锌联合Si-mdm2与P53共表达质粒（Pmp53）抗前列腺癌的实验研究

发布时间：2018-06-19 05:00

本文选题：前列腺癌 + 锌　；参考：《吉林大学》2014年博士论文

【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。
[Abstract]:Background: prostate cancer is one of the most common malignant tumors in men. Surgery and radiotherapy and chemotherapy are the main treatment methods for prostate cancer. Due to their large side effects and easy recurrence, the treatment and prognosis of prostate cancer are seriously affected. In recent years, with the rapid development of drugs and molecular biology, the combination of drug and gene therapy has been used for it. Security and effectiveness have attracted more and more attention.
The growth and death of tumor cells depend largely on the unbalance of oncogene and tumor suppressor gene, and P53 is one of the most important tumor suppressor genes. It is called the name of "gene guard". It plays an important role in regulating the process of normal cell cycle, apoptosis, DNA repair and homeostasis of metabolic environment. 50% of human tumors have P53 mutations and more than 2000 P53 mutations have been identified in various types of tumors. The P53 function of reversing defects is an ideal treatment strategy for reducing the occurrence of tumors and improving tumor resistance. There are many reasons for the inactivation of P53, of which two are important: (1) the ubiquitin of the proto oncogene MDM2 to P53 The degradation of.MDM2 is induced by P53, and P53 and MDM2 form a negative feedback regulation loop, which restrict the activity of.MDM2 E3 ubiquitin ligase, which can specifically catalyze the ubiquitination of P53 and mediate its nucleation, and degrade in the 26S protease complex. Moreover, MDM2 can directly inhibit the activation function of P53, so that MDM2-P53 is cut off. The feedback loop is very important for enhancing the tumor suppressor function of P53 protein. (2) the deletion of zinc also affects the function of P53. Zinc is a coenzyme of more than 200 metalloenzymes in the body. It plays an important role in gene expression and gene stability. The binding site of the.P53 protein DNA binding domain can stabilize the spatial conformation of the P53 protein after zinc. In the cell culture of the wild type P53 protein, the zinc deficiency environment induced by the zinc chelating agent TPEN can destroy the P53 wild type conformation and lose its binding activity with the target gene DNA. In addition, zinc is one of the essential trace elements involved in the synthesis of prostate fluid. The zinc content in the normal adult gland fluid is 720ug/ml, and the other tissues are only The concentration of zinc in the serum and tissue of prostate cancer is decreased by 60%-70%, and the zinc concentration is further decreased with the progression of prostate cancer. The loss of zinc may be one of the main reasons for the loss of P53 function in prostate cancer and the tolerance to radiotherapy and chemotherapy in prostate cancer.
Therefore, on the basis of the construction of the co expression plasmid Pmp53 linked to the MDM2 specific siRNA and wild type P53, the scheme for the treatment of prostate cancer with co expression of zinc co expression plasmid Pmp53 is first proposed. The purpose of this study is to restore the P53 state of the deletion or mutation in prostate cancer and to better play the function of P53 as a tumor suppressor gene, on the other hand. Objective: to improve the state of zinc deficiency in serum and tissues of prostate cancer. Objective: to construct pGCsilencer-mdm2 (Si-mdm2) plasmids and pcDNA3.1-U6si-mdm2-p53 co expression plasmids (Pmp53) by gene recombination technology, and to explore the synergistic effect and possible mechanism of zinc co expression plasmid Pmp53 on prostate cancer cell line PC-3 and DU145 and prostate cancer transplantation tumor. To provide new theoretical and experimental basis for the combined treatment of prostate cancer. Methods: referring to the previous work basis of the laboratory, according to the gene sequence of P53 and MDM2 and the basic principles of siRNA design, pGCsilencerTMU6/Neo/GFP/RNAi and PCDNA3.1 vectors were used to construct plasmid Si-mdm2 and Pmp53 by enzyme cutting and other techniques. RT-PCR and Western were used. Blot method was used to detect the expression of MDM2 and P53 gene and protein. The effect of plasmid on apoptosis of PC-3 cells was detected by flow cytometry.
In vitro experiments were conducted with prostate cancer cell line PC-3 (deletion P53) and DU145 (mutant P53) cells as the research object. In vitro experiment group: control group, Zn group, TPEN group, Pmp53 group, Pmp53+Zn group, Pmp53+TPEN group and Pmp53+Zn+TPEN group.MTT method to detect the effect of zinc combined Pmp53 plasmids on the proliferation and proliferation activity; flow cytometry The effect of zinc combined with Pmp53 plasmid on the cell cycle of PC-3 and DU145; flow cytometry and TUNEL method to detect the effect of zinc combined with Pmp53 plasmid on the apoptosis of PC-3 and DU145 cells; Luo Danming 123 observed the effect of zinc combined with Pmp53 plasmids on the mitochondrial membrane potential of PC-3 cells; qPCR, RT-PCR, and Western The expression of P53 protein conformation was detected by immuno coprecipitation (Co-Immunoprecipatation); the luciferase reporter gene (Luciferase Reporter Activity) detected the activity of the downstream target gene by P53 transcription, and chromatin immunoprecipitation (Chromatin Immunoprecipatation) was used to detect the binding ability of P53 to its downstream gene p21 and Bax promoter.
In vivo, the model of prostate cancer xenografts in nude mice was constructed. The co expression plasmid Pmp53 was carried by the tumor targeting Salmonella (Ty21a), and the zinc and zinc inhibitor TPEN was given by combined gavage. The effects on the growth of the prostate cancer transplanted tumor were observed and the related molecular mechanisms were discussed. In vivo experiment group: control group, Pm Group p53+TPEN, group Pmp53 and group Pmp53+Zn. The aim of this group is to create a low zinc, normal zinc and zinc supplementation in nude mice. QPCR and Western blot were used to detect the expression of P53 related genes and proteins in tumor tissues; the conformation of P53 protein was detected by immunoprecipitation; flow cytometry and TUNEL detected cell apoptosis. HE and immunohistochemistry were used to detect tumor histomorphology and PCNA expression.
Results: the co expression plasmid Pmp53.PCR of siRNA-mdm2 and p53 was successfully constructed by enzyme digestion. The results of Western blot and flow cytometry showed that the expression of MDM2 gene was interfered after transfection of Pmp53 to the prostate cancer cells, and the wild type P53 was highly expressed, and the P53 suppressor function was significantly enhanced.
In vitro experiments showed that zinc combined with Pmp53 group significantly enhanced the expression of PAB1620 and stabilized the wild type conformation of P53. Zinc combined with Pmp53 group induced cell cycle arrest in GO-G1 phase, and its mechanism may be related to up regulation of P21 expression and down regulation of CDK4, CDK6, CyclinD1 expression. Zinc combined with Pmp53 group inhibited proliferation and induced cells to occur. The mechanism of apoptosis may be related to upregulation of P53, Bax, Caspase-8, Caspase-9, Caspase-3 expression and down regulation of Bcl-2, PCNA, MMP2 and MMP9.
In vivo experiments proved that the Pmp53 plasmid combined with zinc was used to treat the tumor of prostate cancer. Compared with the control group, the volume of the tumor in the group Pmp53+Zn was significantly reduced, the weight of the Pmp53+Zn group was significantly reduced, the apoptosis rate in the tumor tissue increased, and the difference was statistically significant. The results of immunoprecipitation showed that the Pmp53+Zn group was significantly increased. The expression of PAB1620 was stronger than that in the experimental results in vitro..HE results showed that there was a large area of non structural necrosis in Pmp53+Zn group. The expression of PCNA and MDM2 protein in tumor tissue was down regulated by immunohistochemistry and the expression of P53 protein was up regulated. Conclusion: this experiment successfully constructed a co expression plasmid Pmp53. in vivo and in vivo and in vitro. Co expression plasmid Pmp53 has a significant therapeutic effect on prostate cancer, and the therapeutic effect of combined therapy is better than single treatment. Its mechanism may, on one hand, inhibit the expression of MDM2 directly with co expression plasmid Pmp53, to a certain extent, relieving the negative feedback inhibition and degradation of P53 by MDM2, and high expression of wild type P53 at the same time, which both enhanced both the wild type and the wild type. P53 inhibits the role of tumor growth; on the other hand, zinc may further stabilize the P53 wild type conformation due to the introduction of Pmp53 plasmids, enhance the activation of P53 transcriptional activation, improve the expression level of the target gene p21 and Bax, and induce the cell cycle arrest and apoptosis of prostate cancer cells.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.25

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