【摘要】：目的胰腺癌增殖速度快,侵襲轉(zhuǎn)移發(fā)生早,對(duì)化療藥物不敏感,被稱為“癌中之王”。傳統(tǒng)的胰腺癌研究模式將焦點(diǎn)集中于胰腺癌本身,而忽略了腫瘤微環(huán)境對(duì)胰腺癌細(xì)胞的影響,這可能是現(xiàn)階段胰腺癌治療效果欠佳的原因之一。本研究通過檢測胰腺星狀細(xì)胞對(duì)胰腺癌細(xì)胞侵襲轉(zhuǎn)移的影響,對(duì)HGF/c-met信號(hào)通路在其中的作用及機(jī)制進(jìn)行初步的探討,旨在揭示腫瘤微環(huán)境在腫瘤惡性行為中的促進(jìn)作用。方法1.測定PSCs對(duì)胰腺癌細(xì)胞株P(guān)anc-1,SW1990侵襲轉(zhuǎn)移力的影響。收集PSCs的上清液作為條件培養(yǎng)基(PSC-CM),并通過細(xì)胞劃痕實(shí)驗(yàn)測定其對(duì)兩株胰腺癌細(xì)胞轉(zhuǎn)移能力的影響;利用Transwell共培養(yǎng)體系將PSCs與胰腺癌細(xì)胞進(jìn)行共培養(yǎng),測定PSCs對(duì)胰腺癌細(xì)胞的侵襲轉(zhuǎn)移的影響;2.測定PSCs對(duì)胰腺癌細(xì)胞上皮間質(zhì)轉(zhuǎn)換(Epithelial mesenchymal transition,EMT)的影響。將PSC-CM作用于胰腺癌細(xì)胞Panc-1,SW1990后,光鏡下觀察其形態(tài)的變化;利用RT-PCR,Western Blot法分別檢測兩株胰腺癌細(xì)胞E-cadherin、Vimentin的表達(dá)變化情況;利用細(xì)胞免疫熒光技術(shù)檢測E-cadherin、Vimentin在胰腺癌細(xì)胞內(nèi)的表達(dá)變化情況;3.檢測HGF對(duì)胰腺癌細(xì)胞的EMT以及侵襲轉(zhuǎn)移的影響。運(yùn)用ELISA法分別檢測胰腺星狀細(xì)胞與胰腺癌細(xì)胞上清液中HGF的含量;運(yùn)用RT-PCR,Western Blot法檢測胰腺星狀細(xì)胞與胰腺癌細(xì)胞胞內(nèi)HGF表達(dá)的差異;采用si RNA技術(shù)干擾胰腺星狀細(xì)胞胞內(nèi)中HGF的表達(dá),收集其上清液并測定其對(duì)胰腺癌細(xì)胞侵襲轉(zhuǎn)移的影響;將胰腺星狀細(xì)胞上清液中加入HGF-antibody,隨后用其培養(yǎng)胰腺癌細(xì)胞,觀察其對(duì)胰腺癌細(xì)胞EMT和侵襲轉(zhuǎn)移的影響;將外源性HGF加入到胰腺癌細(xì)胞培養(yǎng)基內(nèi),觀察胰腺癌細(xì)胞侵襲轉(zhuǎn)移力的變化;4.檢測HGF對(duì)胰腺癌細(xì)胞survivin表達(dá)的影響。將PSC-CM作用于胰腺癌細(xì)胞后,采用RT-PCR法、Western Blot法檢測胰腺癌細(xì)胞內(nèi)survivin基因的表達(dá)水平變化;在胰腺星狀細(xì)胞條件培養(yǎng)基中加入HGF-antibody并培養(yǎng)胰腺癌細(xì)胞,觀察癌細(xì)胞內(nèi)survivin基因的表達(dá)水平變化;采用si RNA技術(shù)干擾胰腺癌細(xì)胞內(nèi)survivin基因的表達(dá),隨后將其與胰腺星狀細(xì)胞共培養(yǎng),觀察后者對(duì)其侵襲轉(zhuǎn)移的促進(jìn)作用是否被抑制;5.檢測HGF對(duì)其受體c-Met表達(dá)的影響。將PSC-CM作用于胰腺癌細(xì)胞后,采用RT-PCR法、Western Blot法檢測胰腺癌細(xì)胞內(nèi)c-Met、p-Met基因的表達(dá)水平變化;在胰腺星狀細(xì)胞條件培養(yǎng)基中加入HGF-antibody并培養(yǎng)胰腺癌細(xì)胞,觀察癌細(xì)胞內(nèi)c-Met、p-Met基因的表達(dá)水平變化;將胰腺星狀細(xì)胞條件培養(yǎng)基中加入c-Met特異性抑制劑SU11274,隨后培養(yǎng)胰腺癌細(xì)胞,觀察癌細(xì)胞內(nèi)c-Met、p-Met、survivin基因的表達(dá)水平變化;6.檢測P53/P21對(duì)HGF/c-Met/survivin信號(hào)通路的影響。采用RT-PCR法、Western Blot法檢測兩株胰腺癌細(xì)胞內(nèi)P53,P21的表達(dá)水平差異;采用P53抑制劑pifithrin-α干擾癌細(xì)胞內(nèi)P53基因的表達(dá),檢測癌細(xì)胞內(nèi)P21、c-Met、p-Met、survivin基因的表達(dá)水平變化;構(gòu)建P53過表達(dá)質(zhì)粒轉(zhuǎn)染癌細(xì)胞使其胞內(nèi)P53基因過表達(dá),檢測癌細(xì)胞內(nèi)P21、c-Met、p-Met、survivin基因的表達(dá)水平變化;采用si RNA-P21干擾癌細(xì)胞內(nèi)P21的表達(dá),隨后檢測c-Met的表達(dá)水平變化;構(gòu)建P21過表達(dá)質(zhì)粒轉(zhuǎn)染癌細(xì)胞使其胞內(nèi)P21基因過表達(dá),檢測c-Met表達(dá)水平變化。結(jié)果胰腺星狀細(xì)胞能夠促進(jìn)胰腺癌細(xì)胞(Panc-1,SW1990)發(fā)生上皮間質(zhì)轉(zhuǎn)換現(xiàn)象,并能促進(jìn)胰腺癌細(xì)胞株P(guān)anc-1的侵襲轉(zhuǎn)移,但對(duì)SW1990的侵襲轉(zhuǎn)移能力無明顯促進(jìn)作用;胰腺星狀細(xì)胞胞內(nèi)以及其上清液中均能夠檢測到HGF的大量存在,而胰腺癌細(xì)胞胞內(nèi)以及其上清液中則幾乎沒有,特異性阻斷胰腺星狀細(xì)胞內(nèi)HGF的表達(dá)可以減弱胰腺星狀細(xì)胞對(duì)Panc-1細(xì)胞侵襲轉(zhuǎn)移的促進(jìn)作用;外源性的HGF則可以誘導(dǎo)Panc-1細(xì)胞侵襲轉(zhuǎn)移,而對(duì)SW1990無作用;HGF作用于癌細(xì)胞后,癌細(xì)胞內(nèi)特異性受體c-Met表達(dá)水平無明顯變化,而其磷酸化水平(p-Met)表達(dá)水平明顯上升,特異性阻斷HGF后,p-Met表達(dá)水平下降;運(yùn)用SU11274干擾c-Met表達(dá)后,其磷酸化水平p-Met表達(dá)水平隨之下降,survivin基因的表達(dá)亦隨之下降;運(yùn)用si RNA技術(shù)干擾癌細(xì)胞內(nèi)survivin基因的表達(dá)后,胰腺星狀細(xì)胞對(duì)胰腺癌細(xì)胞侵襲轉(zhuǎn)移促進(jìn)作用較之前明顯下降;Panc-1細(xì)胞內(nèi)P53,P21缺失,SW1990內(nèi)P53,P21表達(dá)水平較高,將Panc-1細(xì)胞轉(zhuǎn)染P53過表達(dá)質(zhì)粒后,P21表達(dá)上調(diào),c-Met表達(dá)水平下調(diào);運(yùn)用si RNA技術(shù)干擾SW1990細(xì)胞內(nèi)P53的表達(dá)后,P21表達(dá)減弱,c-Met表達(dá)水平上調(diào)。將pifithrin-α處理后的的SW1990細(xì)胞與胰腺星狀細(xì)胞共培養(yǎng)后,其侵襲轉(zhuǎn)移能力較之前明顯加強(qiáng)。結(jié)論胰腺星狀細(xì)胞通過HGF/c-Met/survivin信號(hào)通路促進(jìn)胰腺癌細(xì)胞的侵襲轉(zhuǎn)移過程,這一信號(hào)通路受到P53/P21軸的負(fù)性調(diào)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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.9

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