【摘要】：背景與目的:胰腺神經(jīng)內(nèi)分泌瘤(Pancreatic Neuroendocrine Tumors,PNETs)是我國最常見的神經(jīng)內(nèi)分泌瘤類型。雖然PNETs發(fā)病率并不高,但因其前期癥狀隱匿,能及時發(fā)現(xiàn)并接受早期手術(shù)治療的患者很少,而后期常伴有肝臟及其他部位轉(zhuǎn)移,外科治療效果不佳,而PNETs對放、化療治療不敏感。PNETs是一種富血管的腫瘤,血管生成在其發(fā)生發(fā)展中起到了非常重要的作用,抗血管生成逐漸成為其重要的治療策略之一。分子靶向藥物舒尼替尼已被批準(zhǔn)用于PNETs的治療。但臨床觀察顯示,患者使用舒尼替尼,在獲得短暫受益后,往往出現(xiàn)耐藥,腫瘤侵襲能力反而增強,更易發(fā)生轉(zhuǎn)移,提示有必要尋找新的抗血管生成靶點。神經(jīng)纖毛蛋白2(neuropilin2,NRP2)可表達于黑色素瘤、神經(jīng)母細胞瘤和膠質(zhì)母細胞瘤等腫瘤細胞。目前有研究報道,NRP2在損傷缺血部位的表達上調(diào),可能參與生理性血管的修復(fù);在如風(fēng)濕性關(guān)節(jié)炎等一些以血管新生活動增強為特點的疾病中也發(fā)現(xiàn)NRP2高表達,提示其參與病理性血管生成的可能。然而,在富血管的胰腺神經(jīng)內(nèi)分泌腫瘤中NRP2的表達情況及其對血管生成的影響尚未見報道。本研究擬對PNETs中NRP2的表達水平及促血管生成作用行初步探索,希望為PNETs的抗血管治療提供新的思路。方法:1.免疫組化檢測人胰腺神經(jīng)內(nèi)分泌瘤組織標(biāo)本中NRP2和血管標(biāo)志物CD31的表達。2.建立胰腺神經(jīng)內(nèi)分泌腫瘤BON-1細胞NRP2缺失的細胞系,Western blot檢測BON-1CON(對照組)和BON-1 NRP2KD(干擾組)細胞中NRP2的表達情況。3.細胞增殖實驗檢測對照組和干擾組條件上清對內(nèi)皮細胞增殖的影響。4.細胞遷移實驗檢測對照組和干擾組條件上清對內(nèi)皮細胞遷移的影響。5.小管形成實驗檢測對照組和干擾組條件上清對內(nèi)皮細胞成管能力的影響。結(jié)果:1.免疫組化染色結(jié)果顯示,NRP2在人胰腺神經(jīng)內(nèi)分泌腫瘤組織中高表達,在癌旁組織中低表達。NRP2的表達與微血管標(biāo)志物CD31呈正相關(guān)。2.CCK-8檢測顯示對照組和干擾組培養(yǎng)上清處理的HUVEC細胞組間增殖無統(tǒng)計學(xué)差異(p0.05):對照組吸光度為0.35±0.04,干擾組為0.32±0.04。3.細胞遷移實驗結(jié)果顯示干擾組細胞培養(yǎng)上清處理的HUVEC細胞遷移能力較對照組減弱。(1)Transwell實驗結(jié)果:對照組(203±13)個/孔,干擾組(100±10)個/孔(P0.01)。(2)劃痕實驗結(jié)果:對照組愈合距離為654±29,干擾組為367±24(P0.01)。4.小管形成實驗結(jié)果顯示干擾組細胞培養(yǎng)上清處理的HUVEC細胞小管形成減少,對照組為40±5,干擾組為24±3(P0.01)。結(jié)論:1.研究結(jié)果顯示,NRP2在胰腺神經(jīng)內(nèi)分泌腫瘤組織中的表達明顯高于癌旁胰腺組織,其表達與血管密度呈正相關(guān),提示PNETs的發(fā)展與血管生成密切相關(guān);2.進一步研究顯示,PNETs中NRP2的缺失對腫瘤相關(guān)血管內(nèi)皮細胞的增殖無影響,但能抑制其遷移、成管能力,抑制PNETs的血管生成。其作用機制有待更多實驗證據(jù)證實。綜上,我們認為,NRP2可作為新的診療靶點。通過抑制PNETs中NRP2的表達,抑制血管生成,進而抑制腫瘤生長。
[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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.9

【參考文獻】

相關(guān)期刊論文 前2條

1 郭林杰;唐承薇;;中國胃腸胰神經(jīng)內(nèi)分泌腫瘤臨床研究現(xiàn)狀分析[J];胃腸病學(xué);2012年05期

2 周曉軍;樊祥山;;解讀2010年消化系統(tǒng)腫瘤WHO分類(Ⅰ)[J];臨床與實驗病理學(xué)雜志;2011年04期

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