Notch-1通路在人胰腺癌细胞上皮—间质转化和肿瘤干性中的作用机制研究

发布时间：2018-10-25 19:34

【摘要】：第一部分Notch-1基因在吉西他滨诱导人胰腺癌细胞上皮-间质转化中的表达及作用机制研究目的：探讨Notch-1在吉西他滨(GEM)诱导的人胰腺癌Panc-1细胞上皮间叶样表型转化(EMT)中的表达及作用机制。方法：低剂量、较长时间GEM处理胰腺癌Panc-1细胞,观察细胞形态学改变,Western Blot和RT-PCR法检测EMT标志物、Notch-1及相关转录因子的表达,免疫荧光技术检测E-cadherin及vimentin的表达,Transwell小室检测EMT细胞的迁移和侵袭能力。结果：GEM诱导胰腺癌Panc-1细胞的EMT最适药物浓度为5-10mM,最适时间为9天；EMT标志物E-cadherin表达下调,vimentin表达上调,Notch-1和相关转录因子的表达上调。EMT细胞迁移和侵袭能力明显增强。结论：适当浓度的GEM持续作用于人胰腺癌Panc-1细胞,可诱导Panc-1细胞EMT, Notch-1是该过程的重要信号通路,且胰腺癌细胞发生EMT改变后迁移和侵袭能力明显增强。第二部分侧群细胞及ABC转运体家族在吉西他滨诱导人胰腺癌细胞上皮-间质转化中的作用机制研究目的：探讨侧群细胞(SP)及ABCG2在吉西他滨诱导人胰腺癌细胞EMT中的作用机制研究。方法：低剂量、较长时间GEM处理胰腺癌Panc-1细胞,Western Blot和RT-PCR法检测EMT改变的Panc-1细胞中ABC转运体家族ABCB1、ABCC1及ABCG2的表达。荧光激活细胞分选法(FACS)分离SP细胞,对比EMT改变前后SP细胞的含量变化。结果：胰腺癌Panc-1细胞EMT后ABC转运体家族ABCB1、ABCC1、ABCG2表达明显上调,且发生EMT改变后SP细胞含量显著增加(P0.05)。结论：人胰腺癌细胞EMT可使肿瘤CSC相关蛋白表达上调,同时可增加胰腺癌CSC的含量,有助于胰腺癌细胞的增殖、侵袭及耐药。第三部分Notch-1 siRNA在人胰腺癌细胞问质-上皮转化中的作用及侧群细胞、ABC转运体家族的表达机制研究目的：探讨Notch-1 siRNA对人胰腺癌细胞间质-上皮转化(MET)的作用,以及SP细胞和ABCG2的表达机制研究。方法：Notch-1 siRNA转染EMT改变的人胰腺癌Panc-1细胞,Western Blot和RT-PCR法检测EMT标志物E-cadherin、vimentin及相关转录因子的表达,同时检测ABC转运体家族成员的表达,FACS检测SP细胞的改变,Transwell小室检测MET细胞迁移和侵袭能力的改变。结果：Notch-1 siRNA可使EMT改变的Panc-1细胞发生MET, EMT标志物E-cadherin表达上调,vimentin及相关转录因子表达下调,MET细胞迁移和侵袭能力下降,同时,ABC转运体家族成员表达下调,SP细胞含量明显减少。结论：Notch-1 siRNA可抑制Notch-1信号通路,Notch-1是Panc-1细胞EMT的重要信号通路；EMT可促进Panc-1细胞侵袭、转移及CSC特性的表达,抑制Notch-1信号通路可阻止胰腺癌细胞EMT,从而抑制其侵袭、转移及耐药等特性。
[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,.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.9

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