肝细胞生长因子HGF介导的胰腺星状细胞促胰腺癌细胞侵袭转移作用及其机制研究
发布时间：2018-08-19 18:10
【摘要】：目的胰腺癌增殖速度快,侵袭转移发生早,对化疗药物不敏感,被称为“癌中之王”。传统的胰腺癌研究模式将焦点集中于胰腺癌本身,而忽略了肿瘤微环境对胰腺癌细胞的影响,这可能是现阶段胰腺癌治疗效果欠佳的原因之一。本研究通过检测胰腺星状细胞对胰腺癌细胞侵袭转移的影响,对HGF/c-met信号通路在其中的作用及机制进行初步的探讨,旨在揭示肿瘤微环境在肿瘤恶性行为中的促进作用。方法1.测定PSCs对胰腺癌细胞株Panc-1,SW1990侵袭转移力的影响。收集PSCs的上清液作为条件培养基(PSC-CM),并通过细胞划痕实验测定其对两株胰腺癌细胞转移能力的影响;利用Transwell共培养体系将PSCs与胰腺癌细胞进行共培养,测定PSCs对胰腺癌细胞的侵袭转移的影响;2.测定PSCs对胰腺癌细胞上皮间质转换(Epithelial mesenchymal transition,EMT)的影响。将PSC-CM作用于胰腺癌细胞Panc-1,SW1990后,光镜下观察其形态的变化;利用RT-PCR,Western Blot法分别检测两株胰腺癌细胞E-cadherin、Vimentin的表达变化情况;利用细胞免疫荧光技术检测E-cadherin、Vimentin在胰腺癌细胞内的表达变化情况;3.检测HGF对胰腺癌细胞的EMT以及侵袭转移的影响。运用ELISA法分别检测胰腺星状细胞与胰腺癌细胞上清液中HGF的含量;运用RT-PCR,Western Blot法检测胰腺星状细胞与胰腺癌细胞胞内HGF表达的差异;采用si RNA技术干扰胰腺星状细胞胞内中HGF的表达,收集其上清液并测定其对胰腺癌细胞侵袭转移的影响;将胰腺星状细胞上清液中加入HGF-antibody,随后用其培养胰腺癌细胞,观察其对胰腺癌细胞EMT和侵袭转移的影响;将外源性HGF加入到胰腺癌细胞培养基内,观察胰腺癌细胞侵袭转移力的变化;4.检测HGF对胰腺癌细胞survivin表达的影响。将PSC-CM作用于胰腺癌细胞后,采用RT-PCR法、Western Blot法检测胰腺癌细胞内survivin基因的表达水平变化;在胰腺星状细胞条件培养基中加入HGF-antibody并培养胰腺癌细胞,观察癌细胞内survivin基因的表达水平变化;采用si RNA技术干扰胰腺癌细胞内survivin基因的表达,随后将其与胰腺星状细胞共培养,观察后者对其侵袭转移的促进作用是否被抑制;5.检测HGF对其受体c-Met表达的影响。将PSC-CM作用于胰腺癌细胞后,采用RT-PCR法、Western Blot法检测胰腺癌细胞内c-Met、p-Met基因的表达水平变化;在胰腺星状细胞条件培养基中加入HGF-antibody并培养胰腺癌细胞,观察癌细胞内c-Met、p-Met基因的表达水平变化;将胰腺星状细胞条件培养基中加入c-Met特异性抑制剂SU11274,随后培养胰腺癌细胞,观察癌细胞内c-Met、p-Met、survivin基因的表达水平变化;6.检测P53/P21对HGF/c-Met/survivin信号通路的影响。采用RT-PCR法、Western Blot法检测两株胰腺癌细胞内P53,P21的表达水平差异;采用P53抑制剂pifithrin-α干扰癌细胞内P53基因的表达,检测癌细胞内P21、c-Met、p-Met、survivin基因的表达水平变化;构建P53过表达质粒转染癌细胞使其胞内P53基因过表达,检测癌细胞内P21、c-Met、p-Met、survivin基因的表达水平变化;采用si RNA-P21干扰癌细胞内P21的表达,随后检测c-Met的表达水平变化;构建P21过表达质粒转染癌细胞使其胞内P21基因过表达,检测c-Met表达水平变化。结果胰腺星状细胞能够促进胰腺癌细胞(Panc-1,SW1990)发生上皮间质转换现象,并能促进胰腺癌细胞株Panc-1的侵袭转移,但对SW1990的侵袭转移能力无明显促进作用;胰腺星状细胞胞内以及其上清液中均能够检测到HGF的大量存在,而胰腺癌细胞胞内以及其上清液中则几乎没有,特异性阻断胰腺星状细胞内HGF的表达可以减弱胰腺星状细胞对Panc-1细胞侵袭转移的促进作用;外源性的HGF则可以诱导Panc-1细胞侵袭转移,而对SW1990无作用;HGF作用于癌细胞后,癌细胞内特异性受体c-Met表达水平无明显变化,而其磷酸化水平(p-Met)表达水平明显上升,特异性阻断HGF后,p-Met表达水平下降;运用SU11274干扰c-Met表达后,其磷酸化水平p-Met表达水平随之下降,survivin基因的表达亦随之下降;运用si RNA技术干扰癌细胞内survivin基因的表达后,胰腺星状细胞对胰腺癌细胞侵袭转移促进作用较之前明显下降;Panc-1细胞内P53,P21缺失,SW1990内P53,P21表达水平较高,将Panc-1细胞转染P53过表达质粒后,P21表达上调,c-Met表达水平下调;运用si RNA技术干扰SW1990细胞内P53的表达后,P21表达减弱,c-Met表达水平上调。将pifithrin-α处理后的的SW1990细胞与胰腺星状细胞共培养后,其侵袭转移能力较之前明显加强。结论胰腺星状细胞通过HGF/c-Met/survivin信号通路促进胰腺癌细胞的侵袭转移过程,这一信号通路受到P53/P21轴的负性调控。
[Abstract]:Objective Pancreatic cancer is known as the "king of cancer" because of its rapid proliferation, early invasion and metastasis, and insensitivity to chemotherapeutics.The traditional model of pancreatic cancer research focuses on pancreatic cancer itself, but ignores the effect of tumor microenvironment on pancreatic cancer cells, which may be one of the reasons for the poor therapeutic effect of pancreatic cancer at this stage. To explore the role and mechanism of HGF/c-met signaling pathway in the invasion and metastasis of pancreatic cancer cells by detecting the effect of pancreatic stellate cells on the invasion and metastasis of pancreatic cancer cells, so as to reveal the role of tumor microenvironment in the malignant behavior of tumor. Methods 1. To determine the effect of PSCs on the invasion and metastasis of pancreatic cancer cell lines Panc-1, SW1990. The supernatant was used as conditioned medium (PSC-CM), and the effect of PSCs on the metastasis ability of two pancreatic cancer cells was determined by cell scratch test; PSCs were co-cultured with pancreatic cancer cells in Transwell co-culture system to determine the effect of PSCs on the invasion and metastasis of pancreatic cancer cells; 2. The effect of PSCs on epithelial-mesenchymal transition (Ep) of pancreatic cancer cells was determined. After PSC-CM was applied to pancreatic cancer cell Panc-1, SW1990, the morphological changes were observed under light microscope; the expressions of E-cadherin and Vimentin in two pancreatic cancer cells were detected by RT-PCR and Western Blot respectively; and the expression of E-cadherin and Vimentin in pancreatic cancer cells was detected by immunofluorescence technique. The expression of HGF in the supernatant of pancreatic stellate cells and pancreatic cancer cells was detected by ELISA, and the expression of HGF in the supernatant of pancreatic stellate cells and pancreatic cancer cells was detected by RT-PCR and Western Blot. The expression of HGF in pancreatic stellate cells was disturbed, and the supernatant was collected to determine the effect of HGF on invasion and metastasis of pancreatic cancer cells. The expression of survivin gene in pancreatic cancer cells was detected by RT-PCR and Western Blot after PSC-CM was applied to pancreatic cancer cells. HGF-antibody was added to pancreatic stellate cell conditioned medium and cultured. To observe the expression of survivin gene in cultured pancreatic cancer cells, we used Si RNA technology to interfere with the expression of survivin gene in pancreatic cancer cells, and then co-cultured with pancreatic stellate cells to observe whether the latter promotes the invasion and metastasis of pancreatic cancer cells was inhibited. 5. To detect the effect of HGF on the expression of c-Met receptor. The expression levels of c-Met and p-Met genes in pancreatic cancer cells were detected by RT-PCR and Western Blot, and the expression levels of c-Met and p-Met genes in pancreatic stellate cell conditioned medium were observed by adding HGF-antibody to the conditioned medium. The expression levels of c-Met, p-Met and Survivin genes in pancreatic cancer cells were observed after the addition of c-Met specific inhibitor SU11274. 6. The effect of P53/P21 on HGF/c-Met/survivin signaling pathway was detected. The expression levels of P53 and P21 in pancreatic cancer cells were detected by RT-PCR and Western Blot respectively. Fihrin-alpha interfered with the expression of P53 gene in cancer cells, and detected the expression level of P21, c-Met, p-Met, survivin gene in cancer cells; P53 overexpression plasmid was constructed and transfected into cancer cells to overexpress P53 gene, and the expression level of P21, c-Met, p-Met, survivin gene in cancer cells was detected; RNA-P21 interfered with P21 in cancer cells. Results Pancreatic stellate cells could promote the epithelial-mesenchymal transition of pancreatic cancer cells (Panc-1, SW1990) and promote the invasion and metastasis of pancreatic cancer cell line Panc-1. HGF could be detected in pancreatic stellate cells and their supernatants, but not in pancreatic cancer cells and their supernatants. Specific blockade of HGF expression in pancreatic stellate cells could attenuate the invasion and metastasis of pancreatic stellate cells to Panc C-1 cells. Exogenous HGF could induce the invasion and metastasis of Panc-1 cells, but had no effect on SW1990. HGF could induce the expression of specific receptor c-Met in cancer cells, but its phosphorylation level (p-Met) increased significantly, and the expression of p-Met decreased after specific blockade of HGF. After the expression of c-Met, the phosphorylation level of p-Met decreased, and the expression of survivin gene also decreased. After interfering with the expression of survivin gene in cancer cells by using Si RNA technology, the effect of pancreatic stellate cells on the invasion and metastasis of pancreatic cancer cells was significantly lower than that before; the expression of P53 and P21 in Panc-1 cells was deleted, and the expression of P53 and P21 in SW1990 was decreased. When Panc-1 cells were transfected with P53 overexpression plasmid, P21 expression was up-regulated and c-Met expression was down-regulated. After interfering with P53 expression in SW1990 cells by Si RNA, P21 expression was down-regulated and c-Met expression was up-regulated. Conclusion Pancreatic stellate cells promote the invasion and metastasis of pancreatic cancer cells through HGF/c-Met/survivin signaling pathway, which is negatively regulated by P53/P21 axis.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.9
本文编号：2192427
[Abstract]:Objective Pancreatic cancer is known as the "king of cancer" because of its rapid proliferation, early invasion and metastasis, and insensitivity to chemotherapeutics.The traditional model of pancreatic cancer research focuses on pancreatic cancer itself, but ignores the effect of tumor microenvironment on pancreatic cancer cells, which may be one of the reasons for the poor therapeutic effect of pancreatic cancer at this stage. To explore the role and mechanism of HGF/c-met signaling pathway in the invasion and metastasis of pancreatic cancer cells by detecting the effect of pancreatic stellate cells on the invasion and metastasis of pancreatic cancer cells, so as to reveal the role of tumor microenvironment in the malignant behavior of tumor. Methods 1. To determine the effect of PSCs on the invasion and metastasis of pancreatic cancer cell lines Panc-1, SW1990. The supernatant was used as conditioned medium (PSC-CM), and the effect of PSCs on the metastasis ability of two pancreatic cancer cells was determined by cell scratch test; PSCs were co-cultured with pancreatic cancer cells in Transwell co-culture system to determine the effect of PSCs on the invasion and metastasis of pancreatic cancer cells; 2. The effect of PSCs on epithelial-mesenchymal transition (Ep) of pancreatic cancer cells was determined. After PSC-CM was applied to pancreatic cancer cell Panc-1, SW1990, the morphological changes were observed under light microscope; the expressions of E-cadherin and Vimentin in two pancreatic cancer cells were detected by RT-PCR and Western Blot respectively; and the expression of E-cadherin and Vimentin in pancreatic cancer cells was detected by immunofluorescence technique. The expression of HGF in the supernatant of pancreatic stellate cells and pancreatic cancer cells was detected by ELISA, and the expression of HGF in the supernatant of pancreatic stellate cells and pancreatic cancer cells was detected by RT-PCR and Western Blot. The expression of HGF in pancreatic stellate cells was disturbed, and the supernatant was collected to determine the effect of HGF on invasion and metastasis of pancreatic cancer cells. The expression of survivin gene in pancreatic cancer cells was detected by RT-PCR and Western Blot after PSC-CM was applied to pancreatic cancer cells. HGF-antibody was added to pancreatic stellate cell conditioned medium and cultured. To observe the expression of survivin gene in cultured pancreatic cancer cells, we used Si RNA technology to interfere with the expression of survivin gene in pancreatic cancer cells, and then co-cultured with pancreatic stellate cells to observe whether the latter promotes the invasion and metastasis of pancreatic cancer cells was inhibited. 5. To detect the effect of HGF on the expression of c-Met receptor. The expression levels of c-Met and p-Met genes in pancreatic cancer cells were detected by RT-PCR and Western Blot, and the expression levels of c-Met and p-Met genes in pancreatic stellate cell conditioned medium were observed by adding HGF-antibody to the conditioned medium. The expression levels of c-Met, p-Met and Survivin genes in pancreatic cancer cells were observed after the addition of c-Met specific inhibitor SU11274. 6. The effect of P53/P21 on HGF/c-Met/survivin signaling pathway was detected. The expression levels of P53 and P21 in pancreatic cancer cells were detected by RT-PCR and Western Blot respectively. Fihrin-alpha interfered with the expression of P53 gene in cancer cells, and detected the expression level of P21, c-Met, p-Met, survivin gene in cancer cells; P53 overexpression plasmid was constructed and transfected into cancer cells to overexpress P53 gene, and the expression level of P21, c-Met, p-Met, survivin gene in cancer cells was detected; RNA-P21 interfered with P21 in cancer cells. Results Pancreatic stellate cells could promote the epithelial-mesenchymal transition of pancreatic cancer cells (Panc-1, SW1990) and promote the invasion and metastasis of pancreatic cancer cell line Panc-1. HGF could be detected in pancreatic stellate cells and their supernatants, but not in pancreatic cancer cells and their supernatants. Specific blockade of HGF expression in pancreatic stellate cells could attenuate the invasion and metastasis of pancreatic stellate cells to Panc C-1 cells. Exogenous HGF could induce the invasion and metastasis of Panc-1 cells, but had no effect on SW1990. HGF could induce the expression of specific receptor c-Met in cancer cells, but its phosphorylation level (p-Met) increased significantly, and the expression of p-Met decreased after specific blockade of HGF. After the expression of c-Met, the phosphorylation level of p-Met decreased, and the expression of survivin gene also decreased. After interfering with the expression of survivin gene in cancer cells by using Si RNA technology, the effect of pancreatic stellate cells on the invasion and metastasis of pancreatic cancer cells was significantly lower than that before; the expression of P53 and P21 in Panc-1 cells was deleted, and the expression of P53 and P21 in SW1990 was decreased. When Panc-1 cells were transfected with P53 overexpression plasmid, P21 expression was up-regulated and c-Met expression was down-regulated. After interfering with P53 expression in SW1990 cells by Si RNA, P21 expression was down-regulated and c-Met expression was up-regulated. Conclusion Pancreatic stellate cells promote the invasion and metastasis of pancreatic cancer cells through HGF/c-Met/survivin signaling pathway, which is negatively regulated by P53/P21 axis.
【学位授予单位】：青岛大学
【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
1 Daniel Delitto;Eva Vertes-George;Steven J Hughes;Kevin E Behrns;Jose G Trevino;;c-Met signaling in the development of tumorigenesis and chemoresistance: Potential applications in pancreatic cancer[J];World Journal of Gastroenterology;2014年26期
2 ;Pancreatic stellate cells promote proliferation and invasiveness of human pancreatic cancer cells via galectin-3[J];World Journal of Gastroenterology;2008年13期
，本文编号：2192427
本文链接：https://www.wllwen.com/yixuelunwen/zlx/2192427.html
