【摘要】：第一部分Notch-1基因在吉西他濱誘導人胰腺癌細胞上皮-間質(zhì)轉(zhuǎn)化中的表達及作用機制研究目的：探討Notch-1在吉西他濱(GEM)誘導的人胰腺癌Panc-1細胞上皮間葉樣表型轉(zhuǎn)化(EMT)中的表達及作用機制。方法：低劑量、較長時間GEM處理胰腺癌Panc-1細胞,觀察細胞形態(tài)學改變,Western Blot和RT-PCR法檢測EMT標志物、Notch-1及相關轉(zhuǎn)錄因子的表達,免疫熒光技術檢測E-cadherin及vimentin的表達,Transwell小室檢測EMT細胞的遷移和侵襲能力。結(jié)果：GEM誘導胰腺癌Panc-1細胞的EMT最適藥物濃度為5-10mM,最適時間為9天；EMT標志物E-cadherin表達下調(diào),vimentin表達上調(diào),Notch-1和相關轉(zhuǎn)錄因子的表達上調(diào)。EMT細胞遷移和侵襲能力明顯增強。結(jié)論：適當濃度的GEM持續(xù)作用于人胰腺癌Panc-1細胞,可誘導Panc-1細胞EMT, Notch-1是該過程的重要信號通路,且胰腺癌細胞發(fā)生EMT改變后遷移和侵襲能力明顯增強。第二部分側(cè)群細胞及ABC轉(zhuǎn)運體家族在吉西他濱誘導人胰腺癌細胞上皮-間質(zhì)轉(zhuǎn)化中的作用機制研究目的：探討側(cè)群細胞(SP)及ABCG2在吉西他濱誘導人胰腺癌細胞EMT中的作用機制研究。方法：低劑量、較長時間GEM處理胰腺癌Panc-1細胞,Western Blot和RT-PCR法檢測EMT改變的Panc-1細胞中ABC轉(zhuǎn)運體家族ABCB1、ABCC1及ABCG2的表達。熒光激活細胞分選法(FACS)分離SP細胞,對比EMT改變前后SP細胞的含量變化。結(jié)果：胰腺癌Panc-1細胞EMT后ABC轉(zhuǎn)運體家族ABCB1、ABCC1、ABCG2表達明顯上調(diào),且發(fā)生EMT改變后SP細胞含量顯著增加(P0.05)。結(jié)論：人胰腺癌細胞EMT可使腫瘤CSC相關蛋白表達上調(diào),同時可增加胰腺癌CSC的含量,有助于胰腺癌細胞的增殖、侵襲及耐藥。第三部分Notch-1 siRNA在人胰腺癌細胞問質(zhì)-上皮轉(zhuǎn)化中的作用及側(cè)群細胞、ABC轉(zhuǎn)運體家族的表達機制研究目的：探討Notch-1 siRNA對人胰腺癌細胞間質(zhì)-上皮轉(zhuǎn)化(MET)的作用,以及SP細胞和ABCG2的表達機制研究。方法：Notch-1 siRNA轉(zhuǎn)染EMT改變的人胰腺癌Panc-1細胞,Western Blot和RT-PCR法檢測EMT標志物E-cadherin、vimentin及相關轉(zhuǎn)錄因子的表達,同時檢測ABC轉(zhuǎn)運體家族成員的表達,FACS檢測SP細胞的改變,Transwell小室檢測MET細胞遷移和侵襲能力的改變。結(jié)果：Notch-1 siRNA可使EMT改變的Panc-1細胞發(fā)生MET, EMT標志物E-cadherin表達上調(diào),vimentin及相關轉(zhuǎn)錄因子表達下調(diào),MET細胞遷移和侵襲能力下降,同時,ABC轉(zhuǎn)運體家族成員表達下調(diào),SP細胞含量明顯減少。結(jié)論：Notch-1 siRNA可抑制Notch-1信號通路,Notch-1是Panc-1細胞EMT的重要信號通路；EMT可促進Panc-1細胞侵襲、轉(zhuǎn)移及CSC特性的表達,抑制Notch-1信號通路可阻止胰腺癌細胞EMT,從而抑制其侵襲、轉(zhuǎn)移及耐藥等特性。
[Abstract]:Part I expression of Notch-1 gene in gemcitabine-induced epithelial-interstitial transformation of human pancreatic cancer cells objective: to investigate the role of Notch-1 in Panc-1 cells induced by gemcitabine (GEM) Expression and mechanism of mesenchymal phenotypic transformation in (EMT). Methods: Panc-1 cells of pancreatic cancer were treated with GEM at low dose for a long time. The expression of EMT markers, Notch-1 and related transcription factors were detected by, Western Blot and RT-PCR. The expression of E-cadherin and vimentin was detected by immunofluorescence technique, and the migration and invasion of EMT cells were detected by Transwell chamber. Results: the optimal concentration of EMT in Panc-1 cells induced by GEM was 5-10 mm and the optimal time was 9 days. The expression of EMT marker E-cadherin was down-regulated, the expression of vimentin was up-regulated, and the expression of Notch-1 and related transcription factors was up-regulated. The migration and invasion ability of EMT cells was obviously enhanced. Conclusion: the sustained action of GEM at appropriate concentration on human pancreatic cancer Panc-1 cells can induce EMT, Notch-1 of Panc-1 cells to be an important signal pathway in this process, and the migration and invasion ability of pancreatic cancer cells after EMT changes is obviously enhanced. The role of ABC transporter family in the epithelial-interstitial transformation of human pancreatic cancer cells induced by gemcitabine objective: to investigate the role of (SP) and ABCG2 in gemcitabine-induced human pancreatic cancer cells Study on the mechanism of action in EMT. Methods:, Western Blot and RT-PCR were used to detect the expression of ABC transporter family ABCB1,ABCC1 and ABCG2 in Panc-1 cells treated with low dose GEM for a long time. SP cells were isolated by fluorescence activated cell sorting (FACS), and the changes of SP cell content before and after EMT were compared. Results: the expression of ABCB1,ABCC1,ABCG2 of ABC transporter family was up-regulated in Panc-1 cells of pancreatic cancer after EMT, and the content of SP cells increased significantly after EMT change (P0.05). Conclusion: human pancreatic cancer cell line EMT can up-regulate the expression of CSC related protein and increase the content of CSC in pancreatic carcinoma, which is helpful to the proliferation, invasion and drug resistance of pancreatic cancer cells. The role of Notch-1 siRNA in the Transformation of Human Pancreatic Cancer cells into Interstitial epithelium and the expression Mechanism of ABC Transporter Family in Human Pancreatic Cancer cells objective: to investigate the effect of Notch-1 siRNA on the transformation of (MET) into interstitial epithelium of human pancreatic cancer cells. And the expression mechanism of SP cells and ABCG2. Methods: the expression of EMT marker E-cadherin vimentin and related transcription factors was detected by, Western Blot and RT-PCR in Panc-1 cells transfected with EMT by Notch-1 siRNA, and the expression of ABC transporter family members was also detected. FACS was used to detect the change of SP cells, and Transwell chamber was used to detect the changes of migration and invasion ability of MET cells. Results: Notch-1 siRNA up-regulated the expression of MET, EMT marker E-cadherin, down-regulated the expression of vimentin and related transcription factors, decreased the migration and invasion of MET cells, and down-regulated the expression of ABC transporter family members. The content of SP cells decreased significantly. Conclusion: Notch-1 siRNA can inhibit Notch-1 signaling pathway and Notch-1 is an important signal pathway of EMT in Panc-1 cells. EMT can promote the invasion, metastasis and expression of CSC in Panc-1 cells. Inhibiting Notch-1 signaling pathway can inhibit the invasion, metastasis and drug resistance of pancreatic cancer cell EMT,.
【學位授予單位】：武漢大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R735.9

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