白杨素通过抑制自噬增强胰腺癌吉西他滨化疗敏感性的实验研究

发布时间：2018-03-07 19:41

本文选题：白杨素　切入点：胰腺癌　出处：《华中科技大学》2016年博士论文　论文类型：学位论文

【摘要】：第一部分白杨素抑制胰腺癌细胞增殖并诱导其线粒体途径凋亡目的研究黄酮类化合物白杨素对人胰腺癌细胞增殖、凋亡等的影响及其可能的机制。方法细胞增殖活性采用CCK-8法和克隆平板试验检测：细胞凋亡采用Annexin V-FITC/PI双标流式细胞术检测；细胞周期采用PI单标流式细胞术检测；免疫印迹法检测凋亡相关蛋白的表达。结果白杨素能抑制胰腺癌细胞增殖和克隆形成,且这种作用呈时间和浓度依赖性；白杨素能阻滞细胞周期于G0/G1期,并诱导胰腺癌细胞发生凋亡；免疫印迹结果显示,白杨素能引起PARP、Caspase-9、Caspase-3剪切体的激活,同时上调Bax/Bcl-2的比值。结论白杨素能抑制人胰腺癌细胞增殖和克隆形成能力,并阻滞细胞周期于G0/G1期；白杨素能够诱导胰腺癌细胞发生线粒体途径凋亡。第二部分白杨素通过抑制溶酶体酶活性阻断胰腺癌细胞自噬目的研究白杨素对人胰腺癌细胞自噬的影响及其可能的机制。方法构建携带GFP标签的LC3过表达慢病毒载体并稳定转染胰腺癌细胞株,激光共聚焦显微镜观察细胞内GFP-LC3点状聚集的数目：免疫印迹法检测自噬相关蛋白的表达情况：免疫荧光法检测自噬底物p62蛋白的表达;采用透射电子显微镜观察细胞超微结构的变化；构建携带GFP和mRPF双标签的LC3过表达慢病毒载体并稳定转染胰腺癌细胞株,激光共聚焦显微镜观察细胞内GFP-LC3和mRFP-LC3共定位关系;利用溶酶体探针LysoTracker Red检测溶酶体与GFP-LC3之间的空间定位关系；采用AO(吖啶橙)染色观察细胞内酸性自噬泡的数目；采用组织蛋白酶活性测定试剂盒检测细胞内组织蛋白酶的活性情况。结果共聚焦结果显示,白杨素能导致稳定转染GFP-LC3过表达慢病毒的胰腺癌细胞内出现大量GFP-LC3点状聚集,且这种作用呈浓度和时间依赖性;同时,这种GFP-LC3绿色荧光与LysoTracker Red红色荧光具有共定位关系：免疫印迹和免疫荧光结果显示,白杨素可诱导胰腺癌细胞自噬关键蛋白LC3-II的表达增加,而自噬底物蛋白p62表达也增加；电镜结果显示,白杨素能诱导胰腺癌细胞胞质内形成大量自噬泡；免疫印迹结果显示,溶酶体抑制剂CQ与白杨素联合作用后,LC3-II的表达较单用CQ组无明显增加；与CQ作用相似,白杨素能诱导稳定转染GFP-mRPF-LC3过表达慢病毒的胰腺癌细胞内GFP-LC3和mRFP-LC3荧光共定位,而自噬激活剂Rap作用后mRFP-LC3荧光显著高于GFP-LC3;AO染色结果显示,白杨素与CQ均能显著增加胰腺癌细胞内酸性自噬泡的数量；组织蛋白酶活性检测和免疫印迹结果显示,白杨素能抑制胰腺癌细胞内组织蛋白酶B和组织蛋白酶D的活性。结论白杨素通过抑制溶酶体酶活性阻断胰腺癌细胞自噬。第三部分白杨素通过抑制自噬增强胰腺癌细胞对吉西他滨化疗敏感性目的探讨白杨素联合吉西他滨对胰腺癌细胞增殖、凋亡的影响及其机制。方法细胞增殖活性采用CCK-8法检测；细胞凋亡采用Annexin V-FITC/PI双标流式细胞术检测;倒置显微镜观察药物单用或联用对细胞形态的影响；免疫印迹法检测自噬和凋亡相关蛋白的表达;采用RNAi技术沉默自噬关键基因Beclin1、ATG5、ATG7后检测吉西他滨对胰腺癌细胞增殖、凋亡作用的变化。结果吉西他滨能通过浓度和时间依赖性方式部分抑制胰腺癌细胞增殖;免疫印迹结果显示,吉西他滨可诱导胰腺癌细胞LC3-Ⅱ的表达增加,而自噬底物蛋白p62则表达减少；吉西他滨与溶酶体抑制剂CQ合用后,LC3-Ⅱ的表达较单用CQ显著增加；无论是联合自噬抑制剂CQ或是沉默自噬关键基因Beclin1、ATG5、ATG7后,均能显著增加吉西他滨对胰腺癌细胞的杀伤效果；当吉西他滨与白杨素联合使用后,LC3-Ⅱ的表达较二者单用显著增加；同时,两药联用较单药使用更能抑制胰腺癌细胞活性并诱导细胞凋亡,而在联用基础上加入CQ后并未增加细胞杀伤效果；免疫印迹结果显示,两药联用较单药使用更能增加PARP、Caspase-9、Caspase-3剪切体的激活,并进一步上调Bax/Bcl-2的比值。结论吉西他滨部分抑制胰腺癌细胞增殖并诱导细胞发生保护性自噬：白杨素通过抑制自噬增强胰腺癌细胞对吉西他滨的化疗敏感性。
[Abstract]:The first part of chrysin inhibits the proliferation of pancreatic cancer cells and on human pancreatic cancer cell proliferation and induce the apoptosis of mitochondrial pathway of flavonoids in poplar, apoptosis and its possible mechanism. The activity of cell proliferation by CCK-8 method and plate cloning test: cell apoptosis by Annexin V-FITC/PI double staining flow cytometry; cells cycle using PI single labeled flow cytometry; detect the expression of apoptosis related proteins by Western blotting. Results of chrysin can inhibit pancreatic cancer cell proliferation and colony formation, and this effect was time and dose-dependent; chrysin can block the cell cycle in G0/G1 phase, and induced apoptosis of pancreatic cancer cells; Western blot the results show that chrysin can cause PARP, Caspase-9, activation of Caspase-3 shear body, while raising the ratio of Bax/Bcl-2. Conclusion chrysin inhibits The proliferation of human pancreatic cancer cells and cloning formation ability and cell cycle arrest in G0/G1 phase; chrysin can induce pancreatic cancer cell apoptosis of mitochondrial pathway. The second part chrysin by blocking the effects of pancreatic cancer cell autophagy objective of chrysin on autophagy in human pancreatic adenocarcinoma cells inhibit the activity of lysosomal enzyme and its possible mechanisms. Methods of construction with GFP tag LC3 overexpression lentivirus vector and stably transfected into pancreatic cancer cell line, observe the number of intracellular GFP-LC3 punctate aggregation of laser confocal microscopy: to detect the expression of autophagy related protein by Western blotting to detect the expression of p62 protein: autophagy substrates immunofluorescence; the changes of ultrastructure were observed by transmission electron microscope; construct GFP and mRPF double labeling of LC3 overexpression lentivirus vector and stably transfected into pancreatic cancer cell line, confocal laser display Micro mirror observation of intracellular GFP-LC3 and mRFP-LC3 Co located; using spatial location relationship between probe LysoTracker Red detection of lysosomes and lysosomal GFP-LC3; using AO (acridine orange) staining was used to observe the number of intracellular acidic autophagic vacuoles; the cathepsin activity determination kit for detecting intracellular cathepsin activity. The results of confocal results show that chrysin can lead to the emergence of pancreatic cancer cells transfected with GFP-LC3 overexpression lentivirus in GFP-LC3 punctate aggregation, and this effect is dose and time dependent; at the same time, the GFP-LC3 green fluorescence and LysoTracker Red red fluorescence with CO localization between Western blotting and immunofluorescence results showed that increased expression of chrysin induced pancreatic cancer autophagy protein LC3-II, and p62 also increased the expression of autophagy protein substrate; electron microscopy showed that chrysin Can induce pancreatic cancer cells in the cytoplasm of the formation of a large number of autophagic vacuoles; Western blotting showed that the lysosomal inhibitor CQ and the combined effect of chrysin, the expression of LC3-II was no significant increase in CQ group; similar to the CQ effect of chrysin induced pancreatic cancer cell can be stably transfected with lentivirus expressing GFP-mRPF-LC3 in GFP-LC3 and mRFP-LC3 fluorescence co localization, and activate autophagy after Rap mRFP-LC3 fluorescence was significantly higher than that of GFP-LC3; AO staining showed that chrysin and CQ significantly increased the number of pancreatic cancer cells in acidic autophagic vacuoles; cathepsin activity test and Western blotting showed that chrysin inhibits pancreatic cancer cells, cathepsin B and cathepsin D conclusion chrysin. Activity by inhibiting the activity of lysosomal enzyme blocking autophagy in pancreatic cancer cells. The third part of chrysin through inhibition of autophagy enhances pancreatic cancer cells in Kyrgyzstan Objective to investigate the chemosensitivity to gemcitabine chrysin combined with gemcitabine on pancreatic cancer cell proliferation, apoptosis and its mechanism. The cell proliferation activity was detected by CCK-8 method; cell apoptosis by Annexin V-FITC/PI double staining flow cytometry; inverted microscope observation of the drug alone or in combination with effects on cell morphology; expression of autophagy and apoptosis detection by Western blotting; using RNAi technique to silence autophagy key genes Beclin1, ATG5, detected the proliferation of gemcitabine on pancreatic cancer ATG7 cells, changes of apoptosis. Results of gemcitabine by time and concentration dependent manner inhibited the proliferation of pancreatic cancer cells; Western blotting showed that increased expression of gemcitabine induced pancreatic cancer cell LC3- II, p62 and autophagy substrate protein expression decreased; gemcitabine and lysosomal inhibitor CQ with LC3- II. The expression of CQ was significantly increased compared with single; whether autophagy with CQ or silent key autophagy genes Beclin1, ATG5, ATG7, significantly increased the antitumor effect of gemcitabine in pancreatic cancer cells; when combined with gemcitabine and chrysin after use, the expression of LC3- II is the two single by increased significantly; at the same time, with the combination of the two drugs with a single drug use can inhibit the activity of pancreatic cancer cells and induce cell apoptosis, and in combination on the basis of after joining the CQ did not increase the cell killing effect; Western blotting showed that the two drugs combined with a single drug can increase PARP, Caspase-9, activation of Caspase-3 shear body, and further increase the ratio of Bax/Bcl-2. Conclusion gemcitabine partially inhibited and apoptosis induced by protective autophagy in pancreatic cancer cell proliferation: inhibition of autophagy by chrysin enhances chemosensitivity to gemcitabine in pancreatic cancer cells.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.9

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