本文選題：前列腺癌 + 鋅�。� 參考：《吉林大學(xué)》2014年博士論文

【摘要】：背景：前列腺癌是男性最常見(jiàn)的惡性腫瘤之一，手術(shù)和放化療是前列腺癌主要的治療手段，由于其副作用大，容易復(fù)發(fā)等原因，嚴(yán)重影響前列腺癌的治療和預(yù)后。近年來(lái)，隨著藥物和分子生物學(xué)的迅猛發(fā)展，藥物聯(lián)合基因治療方法以它的安全性和有效性得到了越來(lái)越多的關(guān)注。 腫瘤細(xì)胞的生長(zhǎng)和死亡很大程度上取決于癌基因和抑癌基因作用不平衡所導(dǎo)致，其中P53是最主要的抑癌基因之一，被冠以“基因衛(wèi)士”之美名，它在調(diào)節(jié)正常細(xì)胞周期進(jìn)程，凋亡發(fā)生，DNA修復(fù)及代謝環(huán)境穩(wěn)態(tài)等方面發(fā)揮重要作用。大約有50%的人類腫瘤中存在P53突變，而且在各類腫瘤中已經(jīng)鑒定出2000多種P53突變類型。逆轉(zhuǎn)缺陷的P53功能對(duì)于減少腫瘤發(fā)生，改善腫瘤耐藥將是一項(xiàng)理想的治療策略。引起P53失活的原因很多，其中重要的有兩個(gè)：（1）原癌基因MDM2對(duì)P53的泛素化降解。MDM2由P53誘導(dǎo)產(chǎn)生，P53與MDM2形成一種負(fù)反饋的調(diào)節(jié)環(huán)路，相互制約。MDM2的E3泛素連接酶的活性可以特異性的催化P53的泛素化及介導(dǎo)其出核，并在26S蛋白酶復(fù)合體中降解。此外MDM2可直接抑制P53的轉(zhuǎn)錄激活功能。所以切斷MDM2-P53的反饋環(huán)路，對(duì)于增強(qiáng)P53蛋白的抑癌功能至關(guān)重要。（2）鋅的缺失同樣會(huì)影響P53功能。鋅是體內(nèi)200多種金屬酶的輔酶，在基因表達(dá)和基因穩(wěn)定性方面起著重要作用。P53蛋白DNA結(jié)合結(jié)構(gòu)域具有鋅的結(jié)合位點(diǎn)，結(jié)合鋅以后能夠穩(wěn)定P53蛋白空間構(gòu)象。在表達(dá)野生型P53蛋白的細(xì)胞培養(yǎng)中，應(yīng)用金屬鋅螯合劑TPEN誘導(dǎo)的缺鋅環(huán)境能夠破壞P53野生型構(gòu)象，失去其與靶基因DNA結(jié)合的活性。此外鋅是參與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鋅含量為720ug/ml，而其他組織僅為80ug/ml，而前列腺癌血清和組織中的鋅濃度下降60%-70%，并且隨著前列腺癌的進(jìn)展，鋅的濃度進(jìn)一步下降，鋅的缺失可能也是前列腺癌中P53功能喪失和對(duì)放化療產(chǎn)生耐受的主要原因之一。 因此，本課題在構(gòu)建MDM2特異siRNA與野生型P53相連接的共表達(dá)質(zhì)粒Pmp53基礎(chǔ)上，首次提出鋅聯(lián)合共表達(dá)質(zhì)粒Pmp53治療前列腺癌的方案。其目的一方面可以恢復(fù)前列腺癌中缺失或突變的P53狀態(tài)，更好的發(fā)揮P53作為抑癌基因的功能，另一方面改善前列腺癌血清和組織中的缺鋅狀態(tài)。目的：通過(guò)基因重組技術(shù)構(gòu)建pGCsilencer-mdm2（Si-mdm2）質(zhì)粒與pcDNA3.1-U6si-mdm2-p53共表達(dá)質(zhì)粒（Pmp53），探討鋅聯(lián)合共表達(dá)質(zhì)粒Pmp53對(duì)前列腺癌細(xì)胞株P(guān)C-3和DU145及前列腺癌移植瘤協(xié)同治療效應(yīng)及可能機(jī)制，為前列腺癌的聯(lián)合治療提供新的理論和實(shí)驗(yàn)依據(jù)。方法：參考實(shí)驗(yàn)室以前的工作基礎(chǔ)，根據(jù)P53和MDM2的基因序列以及siRNA設(shè)計(jì)的基本原理，使用pGCsilencerTMU6/Neo/GFP/RNAi和PCDNA3.1載體，利用酶切等技術(shù)，，構(gòu)建質(zhì)粒Si-mdm2和Pmp53，運(yùn)用RT-PCR和Western blot方法檢測(cè)MDM2和P53基因和蛋白的表達(dá)，流式細(xì)胞術(shù)檢測(cè)各組質(zhì)粒對(duì)PC-3細(xì)胞凋亡的影響。 體外實(shí)驗(yàn)以前列腺癌細(xì)胞株P(guān)C-3（缺失性P53）和DU145（突變性P53）細(xì)胞為研究對(duì)象。體外實(shí)驗(yàn)的分組：對(duì)照組，Zn組，TPEN組，Pmp53組，Pmp53+Zn組，Pmp53+TPEN組和Pmp53+Zn+TPEN組。MTT法檢測(cè)鋅聯(lián)合Pmp53質(zhì)粒對(duì)PC-3和DU145細(xì)胞增殖活性的影響；流式細(xì)胞術(shù)檢測(cè)鋅聯(lián)合Pmp53質(zhì)粒對(duì)PC-3和DU145細(xì)胞周期的影響；流式細(xì)胞術(shù)及TUNEL法檢測(cè)鋅聯(lián)合Pmp53質(zhì)粒對(duì)PC-3和DU145細(xì)胞凋亡的影響；羅丹明123觀察鋅聯(lián)合Pmp53質(zhì)粒對(duì)PC-3細(xì)胞線粒體膜電位的影響；qPCR, RT-PCR和Western blot檢測(cè)與p53相關(guān)的靶基因和蛋白的表達(dá)；免疫共沉淀（Co-Immunoprecipatation）檢測(cè)P53蛋白構(gòu)象的變化；熒光素酶報(bào)告基因（Luciferase Reporter Activity）檢測(cè)P53轉(zhuǎn)錄激活下游靶基因的活性；染色質(zhì)免疫共沉淀（Chromatin Immunoprecipatation）檢測(cè)P53與其下游基因p21和bax啟動(dòng)子結(jié)合能力。 體內(nèi)實(shí)驗(yàn)構(gòu)建裸鼠前列腺癌移植瘤模型，運(yùn)用具有腫瘤靶向性的減毒沙門氏菌（Ty21a）攜帶共表達(dá)質(zhì)粒Pmp53，聯(lián)合灌胃方式給予鋅及鋅的抑制劑TPEN，觀察其對(duì)前列腺癌移植瘤生長(zhǎng)的影響及探討其相關(guān)的分子機(jī)制。體內(nèi)實(shí)驗(yàn)分組：對(duì)照組，Pmp53+TPEN組，Pmp53組和Pmp53+Zn組。這樣分組的目是為了給裸鼠體內(nèi)營(yíng)造一種低鋅，正常鋅和補(bǔ)鋅的環(huán)境。應(yīng)用qPCR和Western blot檢測(cè)腫瘤組織P53相關(guān)基因和蛋白的表達(dá)；應(yīng)用免疫共沉淀檢測(cè)P53蛋白構(gòu)象的變化；流式細(xì)胞術(shù)和TUNEL檢測(cè)細(xì)胞凋亡變化；HE和免疫組化檢測(cè)腫瘤組織形態(tài)學(xué)及PCNA的表達(dá)。 結(jié)果：經(jīng)酶切鑒定，成功構(gòu)建了siRNA-mdm2與p53的共表達(dá)質(zhì)粒Pmp53。PCR，Western blot和流式細(xì)胞術(shù)結(jié)果顯示前列腺癌細(xì)胞經(jīng)轉(zhuǎn)染Pmp53后，MDM2基因表達(dá)被干涉，同時(shí)高表達(dá)野生型P53，顯著增強(qiáng)P53抑癌功能。 體外實(shí)驗(yàn)證明：與鋅和Pmp53組相比，鋅聯(lián)合Pmp53組顯著增強(qiáng)了PAB1620的表達(dá)，穩(wěn)定了P53的野生型構(gòu)象。鋅聯(lián)合Pmp53組誘導(dǎo)細(xì)胞周期阻滯于GO-G1期，其機(jī)制可能與上調(diào)P21表達(dá)，下調(diào)CDK4，CDK6，CyclinD1表達(dá)有關(guān)。鋅聯(lián)合Pmp53組抑制增殖，誘導(dǎo)發(fā)生細(xì)胞凋亡，其機(jī)制可能與上調(diào)P53，Bax，Caspase-8, Caspase-9，Caspase-3表達(dá)，下調(diào)Bcl-2，PCNA，MMP2，MMP9表達(dá)有關(guān)。 體內(nèi)實(shí)驗(yàn)證明：應(yīng)用減毒沙門氏菌攜帶Pmp53質(zhì)粒聯(lián)合鋅治療前列腺癌移植瘤。與對(duì)照，Pmp53+TPEN和Pmp53組相比，Pmp53+Zn組腫瘤體積顯著縮小，重量顯著減輕，腫瘤組織內(nèi)凋亡率上升，差異具有統(tǒng)計(jì)學(xué)意義。免疫共沉淀結(jié)果顯示Pmp53+Zn組顯著增強(qiáng)了PAB1620的表達(dá)，與體外實(shí)驗(yàn)結(jié)果一致。HE結(jié)果顯示Pmp53+Zn組出現(xiàn)大片無(wú)結(jié)構(gòu)壞死灶。免疫組化檢測(cè)到腫瘤組織PCNA和MDM2蛋白表達(dá)下調(diào)，P53蛋白表達(dá)上調(diào)，差異有統(tǒng)計(jì)學(xué)意義。結(jié)論：本實(shí)驗(yàn)成功構(gòu)建了共表達(dá)質(zhì)粒Pmp53。體內(nèi)外實(shí)驗(yàn)證明鋅聯(lián)合共表達(dá)質(zhì)粒Pmp53對(duì)前列腺癌具有顯著治療作用，且聯(lián)合治療的療效優(yōu)于單一治療。其機(jī)制可能一方面與共表達(dá)質(zhì)粒Pmp53直接抑制MDM2的表達(dá)，一定程度解除了MDM2對(duì)P53的負(fù)反饋抑制和降解作用，同時(shí)高表達(dá)野生型P53，既雙重增強(qiáng)了野生型P53抑制腫瘤生長(zhǎng)的作用有關(guān)；另一方面可能與鋅進(jìn)一步穩(wěn)定了因?qū)隤mp53質(zhì)粒而恢復(fù)的P53野生型構(gòu)象，增強(qiáng)了P53轉(zhuǎn)錄激活活性，提高了其靶基因p21和bax的表達(dá)水平，誘導(dǎo)前列腺癌細(xì)胞周期阻滯和凋亡發(fā)生有關(guān)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.25

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