【作者】 张哲伟；

【导师】 陈昭典；

【作者基本信息】 浙江大学 ， 外科学， 2011， 博士

【摘要】 第一部分观察转胶蛋白对肌动蛋白更新的影响研究目的研究转胶蛋白对肌动蛋白更新、细胞ROS水平的影响,并进一步观察其对DNA损伤传导途径和细胞衰老的影响。材料和方法将转胶蛋白转染至人前列腺癌细胞株LNCaP,检测细胞丝状肌动蛋白(F-actin)和球状肌动蛋白(G-actin)的变化,并检测细胞内ROS水平的变化。通过8-OH-dG、phospho-ATM和γH2AX免疫荧光染色观察DNA损伤情况,进一步通过Western blotting检测DNA损伤传导途径ATM (Ataxia-Telangiectasia Mutated)-Chk2 (checkpoint kinase 2)-p53的变化。通过Heterochromatin protein1 (HP1)免疫荧光染色、western blotting和半乳糖苷酶细胞(β-gal)化学染色等方法,观察外源性转胶蛋白和内源性转胶蛋白分别对LNCaP和MRC5细胞染色质重构及细胞衰老的影响。同时,通过SiRNA抑制人骨肉瘤细胞株U2OS内源性转胶蛋白的表达,观察细胞F-actin、G-actin的变化和ROS的变化。结果转染转胶蛋白至LNCaP细胞后24h,细胞F-actin增加明显,G-actin无明显变化,细胞内ROS水平升高；转染U20S细胞Si-transgelin RNA48h后,转胶蛋白水平明显下降,细胞F-actin下降,ROS水平下降。LNCaP转染转胶蛋白后,免疫荧光染色提示细胞核出现8-OH-dG阳性染色,phospho-ATM荧光强度增加及yH2AX簇集的增多。Western blotting提示ATM-Chk2-p53途径的激活。HP1免疫荧光染色提示LNCaP细胞转染转胶蛋白后,细胞衰老相关的异染色质簇集(Senescence-associated heterochromatin Foci, SAHF)增多,western blotting提示HP1γ表达水平上升较明显,acetyl-H3表达下降。β-半乳糖苷酶细胞化学染色提示转染转胶蛋白36h后,阳性细胞数增多。HP1免疫荧光染色和western blotting提示MRC5高代细胞转胶蛋白表达量较低代细胞多,同时SAHF也较低代细胞多。低代MRC5细胞转染转胶蛋白后,p-半乳糖苷酶阳性细胞增加。结论转胶蛋白通过促进LNCaP细胞F-actin聚集,可使细胞发生DNA氧化损伤和DNA断裂,激活DNA损伤信号传导通路。转染转胶蛋白使LNCaP细胞发生细胞衰老。内源性转胶蛋白和MRC5细胞衰老有关,若使MRC5过度表达转胶蛋白,可导致细胞衰老。第二部分观察转胶蛋白和p53之间的相互作用研究目的研究转染转胶蛋白至LNCaP细胞后,p53的表达及其分布的变化,并且研究转胶蛋白和p53之间的相互作用。材料和方法转染转胶蛋白至LNCaP细胞,在不同时间点提取全细胞裂解产物和胞浆、胞核细胞裂解产物进行western blotting检测p53表达情况,并使用转胶蛋白和p53免疫荧光染色检测p53分布情况。随后,我们采用哺乳类细胞双杂交和免疫共沉淀方法研究转胶蛋白和p53蛋白之间的相互作用。结果转染转胶蛋白后,Western blotting提示LNCaP细胞p53表达增加,磷酸化p53(Ser46)也相应增加；胞浆中p53增加,而胞核中p53变化不明显。转胶蛋白和p53双免疫荧光染色也提示胞浆中p53水平增加。哺乳类细胞(人前列腺癌PC-3细胞)双杂交实验提示转胶蛋白和p53相互结合,免疫共沉淀实验显示外源性、内源性转胶蛋白和p53分别在LNCaP细胞和BJ细胞(成纤维细胞)共沉淀。结论转染转胶蛋白使LNCaP细胞p53表达增高,而且p53胞浆转位增加。转胶蛋白和p53在哺乳类细胞内相互作用,并稳定p53蛋白。转胶蛋白和p53免疫共沉淀。第三部分转胶蛋白对p53下游基因及内源性凋亡途径的影响研究目的研究转胶蛋白对p53下游基因表达的影响,以及转胶蛋白诱导LNCaP细胞凋亡的机理。材料和方法转染转胶蛋白至LNCaP细胞,在不同时间点收集细胞裂解产物,使用western blotting检测PUMA (p53 upregulated modulator of apoptosis)、Bax、Bcl-2和caspase-3的表达情况,然后分别将转胶蛋白转染HCT116 p53-/-和p53+/+细胞株,western blotting检测p53下游PUMA的表达情况。最后,采用细胞计数、脱氧核苷酸末端转移酶dUTP标记方法(Terminal deoxynucleotidyl transferase dUTP nick end labeling,TUNEL)和流式细胞计数等方法观察转胶蛋白对LNCaP细胞凋亡的影响。结果转染转胶蛋白后,LNCaP细胞促凋亡因子Bax和PUMA表达量升高,而抗凋亡因子Bcl-2表达水平下降,caspase-3的表达上升。分别转染转胶蛋白至HCT116p53-/-和p53+/+细胞株,p53阴性细胞株PUMA无明显变化；而p53阳性细胞株PUMA表达升高。细胞计数提示转染转胶蛋白后细胞活力下降,TUNEL染色提示转胶蛋白导致TUNEL阳性细胞数量升高,而流式细胞计数亦提示转染转胶蛋白后早期细胞凋亡增多。结论转胶蛋白通过上调促凋亡因子(PUMA、Bax)及抑制抗凋亡因子Bcl-2诱导内源性凋亡途径的激活；而且,转胶蛋白激活PUMA的作用依赖于p53。因此,转胶蛋白激活内源性凋亡途径依赖p53的功能。细胞计数、TUNEL染色和流式细胞计数也证实了转胶蛋白诱导LNCaP细胞凋亡。更多还原

【Abstract】 Part I Transgelin impacts turnover of actinActin turnover is influenced by various actin-binding proteins (ABPs), and it confers crucial effects on cellular ROS (Reactive oxygen species) and apoptosis. Transgelin is the mammalian homologue of Scpl, which can cause inappropriate actin clumping in yeast. To address transgelin affects turnover of actin and cellular ROS in mammalian cell, human prostate cancer LNCaP cells were transfected with pCDNA3.1-transgelin or vector, followed by detecting turnover of F-actin at 24 h after transfection. Simultaneously, cellular ROS was determined by parallel experiment. We have found that ectopic expression of transgelin leads to aggregation of F-actin and increase of cellular ROS. Conversely, knocking down endogenous transgelin of U2OS cells resulted in increase of F-actin turnover and decrease of cellular ROS. To investigate the effects of transgelin on DNA damage response pathway following the induction of ROS,8-OH-dG, phospho-ATM and yH2AX immunofluorescence stained were performed. We found ectopic expression of transgelin caused oxidative stress, characterized by DNA base damage and double strand break. Western blotting indicates activation of DNA damage response, especially ATM (Ataxia-Telangiectasia Mutated)- Chk2 (checkpoint kinase2)-p53 pathway is caused by introduction of ectopic transgelin through elevation of ROS. Finally, immunofluorescence staing for heterochromatin protein 1(HP1), histochemical staing for P-galactosidase (β-gal) and western blotting were carried out to address the induction of chromatin modeling and cell senescence were caused by ectopic expression of transgelin. Our findings reveal that transgelin can lead to decrease of F-actin turnover, and induce cellular ROS, thereby activating ATM-Chk2-p53 pathway and inducing cell senescence.Part II Transgelin interacts with p53Transgelin, known as an ARA54-associated AR inhibitor, can suppress ARA54-enhaced AR transaction in LNCaP cells. Part I experiment suggests that transgelin activates DNA damage response pathway by induction of cellular ROS. In addition to these effects, we aimed to elucidate the pro-apoptotic effects of the protein on LNCaP and its underlying mechanisms, especially the interaction between transgelin and p53. Using western blotting of p53 and double immunofluorescence staining of p53 with transgelin, we have shown that transfection of transgelin resulted in increasing cytoplasmic translocation of p53 and upregulation of p53 expression. We also found an interaction between transgelin and p53 in vivo by mammalian two-hybrid. To futher confirm the interaction, we found both ectopic and endogenous transgelin coimmunoprecipitated with p53 by coimmunoprecipitation assay.Par tⅢEffects of transgelin on p53 downstream target and intrinsic apoptosis pathway The mitochondrial membrane constitutes the battleground on which pro-and anti-apoptotic factors induce or prevent apoptosis. Cytosolic p53 has function to activate intrinsic apoptosis by induction of mitochondrial outer membrane permeabilization (MOMP). PUMA (p53 upregulated modulator of apoptosis), as p53 downstream target, enhances cytosolic p53 function. Using western blotting, we found that ectopic expression of transgelin resulted in downregulation of Bcl-2, and up-regulation of PUMA and Bax, therefore triggering apoptosis. Additionally, we found the effect was dependent of functional p53 by introduction of transgelin to HCT116 p53-/- and p53+/+ cell lines. Futher, Cell counting, flow cytometric analysis and terminal deoxynucleotidyl transferase-dUTP nick-end labeling (TUNEL) assays were applied to measure the pro-apoptotic effect of transgelin. These results suggest the activation of mitochondria-associated apoptosis pathway of LNCaP cells transfected with transgelin. These results are indicative of p53-mediated mitochondria-associated apoptotic effects of transgelin on LNCaP cells in addition to its known suppressive effects on AR pathway.更多还原