【作者】 王萍；

【导师】 李继承；

【作者基本信息】 浙江大学 ， 细胞生物学， 2007， 博士

【摘要】 天花粉蛋白(TCS)具有多种药理学特性,包括中期流产、抗人HIV病毒和诱导多种肿瘤细胞凋亡的抗肿瘤活性。已有研究结果表明,TCS诱导JAR内ROS产生、细胞内钙离子浓度波动、caspase-3活化和凋亡;TCS抑制HSV-1感染的Vero细胞内MAPK和Bcl-2信号通路,进而抑制细胞复制。此外,TCS通过抑制PKC活性而诱导K562细胞凋亡。我们实验室的研究发现,TCS能够诱导Hela细胞形态学改变,包括微绒毛消失、细胞膜发泡,染色质浓缩和凋亡小体形成;TCS抑制细胞增殖的机制与细胞周期S期阻滞和诱导细胞内capsase凋亡通路活化有关。这些研究结果初步证实Hela细胞是TCS的敏感细胞之一,但TCS诱导Hela细胞凋亡过程中的细胞和分子机制仍需做深入的研究。鉴于此,本课题在原有的实验基础上,进一步研究了TCS诱导HeLa细胞凋亡过程中细胞骨架的改变、相关信号转导通路或靶点、转录因子环磷酸腺苷反应结合蛋白(CREB)在调节Bcl-2蛋白表达水平的作用,以便从细胞和分子水平上揭示TCS诱导Hela细胞凋亡的机理,为天花粉蛋白在肿瘤防治中的应用,提供重要的理论和实验依据。研究目的通过研究TCS诱导Hela细胞凋亡过程中细胞骨架的排列、骨架蛋白和基因表达的改变、以及相关信号转导通路或靶点,阐明TCS诱导Hela细胞凋亡的细胞和分子机制。研究方法采用CCK-8的方法,检测TCS对HeLa细胞的抑制作用;在confocal免疫荧光显微镜下,观察TCS诱导Hela细胞内细胞骨架微管(MT)和微丝(MF)的改变;利用western blot检测TCS对细胞骨架蛋白表达的影响,real-timePCR检测TCS对细胞骨架actin和tubulin亚型基因表达的影响;电子显微镜和Annexin/PI双染观察TCS对HeLa细胞诱导凋亡的作用;Fura-4荧光探针检测TCS诱导Hela细胞内钙离子水平的改变;腺苷酸环化酶(AC)、PKA、PKC、ERK、p38-MAPK蛋白激酶抑制剂或激活剂检测这些激酶在TCS抑制HeLa细胞增殖中的作用;用反义环磷酸腺苷反应结合蛋白(CREB)核苷酸敲除法和cAMP反应原件(CRE)诱骗(decoy)寡核苷酸(ODN)法,验证TCS是否通过调节转录因子CREB对Bcl-2蛋白的表达产生影响。研究结果(1)TCS诱导Hela细胞凋亡伴随着MF解聚和质膜下“环状”MT结构的形成;TCS抑制细胞骨架actin和tubulin亚型基因的表达,但不影响actin,α-tubulin和β-tubulin蛋白的表达。(2)钙离子螯合剂破坏TCS诱导的“环状”MT结构,但不影响TCS引起的MF解聚;而且钙离子螯合剂和TCS联合处理,显著性增加LDH的释放。(3)TCS诱导的细胞外钙离子内流,抑制细胞内腺苷酸环化酶的活性和细胞内cAMP的形成;PKC活化剂显著性拮抗TCS抑制的腺苷酸环化酶的活性和细胞内cAMP的水平;TCS抑制的p-CREB表达能够被cAMP异构体拮抗;然而,PKC/ERK/MAPK抑制剂阻滞了cAMP异构体的拮抗效应。(4)反义CREB核苷酸敲除法和CRE诱骗ODN法,有效阻滞了TCS对Bcl-2蛋白的调节作用。结论(1)TCS诱导Hela细胞凋亡伴随着MT“环状”结构的形成和抑制细胞骨架actin和tubulin亚型基因的表达,但不影响actin和tubulin蛋白表达的改变。(2)TCS诱导细胞内钙离子的升高参与调节和维持细胞内MT“环状”结构的形成与凋亡过程中细胞膜的完整性。(3)TCS诱导的细胞内钙离子升高,参与抑制Hela细胞内cAMP/PKC/MAPK/CREB活性,进而抑制细胞增殖。(4)TCS抑制的CREB磷酸化对调控Bcl-2基因表达起主要作用。这些结果为理解TCS诱导凋亡的细胞和分子机制提供了新的思路。更多还原

【Abstract】 Trichosanthin（TCS）possesses a broad spectrum of biological and pharmacological activities,including abortifacient,anti-HIV and anti-tumor activities through apoptosis pathway in some cells.Previous studies showed that TCS induced the apoptosis of JAR cells via ROS production,internal Ca²⁺elevation and caspase-3 activation.TCS suppressed mitogen-activated protein kinase（MAPK）and Bcl-2 signals leading to the replication inhibition in HSV-1 infected Vero cells.Furthermore, K562 cell apoptosis was induced by TCS via PKC inhibition.Additionally,the data obtained from our laboratory indicated that TCS caused Hela cells with markedly morphological changes,including microvilli disappearance or reduction,cell membrane vesiculation,cell shrinkage,condensation of chromosomes and apoptotic bodies with complete membrane.Further studies on the mechanisms of Hela cell apoptosis induced by TCS,showed that TCS-caused a S-phase arrest in cell cycle associated with a time-dependent increase of caspase-3,8, 9 activities.These preliminary results,therefore,indicate that Hela cells are sensitive to TCS.However,these data are insufficient for elucidating the cellular and molecular mechanisms occurring in Hela cell apoptosis induced by TCS.Objective To investigate the effects of TCS on cytoskeletal remodeling,the quantitative changes of actin and tubulin proteins,and subunit cytoskeletal mRNA levels in apoptotic Hela cell.Furthermore,we determined the specific intracellular signaling transduction pathways to explore the underlying cellular and molecular mechanisms of Hela cell apoptosis induced by TCS. Methods The antiproliferative effect of TCS on HeLa cells was measured with CCK-8.Cytoskeletal remodeling,the quantitative changes of actin and tubulin proteins, and subunit cytoskeletal mRNA levels in apoptotic Hela cell were examined by confocal immunofluorescence microscopy observations,western blot and quantitative real-time PCR assays.Electron microscopy and FCM analysis was employed to observe the apoptosis of Hela cells.Fura-4 fluorescence probe was used to check the cytosolic calcium level.Antisense knockdown of CREB gene expression,and blockade the binding of CREB to the Bcl-2 CRE were carded out to examine whether cAMP response element-binding（CREB）was involved in TCS-mediated Bcl-2 expression.Results（1）TCS induced the execution phase of cell apoptosis was a highly coordinated process of cellular reorganization,depolymerized microfilaments accumulated in the coarsened cytoplasm and apoptotic bodies,accompanied by the formation of a ring microtubule structure beneath the plasma membrane.However,no quantitative changes were observed on actin and tubulin proteins.Furthermore, apoptosis occurred by a suppression of actin and tubulin subunit gene expression.（2） Sequestrating the TCS-increased[Ca²⁺]c with EGTA caused less protective of cell viability,which was related to the absence of apoptotic microtubule structure altered plasma membrane permeability.（3）TCS initiated an influx of extracellular Ca²⁺,and it was required for the suppression of adenyl cyclase（AC）activity and intracellular cAMP formation.Furthermore,this inhibition was abolished by preincubation with an activator of PKC rather than PKA.The expression of p-CREB protein expression inhibited by TCS was reversed by cAMP agonists.Nevertheless,blocking the ERK1/2 and p38 MAPK pathway in TCS/FSK treated cells significantly prevented the increase of cell viability.Furthermore,the phosphorylated ERK1/2 and p38 MAPK protein levels were decreased by the inhibitors of PKC.（4）CREB antisense effectively reduced CREB expression levels,and TCS was able to reduce Bcl-2 level in cells treated with CREB sense.The importance of activating the CREB pathway in Hela cell apoptosis induced by TCS was further demonstrated by a CRE decoy oligonucleotide to block the binding of CREB to the Bcl-2 CRE.As expected,the CRE oligonucleotide treatment resulted in the constant expression of Bcl-2 protein.In contrast,the treatment of CRE oligonucleotide failed to affect the phosphorylated CREB protein level.Conclusion These data suggested that（1）Hela cell apoptosis induced by TCS was accompanied by the cytoskeletal remodeling and suppression of cytoskeletal gene expression,but not by quantitative changes of proteins.（2）TCS-increased intracellular calcium exerted the pivotal role on the formation of apoptotic microtubule ring structure,which participated in the regulating of plasma membrane permeability.（3） TCS mediated Hela cell apoptosis via the regulation of cAMP/PKC/MAPK pathway. （4）TCS suppressed Bcl-2 expression through the inhibition of phosphorylated CREB. Therefore,these results provide deep insights for the understanding of the cellular and molecular mechanisms underlying TCS-caused cell apoptosis.更多还原