一氧化氮干预促进胰腺癌侵袭转移的实验研究

发布时间：2019-02-15 08:57

【摘要】：背景和目的:胰腺癌是一种病死率极高的消化系统恶性肿瘤。侵袭转移是胰腺癌的常见现象与局部复发和疼痛有着重要联系。一氧化氮(NO)可调控胰腺癌细胞的生长及侵袭等生物学行为,在维持胰腺癌的恶性生物学行为起到重要作用。NO可上调癌细胞自噬水平为侵袭转移供应能量。神经浸润(PNI)是胰腺癌侵袭转移的主要方式,也是胰腺癌转移的特殊通道。研究目的:(1)观察NO相关蛋白在胰腺癌中的表达及其临床意义,探讨NO相关蛋白与胰腺癌侵袭转移尤其是神经浸润(PNI)的相关性;(2)观察NO供体对胰腺癌细胞增殖、迁移及侵袭的影响及其可能的分子机制。方法:(1)免疫组织化学染色技术检测E-cadherin、iNOS、NQO1及LC-3在胰腺癌组织中的表达,分析其与胰腺癌临床病理特征及PNI之间的相关性。(2)用MTT方法检测不同浓度DETA/NO对胰腺癌细胞株增殖的影响。(3)通过细胞划痕实验及Transwell侵袭实验检测DETA/NO对胰腺癌细胞株迁移及侵袭能力的影响。(4)应用Real-time PCR技术检测DETA/NO干预胰腺癌细胞株后其侵袭相关蛋白mRNA水平的变化。(5)Western blot检测DETA/NO对胰腺癌细胞株自噬相关蛋白表达变化的影响。结果:(1)E-cadherin、iNOS、NQO1、及LC-3在胰腺癌组织中表达率分别是68.9%、63.9%、55.7%、57.4%,E-cadherin表达与LC-3具有相关性,LC-3与胰腺癌局部浸润发生具有相关性,E-cadherin、LC-3表达与胰腺癌神经浸润具有显著相关性。(2)不同浓度DETA/NO对胰腺癌细胞株增殖影响有差异,低浓度促进癌细胞增殖水平,高浓度抑制增殖。(3)低浓度DETA/NO对胰腺癌细胞株进行干预,可促进胰腺癌细胞株迁移及侵袭能力。(4)50μM浓度的DETA/NO干预Bx Pc-3细胞株可上调相关基因iNOS、TNF、MMP-2、MMP-9的mRNA水平,其中MMP-2的mRNA水平显著增高;(5)用100 uM的DETA/NO干预1h后,LC-3表达水平即开始升高,而随干预时间延长至8小时即又降至接近正常,而NQO1和Beclin 1无明显差异。结论:NO代谢相关蛋白的表达与胰腺癌恶性生物学特性密切相关,在胰腺癌进展中可能扮演重要角色。低浓度NO促使胰腺癌细胞迁移侵袭能力增强,可能与胰腺癌神经浸润发生相关;NO上调侵袭相关蛋白表达水平可能是改变胰腺癌细胞侵袭能力的分子机制;NO对细胞自噬蛋白表达水平的影响可能与癌细胞维持增殖活性及侵袭能力有关。本研究可为NO代谢途径导致胰腺癌神经浸润研究提供前期基础。
[Abstract]:Background & objective: pancreatic cancer is a malignant tumor of digestive system with high mortality. Invasion and metastasis is a common phenomenon of pancreatic cancer and has an important relationship with local recurrence and pain. Nitric oxide (NO) can regulate the growth and invasion of pancreatic cancer cells and play an important role in maintaining the malignant biological behavior of pancreatic cancer. NO can up-regulate the level of autophagy of cancer cells to supply energy for invasion and metastasis. Neuroinvasive (PNI) is the main invasion and metastasis of pancreatic cancer, and it is also a special channel for pancreatic cancer metastasis. Objective: (1) to investigate the expression and clinical significance of NO related proteins in pancreatic carcinoma, and to explore the relationship between NO related proteins and invasion and metastasis of pancreatic carcinoma, especially (PNI). (2) to observe the effect of NO donor on the proliferation, migration and invasion of pancreatic cancer cells and its possible molecular mechanism. Methods: (1) Immunohistochemical staining was used to detect the expression of E-cadherinia iNOS NQO1 and LC-3 in pancreatic carcinoma. (2) the effect of different concentrations of DETA/NO on proliferation of pancreatic cancer cell line was detected by MTT method. (3) Cell scratch test and Transwell invasion assay were used to detect the effect of DETA/NO on the proliferation of pancreatic cancer cell line. Effects of DETA/NO on migration and invasion ability of pancreatic cancer cell line. (4) Real-time PCR technique was used to detect the changes of mRNA level of invasion-associated protein (mRNA) after DETA/NO intervention. (5) DETA/NO was detected by) Western blot. Effect on the expression of autophagy associated protein in pancreatic cancer cell line. Results: (1) the expression rates of E-cadherin and LC-3 in pancreatic carcinoma were 68.9 and 63.9, respectively. The expression of E-cadherin was correlated with LC-3. The expression of E-cadherinon LC-3 was significantly correlated with the neural invasion of pancreatic carcinoma. (2) the effects of different concentrations of DETA/NO on the proliferation of pancreatic cancer cell line were different. Low concentration of DETA/NO can promote the proliferation of pancreatic cancer cells and inhibit the proliferation of pancreatic cancer cells at high concentration. (3) low concentration of DETA/NO intervention on pancreatic cancer cell lines, (4) 50 渭 M concentration of DETA/NO could up-regulate the mRNA level of iNOS,TNF,MMP-2,MMP-9 gene in Bx Pc-3 cell line, and the mRNA level of MMP-2 increased significantly. (5) the expression of LC-3 began to increase 1 h after the treatment with 100 uM DETA/NO, and then decreased to close to normal as the intervention time extended to 8 hours, but there was no significant difference between NQO1 and Beclin 1. Conclusion: the expression of NO metabolism-related proteins is closely related to the malignant biological characteristics of pancreatic cancer and may play an important role in the progression of pancreatic cancer. Low concentration of NO enhanced the migration and invasion of pancreatic cancer cells, which may be related to the neuroinvasion of pancreatic cancer, and up-regulated the expression level of invasive-associated protein in pancreatic cancer cells, which may be the molecular mechanism that changes the invasion ability of pancreatic cancer cells. The effect of NO on the expression of autophagy protein may be related to the ability of cancer cell to maintain proliferation and invasion. This study may provide a preliminary basis for the study of the neural invasion of pancreatic cancer induced by NO metabolic pathway.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.9

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