. Results: 1. Immunohistochemical staining showed that NRP2 was highly expressed in human pancreatic neuroendocrine neoplasms and low in paracancerous tissues. The expression of NRP2 was positively correlated with the microvascular marker CD31. CCK-8 assay showed no significant difference in proliferation between the control group and the interfering group (p0.05). The absorbance of the control group was 0.35.04, and that of the interfering group was 0.32.04.3. The migration ability of the interfering group was weaker than that of the control group. (2) Scratch test results: The healing distance of control group was 654 + 29, and that of interference group was 367 + 24 (P 0.01). The tubule formation test showed that the tubule formation of HUVEC cells treated with supernatant of cell culture in interference group was reduced, that of control group was 40 + 5, and that of interference group was 24 + 3 (P 0.01). Conclusion: 1. The results showed that the expression of NRP2 in pancreatic neuroendocrine tumors was significantly higher than that in adjacent pancreatic tissues, and its expression was positively correlated with vascular density, suggesting that the development of PNETs was closely related to angiogenesis. 2. Further studies showed that the absence of NRP2 in PNETs had no effect on the proliferation of tumor-related vascular endothelial cells, but could inhibit the proliferation of tumor-related vascular endothelial cells. In conclusion, we believe that NRP2 can be used as a new target for diagnosis and treatment. It can inhibit angiogenesis and tumor growth by inhibiting the expression of NRP2 in PNETs.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.9
【参考文献】
相关期刊论文 前2条
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2 周晓军;樊祥山;;解读2010年消化系统肿瘤WHO分类(Ⅰ)[J];临床与实验病理学杂志;2011年04期
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