【作者】 顾俊莲；

【导师】 李扬； 蔡露；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2014， 博士

【摘要】 背景：前列腺癌是男性最常见的恶性肿瘤之一，手术和放化疗是前列腺癌主要的治疗手段，由于其副作用大，容易复发等原因，严重影响前列腺癌的治疗和预后。近年来，随着药物和分子生物学的迅猛发展，药物联合基因治疗方法以它的安全性和有效性得到了越来越多的关注。肿瘤细胞的生长和死亡很大程度上取决于癌基因和抑癌基因作用不平衡所导致，其中P53是最主要的抑癌基因之一，被冠以“基因卫士”之美名，它在调节正常细胞周期进程，凋亡发生，DNA修复及代谢环境稳态等方面发挥重要作用。大约有50%的人类肿瘤中存在P53突变，而且在各类肿瘤中已经鉴定出2000多种P53突变类型。逆转缺陷的P53功能对于减少肿瘤发生，改善肿瘤耐药将是一项理想的治疗策略。引起P53失活的原因很多，其中重要的有两个：（1）原癌基因MDM2对P53的泛素化降解。MDM2由P53诱导产生，P53与MDM2形成一种负反馈的调节环路，相互制约。MDM2的E3泛素连接酶的活性可以特异性的催化P53的泛素化及介导其出核，并在26S蛋白酶复合体中降解。此外MDM2可直接抑制P53的转录激活功能。所以切断MDM2-P53的反馈环路，对于增强P53蛋白的抑癌功能至关重要。（2）锌的缺失同样会影响P53功能。锌是体内200多种金属酶的辅酶，在基因表达和基因稳定性方面起着重要作用。P53蛋白DNA结合结构域具有锌的结合位点，结合锌以后能够稳定P53蛋白空间构象。在表达野生型P53蛋白的细胞培养中，应用金属锌螯合剂TPEN诱导的缺锌环境能够破坏P53野生型构象，失去其与靶基因DNA结合的活性。此外锌是参与前列腺液合成的必需微量元素之一，正常成年人前列腺液中锌含量为720ug/ml，而其他组织仅为80ug/ml，而前列腺癌血清和组织中的锌浓度下降60%-70%，并且随着前列腺癌的进展，锌的浓度进一步下降，锌的缺失可能也是前列腺癌中P53功能丧失和对放化疗产生耐受的主要原因之一。因此，本课题在构建MDM2特异siRNA与野生型P53相连接的共表达质粒Pmp53基础上，首次提出锌联合共表达质粒Pmp53治疗前列腺癌的方案。其目的一方面可以恢复前列腺癌中缺失或突变的P53状态，更好的发挥P53作为抑癌基因的功能，另一方面改善前列腺癌血清和组织中的缺锌状态。目的：通过基因重组技术构建pGCsilencer-mdm2（Si-mdm2）质粒与pcDNA3.1-U6si-mdm2-p53共表达质粒（Pmp53），探讨锌联合共表达质粒Pmp53对前列腺癌细胞株PC-3和DU145及前列腺癌移植瘤协同治疗效应及可能机制，为前列腺癌的联合治疗提供新的理论和实验依据。方法：参考实验室以前的工作基础，根据P53和MDM2的基因序列以及siRNA设计的基本原理，使用pGCsilencerTMU6/Neo/GFP/RNAi和PCDNA3.1载体，利用酶切等技术，构建质粒Si-mdm2和Pmp53，运用RT-PCR和Western blot方法检测MDM2和P53基因和蛋白的表达，流式细胞术检测各组质粒对PC-3细胞凋亡的影响。体外实验以前列腺癌细胞株PC-3（缺失性P53）和DU145（突变性P53）细胞为研究对象。体外实验的分组：对照组，Zn组，TPEN组，Pmp53组，Pmp53+Zn组，Pmp53+TPEN组和Pmp53+Zn+TPEN组。MTT法检测锌联合Pmp53质粒对PC-3和DU145细胞增殖活性的影响；流式细胞术检测锌联合Pmp53质粒对PC-3和DU145细胞周期的影响；流式细胞术及TUNEL法检测锌联合Pmp53质粒对PC-3和DU145细胞凋亡的影响；罗丹明123观察锌联合Pmp53质粒对PC-3细胞线粒体膜电位的影响；qPCR, RT-PCR和Western blot检测与p53相关的靶基因和蛋白的表达；免疫共沉淀（Co-Immunoprecipatation）检测P53蛋白构象的变化；荧光素酶报告基因（Luciferase Reporter Activity）检测P53转录激活下游靶基因的活性；染色质免疫共沉淀（Chromatin Immunoprecipatation）检测P53与其下游基因p21和bax启动子结合能力。体内实验构建裸鼠前列腺癌移植瘤模型，运用具有肿瘤靶向性的减毒沙门氏菌（Ty21a）携带共表达质粒Pmp53，联合灌胃方式给予锌及锌的抑制剂TPEN，观察其对前列腺癌移植瘤生长的影响及探讨其相关的分子机制。体内实验分组：对照组，Pmp53+TPEN组，Pmp53组和Pmp53+Zn组。这样分组的目是为了给裸鼠体内营造一种低锌，正常锌和补锌的环境。应用qPCR和Western blot检测肿瘤组织P53相关基因和蛋白的表达；应用免疫共沉淀检测P53蛋白构象的变化；流式细胞术和TUNEL检测细胞凋亡变化；HE和免疫组化检测肿瘤组织形态学及PCNA的表达。结果：经酶切鉴定，成功构建了siRNA-mdm2与p53的共表达质粒Pmp53。PCR，Western blot和流式细胞术结果显示前列腺癌细胞经转染Pmp53后，MDM2基因表达被干涉，同时高表达野生型P53，显著增强P53抑癌功能。体外实验证明：与锌和Pmp53组相比，锌联合Pmp53组显著增强了PAB1620的表达，稳定了P53的野生型构象。锌联合Pmp53组诱导细胞周期阻滞于GO-G1期，其机制可能与上调P21表达，下调CDK4，CDK6，CyclinD1表达有关。锌联合Pmp53组抑制增殖，诱导发生细胞凋亡，其机制可能与上调P53，Bax，Caspase-8, Caspase-9，Caspase-3表达，下调Bcl-2，PCNA，MMP2，MMP9表达有关。体内实验证明：应用减毒沙门氏菌携带Pmp53质粒联合锌治疗前列腺癌移植瘤。与对照，Pmp53+TPEN和Pmp53组相比，Pmp53+Zn组肿瘤体积显著缩小，重量显著减轻，肿瘤组织内凋亡率上升，差异具有统计学意义。免疫共沉淀结果显示Pmp53+Zn组显著增强了PAB1620的表达，与体外实验结果一致。HE结果显示Pmp53+Zn组出现大片无结构坏死灶。免疫组化检测到肿瘤组织PCNA和MDM2蛋白表达下调，P53蛋白表达上调，差异有统计学意义。结论：本实验成功构建了共表达质粒Pmp53。体内外实验证明锌联合共表达质粒Pmp53对前列腺癌具有显著治疗作用，且联合治疗的疗效优于单一治疗。其机制可能一方面与共表达质粒Pmp53直接抑制MDM2的表达，一定程度解除了MDM2对P53的负反馈抑制和降解作用，同时高表达野生型P53，既双重增强了野生型P53抑制肿瘤生长的作用有关；另一方面可能与锌进一步稳定了因导入Pmp53质粒而恢复的P53野生型构象，增强了P53转录激活活性，提高了其靶基因p21和bax的表达水平，诱导前列腺癌细胞周期阻滞和凋亡发生有关。更多还原

【Abstract】 Background: Prostate cancer (PCa) is one of the most malignant tumour in menworldwide. Although PCa can be treated with surgery and radiation, there remains ahighly local recurrence and/or metastasis of the tumour. Therefore, development ofnew treatment strategies is urgently needed. As the development of medicines andmolecular biology, combined gene therapy has attracted great attention.A balance between oncogenes and tumor-suppressing genes largely determinesthe production and development of PCa. P53is a well-known tumor suppressor thatplays a master role in the prevention of tumors by regulating cell cycle, apoptosis,DNA repair, antioxidant defense and even metabolic homeostasis. In about50%human tumors, P53mutations have been occured, and more than2000kinds of P53mutation types have identified in all tumors. Loss of P53expression or p53genemutations are common in a number of tumors including PCa, and are also associatedwith increased resistance to chemo-and radiotherapy. Therefore, strategies toreactivate defective P53function will be an ideal therapy. P53inactivation can becaused by a few different mechanisms. For instance,(1) P53can be inactivated isthrough up-regulation of Murine Double Minute2(MDM2). MDM2is induced byP53, and P53-MDM2will form a negative feedback to regulate each other. MDM2functions both as an E3ubiquitin ligase to recognize the N-terminal trans-activationdomain of the P53for degradation by the proteasome and as an inhibitor of p53transcriptional activation. Therefore, disruption of the P53-MDM2association holdsthe promise as a mechanism to reactivate the P53tumor-suppressing pathway.(2) Azinc (Zn) deficiency may also affect P53function. Zinc is the second most abundanttransition metal in the human body. Nearly half of eukaryotic transcription factorsbind zinc and in most of these instances the metal is used to maintain structure. P53isa transcription factor that contains a Zn ion near its DNA binding interface forsite-specific DNA binding and proper transcriptional function. Treating cells with theZn chelator TPEN causes P53to accumulate in the mis-folded conformation, and lose transcription activity. In addition, zinc is one of the essential trace elements insynthesis of prostatic fluid. Zinc content in normal adult prostate fluid is upto720μg/ml, and other organizations accounted for only80μg/ml. Patients with PCa oftenhave low levels of systemic Zn, and importantly prostate Zn levels are decreased inthe developing and progressive prostate cancers. Therefore, Zn deficiency may be acausative of prostate cancer due to inactivation of p53and also a reason for prostatecancer resistant to radio and chemotherapy. These results suggest the potential use ofZn to inhibit tumor growth by reactivating p53normal function.In summary, On the basis of successful construction of Pmp53[a plasmidcontaining both mdm2small interfering RNA (Si-mdm2) and the wild-type p53gene].We have firstly proposed a combined treatment of Pmp53with zinc. The aim, On theone hand, is to restore defective P53activity and function in prostate cancer. On theother hand, is to improve prostate cancer serum and tissue low levels of zinc status.Objective:The co-expression plasmid pcDNA3.1-U6si-mdm2-p53was constructedusing recombinant DNA technology. To evaluate the antitumor effect of combinedtreatment of Pmp53with zinc and explore it’s antitumor underlying mechanism invitro and in vivo.Method: Based on our previous laboratory work, using the gene sequence of P53andMDM2and the principles design of siRNA, we constructed the Pmp53plasmid. ThemRNA and protein expression levels of MDM2, P53and related genes were detectedby RT-PCR and Western blot analysis. Cell apoptosis was detected by FlowCytometry (FCM) analysis.In vitro, we chose PC-3(p53null) and DU145(mutant p53) cells as researchobjects. The cells were divided into seven groups: the control group, Zinc group,TPEN group, Pmp53group, Pmp53+Zn group, Pmp53+TPEN group andPmp53+Zn+TPEN group. The cell proliferation of combined treatment of Pmp53withzinc to cells was evaluated by MTT assay. Cell cycle was detected by PI assay. Cellapoptosis was detected by FCM and TUNEL assay. Mitochondrial membranepotential was monitored by Rh123. RT-PCR, qPCR and Western blot analysis todetect the expression of P53related genes and proteins changes were performed. P53proteins conformation was detected by Immunoprecipitation. P53transcriptionactivity was detected by Luciferase reporter activity. The capacity of P53protein tobind the promoter of p21and bax was detected by Chromatin immunoprecipitation. To study the effects of combined treatment of Pmp53with zinc in vivo, wedeveloped a PC-3xenograft model. Pmp53plasmid was delivered by AttenuatedSalmonella Typhi Ty21a, with zinc or TPEN supplemented by gavage, was to observeits impact on tumor growth. Tumor-bearing mice were divided randomly into fourgroups: the control group, Pmp53+TPEN group, Pmp53group and Pmp53+Zn group.The purpose of grouping was to build a low, normal and high level of zinc status. ThemRNA and protein expression levels of P53and related genes were detected byQRT-PCR and Western Blot analysis. P53proteins conformation was detected byImmunoprecipitation. Tumor cell apoptosis was detected by FCM and TUNELanalysis. The morphology of tumor tissue was assessed by using H&E staining andimmunohistochemical staining for PCNA protein expression.Result: the co-expression plasmid Pmp53was constructed successfully and verifiedby restriction endonuclease digestion. The studies using plasmid-transfected cellsshowed that Pmp53plasmid reduced mdm2mRNA and protein expression andenhanced P53protein expression.The combined treatment of Pmp53with zinc in vitro significantly increasedPAB1620reactive phenotype and restored P53conformation compared with othergroups. Pmp53with Zn led to to a G1-phase cell cycle arrest, and may be related toup-regulation P21expression and down-regulation CDK4, CDK6and CyclinD1expression. The apoptosis was greatly induced by Pmp53with zinc, and may beassociated with up-regulation of P53, Bax, cleaved-Caspase8, Caspase9and Caspase3expression and down-regulation of Bcl-2, PCNA, MMP2and MMP9expression.Nude mice implanted with PC-3cells and treated with the combined treatment ofPmp53with zinc showed significantly reduced average tumor weights and volumes,and increased apoptosis compared with other groups. Wild-type P53conformation andfunction were also restored, consistent with the result in vitro. H&E stainingdemonstrated significantly increase in necrotic area, and IHC showed significantlyincreased expression of P53and decreased expression of PCNA and MDM2.Conclusion: Co-expression plasmid Pmp53was successfully constructed. In vitro andin vivo the combined treatment of Pmp53with zinc plays an significantly inhibitoryeffect on PCa. The combined therapy is better than single one. The mechanism on theone hand may be associated with P53function. Pmp53plasmid through reduced theexpression of oncogenes MDM2and increased the expression of tumor suppressor P53, play the inhibitory effect on PCa. On the other hand may be associated with P53conformation. Zinc supplementation further retained wild-type P53conformation andenhanced its transcriptional regulation of p21and bax gene expression, leading to thedecreased proliferation and increased apoptosis.更多还原