【作者】 李旭；

【导师】 秦仁义；

【作者基本信息】 华中科技大学 ， 外科学， 2013， 博士

【摘要】 目的研究醉茄的活性成分醉茄素A（withaferin A，WA）对人胰腺癌细胞增殖、凋亡等的影响及其可能的作用机制。方法采用CCK-8法检测细胞增殖活性；倒置显微镜观察WA对细胞形态的影响；Hoechst33258荧光染色和Annexin V–FITC/PI双标流式细胞术检测细胞凋亡；JC-1荧光探针检测细胞线粒体膜电位；PI染色流式细胞术检测细胞周期；Western Blot检测凋亡相关蛋白的变化；CM-H2DCFDA荧光探针标记检测细胞内活性氧（Reactiveoxygen species, ROS）水平；最后使用泛Caspase家族激酶抑制剂Z-VAD-FMK和各种抗氧化剂干预细胞后，观察细胞凋亡的变化。结果WA在体外对多种人胰腺癌细胞株均有一定的增殖抑制和凋亡诱导作用，且这种作用呈浓度和时间依赖性，但其对人正常细胞株的增殖抑制作用较小；WA可诱导人胰腺癌细胞线粒体膜电位下降，并阻滞细胞周期于G2/M期；Western Blot结果显示，WA能引起Caspase-3、Caspase-9、PARP1的剪切激活和Bax/Bcl-2比值上调；而Z-VAD-FMK可部分逆转WA所诱导的细胞凋亡效应；同时WA还可显著增加细胞内的ROS水平，但当使用抗氧化剂NAC和Catalase清除细胞内ROS后，细胞凋亡率也明显减少。结论WA在体外可显著抑制人胰腺癌细胞增殖，并阻滞细胞周期于G2/M期；WA能够诱导细胞发生凋亡，且这种凋亡依赖于Caspase家族的激活；另外，WA还可引起细胞内ROS水平显著增加，而WA诱导的细胞凋亡依赖于这种ROS的产生。目的通过体内外实验研究WA是否诱导人胰腺癌细胞发生自噬及其生物学意义。方法采用透射电子显微镜观察细胞超微结构的变化。Western Blot检测自噬关键蛋白LC3B的表达情况。构建pEGFP-LC3质粒稳定转染细胞株，激光共聚焦显微镜观察细胞内绿色GFP-LC3点状阳性细胞数比例。利用溶酶体探针LysoTracker Red DND-99检测GFP-LC3与溶酶体之间的空间定位关系。采用多种自噬抑制剂及RNAi技术沉默自噬关键基因Atg5后检测细胞增殖和凋亡的变化。最后通过建立Panc-1裸鼠皮下移植瘤模型，在动物水平观察单用WA或联合使用自噬阻滞剂氯喹（chloroquine, CQ）后对胰腺癌细胞血管生成、增殖、凋亡和自噬等生物学特性的影响。结果透射电镜结果显示，WA作用后细胞质中可见大量空泡化双层膜结构及自噬体形成；Western Blot结果显示，WA可诱导自噬关键蛋白LC3B-Ⅱ的表达增加；激光共聚焦结果显示，WA可诱导稳定转染pEGFP-LC3质粒的细胞出现大量GFP-LC3点状聚集，并且这种绿色GFP-LC3与红色LysoTracker Red共定位；使用自噬溶酶体抑制剂与WA联合作用后，细胞内绿色GFP-LC3点状聚集进一步增多，且Western Blot显示LC3B-Ⅱ的表达也进一步增加；但另一种自噬阻滞剂3-甲基腺嘌呤(3-methyladenine,3-MA)以及泛Caspase家族激酶抑制剂Z-VAD-FMK却对WA诱导的LC3B-Ⅱ的表达无显著影响；而当自噬溶酶体抑制剂与WA联用后可增强其抑制细胞增殖和诱导凋亡的效应；同样，当使用RNAi技术沉默自噬关键基因Atg5来抑制自噬后，可显著增加WA诱导的细胞凋亡；裸鼠皮下移植瘤模型结果显示：药物干预21天后，单用WA组的肿瘤体积和重量与对照组相比均有一定程度的减小(P<0.05)；而当联合使用自噬阻滞剂CQ后，肿瘤体积和重量均较单用WA组进一步减小(p<0.05)；免疫组化结果显示，单用WA组移植瘤组织血管标记蛋白CD34和肿瘤增殖抗原Ki67的表达较对照组均有一定程度的减少，而自噬相关蛋白p62/SQSTM1和LC3B的表达均明显增加(P<0.05)；当联合使用CQ后CD34和Ki67的表达较单用WA组进一步下降(p<0.05)，而p62/SQSTM1和LC3B的表达则进一步增加(P<0.05)；Tunel检测显示单用WA组较空白对照组细胞凋亡率升高(P<0.05)，而联合使用CQ后细胞凋亡率进一步增加(P<0.05)。结论WA在体内、体外均可诱导人胰腺癌细胞发生自噬，且自噬可能是作为一种保护性应激反应来对抗细胞凋亡；WA诱导的细胞自噬不能被Caspase家族激酶抑制剂Z-VAD-FMK所阻滞，且不依赖于Class Ⅲ PI3K通路；单用WA可部分抑制裸鼠皮下移植瘤生长，而联合自噬阻滞剂CQ后可显著抑制移植瘤生长并促进其凋亡。目的探讨内质网应激反应对WA诱导胰腺癌细胞凋亡和自噬的影响及其分子机制。方法采用Western Blot检测相关蛋白的变化；观察抑制mTOR通路后对WA诱导的细胞凋亡和自噬的影响；采用间接免疫荧光法和透射电镜观察细胞内质网结构的改变；间接免疫荧光法检测泛素化蛋白（ubiquitin）、p62/SQSTM1蛋白和GFP-LC3的共定位；采用免疫共沉淀技术检测p62/SQSTM1与LC3B蛋白之间的相互作用关系；分别观察内质网应激保护剂Salubrinal和内质网应激诱导剂衣霉素（Tunicamycin，TM）对WA诱导的细胞凋亡和自噬的影响；RNAi技术分别沉默相关关键基因CHOP、Atg5、p62/SQSTM1后检测细胞泛素化蛋白、内质网应激相关蛋白以及凋亡和自噬的变化；最后观察抗氧化剂NAC对细胞内质网应激和自噬的影响。结果WA诱导人胰腺癌细胞p62/SQSTM1表达上调，但对其它自噬相关蛋白影响不大；WA能抑制PI3K-AKT-mTOR信号通路，而抑制mTOR通路后能进一步增加细胞自噬并部分逆转WA所诱导的细胞凋亡效应；WA诱导人胰腺癌细胞泛素化蛋白聚集，且聚集的泛素化蛋白与GFP-LC3共定位；WA诱导人胰腺癌细胞发生内质网应激反应，而抑制内质网应激可减少WA诱导的细胞凋亡和自噬；相反，促进内质网应激可增加WA诱导的细胞凋亡和自噬；并且，抑制细胞自噬可增强WA诱导的泛素化蛋白聚集及内质网应激反应。WA上调p62/SQSTM1的基因转录水平，但不影响其蛋白降解，且p62/SQSTM1与LC3B蛋白为相互作用蛋白；下调p62/SQSTM1后可增强WA诱导的细胞凋亡、自噬和内质网应激反应。最后，WA诱导的内质网应激和自噬反应均可被抗氧化剂NAC所阻断。结论ROS介导的内质网应激反应参与了WA诱导的人胰腺癌细胞凋亡和自噬；WA诱导胰腺癌细胞发生自噬依赖于PI3K/AKT/mTOR信号通路；抑制自噬或增加内质网应激均可显著增强WA对胰腺癌细胞的杀伤作用；p62/SQSTM1介导了自噬溶酶体清除泛素化蛋白和错误未折叠蛋白的过程，并增强了胰腺癌细胞对WA的凋亡抵抗作用。更多还原

【Abstract】 Objective To study the effect of withaferin A(WA), an effective component extracted fromWithania somnifera Dunal, on proliferation and apoptosis of human pancreatic cancer cellsand its possible mechanism.Methods CCK-8assay was performed to detect cell viability; Inverted microscope toobserve the influence of WA for cell morphology; Hoechst33258fluorescence staining andAnnexin V/PI double staining flow cytometry to detect cell apoptosis; JC-1fluorescentprobe were used to detect cell mitochondria membrane potential; the cell cycle distributionof cells was were detected by PI staining flow cytometry; The expressions of apoptosisrelated proteins were detected by Western blotting; CM-H2DCFDA fluorescent probe wereused to detect reactive oxygen species (ROS) production; Finally, using general caspaseinhibitor Z-VAD-FMK and various antioxidants interfere cell, then observe the changes ofcell apoptosis.Results WA significantly inhibited the proliferation and induced apoptosis of many kindsof human pancreatic cancer cell lines in a dose-and time-dependent manner in vitro, butthe inhibitory effect is small for human normal cell lines; WAcan induce human pancreaticcancer cells mitochondrial membrane potential decreased, and caused the cell cyclearrested at G2/M phase; Western blotting analysis showed that WA could significantlyincreased the ratio of Bax/Bcl-2, and enhanced the expression of cleaved caspase-3, cleaved caspase-9and cleaved PARP1in pancreatic cancer cell lines; Z-VAD-FMK couldpartially reversed the apoptosis effect induced by WA; Furthermore, WA also significantlyincreased the intracellular ROS level, but when the use of antioxidants NAC and Catalaseclear cell ROS, apoptosis rate is significantly reduced.Conclusion WA could significantly inhibit the proliferation of human pancreatic cancercells in vitro, and caused the cell cycle arrest at G2/M phase; WA also induced cellapoptosis, and the apoptosis is dependent on the activation of the caspase family;Furthermore, WA also significantly increased the intracellular ROS level, and WAinduction of cell apoptosis is dependent on the production of ROS. Objective Study on whether WA induces autophagy in human pancreatic cancer cells andits biological significance through vitro and vivo experiments.Methods The super-microstructural changes were observed by transmission electronmicroscopy after WA treatment; the expressions of cleavage of microtubule-associatedprotein1light chain3isoform B (LC3B-II) was detected by Western blotting; Constructionof pEGFP-LC3plasmid stable transfected cell lines, and the fluorescent autophagy markerGFP-LC3was observed under a confocal microscope; the fusion of lysosomes andGFP-LC3was detected by staining with LysoTracker Red DND-99lysosomal Probe undera confocal microscope; the autophagic activity was inhibited by using variety of autophagyinhibitors or silencing Atg5(a critical component of the autophagic machinery), and thenthe proliferation and apoptosis induced by WA was assessed; Finally, xenograft tumormodel in nude mice was established to observe the effect of using WA alone or combininguse of autophagy inhibitor chloroquine (CQ) on the biological characteristics of pancreatic cancer cells about angiogenesis, proliferation, apoptosis, autophagy and so on.Results Transmission electron microscope showed that a large number of doublemembrane vacuolar autophagosomes were observed in WA-treated cells; simultaneously,Western Blot results show that WA also could significantly increased the expression ofLC3B-II; confocal microscope show that WA could induce pEGFP-LC3plasmid stabletransfected cell lines appeared a large number of GFP-LC3punctate aggregates, and thegreen GFP-LC3and red LysoTracker Red is colocalized; a combined effect of usingautophagic lysosome inhibitors and WA caused GFP-LC3punctate aggregates furtherincreased, and Western Blot showed LC3B-Ⅱ expression is further up-regulation;nevertheless, another autophagy inhibitor3-methyladenine (3-MA) and the genericCaspase family kinase inhibitor Z-VAD had no significant effect on LC3B-Ⅱexpression;Moreover, it could enhanced the effect of inhibition of cell proliferation and induction ofapoptosis when WA combined with autophagic lysosome inhibitors; similarly, when theautophagic activity was inhibited by silencing Atg5, apoptosis index also increased afterWA interference; xenograft tumor model show that:21days later, single WA couldinhibited the tumor growth and reduced the tumor volume compared with control group(P<0.05); when combined with autophagy inhibitor CQ, tumor volume and weight werefurther reduced compared with WAalone (P<0.05); Immunohistochemical analysis showed,in WA alone group, CD34and Ki67expression had a certain degree of reduction thancontrol group, while p62/SQSTM1and LC3B expression were increased significantly(P<0.05); when combined with CQ, the expression of CD34and Ki67were further reducedcompared with WA alone (P<0.05), and the expression of p62/SQSTM1and LC3B werefurther up-regulation (P<0.05);.Tunel staining showed that WAalone may induce apoptosiscompared with control group (P<0.05), when combined with CQ, the apoptosis rate wasincreased significantly compared with control group (P<0.01).Conclusion WAcould induce autophagy in human pancreatic cancer cells in vivo and vitro,and autophagy may serve as a protective response to stress; WA induced autophagy were not blocked by the Caspase kinase inhibitor Z-VAD-FMK, and it also does not depend onthe Class Ⅲ PI3K pathway; A single WA could partially inhibit the growth of xenografttumor in nude mice, while combined with autophagy inhibitor CQ could inhibit tumorgrowth and promote apoptosis significantly. Objective To explore the effect and molecular mechanism of endoplasmic reticulum stressresponse on WA-induced apoptosis and autophagy in human pancreatic cancer cells.Methods The expressions of related proteins were detected by Western blotting; to observethe changes of WA-induced apoptosis and autophagy after inhibition of mTOR pathway;the change of endoplasmic reticulum structure was observed by using indirectimmunofluorescence and transmission electron microscopy; colocalization ofp62/SQSTM1, ubiquitin and GFP-LC3were observed by using indirectimmunofluorescence; The interaction relationship between p62/SQSTM1and LC3Bprotein were detected by co-immunoprecipitation; observed the effects of ER stressprotective agent Salubrinal and ER stress inducer tunicamycin on WA induced apoptosisand autophagy; to observe the changes of apoptosis and autophagy after silencing relatedgene CHOP, Atg5and p62/SQSTM1, then the expression of ubiquitin and ER stress relatedprotein were also detected by Western blot; Finally, using antioxidants NAC interfere cell,then observe the changes of ER stress and autophagy.Results WA treatment increased the expression of p62/SQSTM1, but had little effect onother autophagy-related proteins; WA could inhibit the PI3K-AKT-mTOR signaling pathway, and inhibition of mTOR pathway can partially reverse the effect of cell apoptosisinduced by WA; WA treatment caused ubiquitinated protein accumulated, and theaccumulated ubiquitin colocalized with GFP-LC3; WA was able to induce ER stress, andSalubrinal or CHOP siRNA pretreatment inhibiting ER stress caused the suppression ofWA-induced apoptosis and autophagy; on the contrary, promoted the ER stress couldincrease WA-induced cell apoptosis and autophagy; Moreover, inhibition of autophagycould enhance the aggregation of ubiquitined protein and ER stress reaction induced byWA; WA caused p62/SQSTM1gene transcription level, but had no effect on the proteindegradation; furthermore, p62/SQSTM1interacts with LC3B; down-regulation ofp62/SQSTM1by small interfering RNA could enhance WA-induced apoptosis, autophagyand ER stress; Finally, autophagy and ER stress response which were induced by WA couldbe blocked by the antioxidant NAC.Conclusion ER stress response mediated by ROS is involved in the induction of apoptosisand autophagy by WA; Autophagy is dependent on PI3K/AKT/mTOR signaling pathwayinduced by WA; inhibition of autophagy or increased ER stress could significantlyenhanced the cytotoxic effect of WA on pancreatic cancer cells; p62/SQSTM1involved inWA resistance in human pancreatic cancer cells by clearing ubiquitinated proteins throughthe autophagic lysosome.更多还原