本文選題：腫瘤　切入點：TEM-8　出處：《第三軍醫(yī)大學》2010年碩士論文

【摘要】： 背景和目的: 實體瘤的生長和轉(zhuǎn)移依賴于血管的生成,因此,抗腫瘤血管生成也成為抗腫瘤治療的一種有效手段。目前,針對于VEGF為靶點的被動免疫抗血管生成的藥物在臨床上取得了一定進展后,隨之而來的抗腫瘤血管生成治療也在不斷的深入,以激活細胞毒性T淋巴細胞(cytotoxic T lymphocyte,CTL)為主的主動免疫治療策略在惡性腫瘤的綜合治療中發(fā)揮了重要作用。與其它傳統(tǒng)的治療方法相比較,主動免疫治療能誘導自身的腫瘤特異性免疫應答,不但可以識別和清除腫瘤細胞,而且還可以誘發(fā)免疫記憶,阻止腫瘤的復發(fā)。同時能夠減少持續(xù)給藥給患者帶來的麻煩和嚴重的經(jīng)濟負擔。所以,抗腫瘤血管生成疫苗的運用前景非常具有吸引力。到目前為止,抗腫瘤血管生成的分子疫苗治療中,靶抗原主要是VEGFR-2,VEGF,FGFR,MMP-2分子等,但是這些分子不僅在腫瘤細胞組織中表達豐富,而且在一些正常組織內(nèi)表達量也較高,所以針對類似靶點設計的分子疫苗對自身有一定的潛在危害性。 2000年Croix發(fā)現(xiàn)了9條在結(jié)腸癌血管內(nèi)皮中特異性高表達的基因,分別命名為腫瘤內(nèi)皮標志物1～9(tumor endothelial markers,TEMs)。經(jīng)原位雜交檢測發(fā)現(xiàn),在成年小鼠體內(nèi),TEM-8的mRNA在正常組織中不表達或者微量表達,而在其腫瘤內(nèi)皮中的表達卻非常豐富。在人體內(nèi),TEM-8mRNA和TEM-8蛋白幾乎在所有的結(jié)腸癌、食管癌、膀胱癌、肺癌病例中高表達,而在同一病例的癌旁組織和正常組織中卻不表達,在黃體及傷口愈合過程中的新血管中也沒有檢測出TEM-8蛋白分子。這些研究結(jié)果提示TEM-8是目前最理想的免疫治療候選靶點之一。在后來的分子疫苗研究中證實TEM-8所構(gòu)成的單獨的分子疫苗免疫原性較低,而與其他抗原結(jié)合以后可以有效的激發(fā)免疫反應,后來的學者在臨床上引入DC疫苗后可以有效的提高TEM-8的免疫原性,抗瘤效果明顯,但是存在轉(zhuǎn)運效率不高,操作不方便,費用高等特點。 陽離子肽類DNA轉(zhuǎn)運載體(cationic peptides DNA delivery systems)是進展較快的領(lǐng)域之一。該類DNA轉(zhuǎn)運載體為一些富含正電荷氨基酸的多肽,如多聚賴氨酸(ploylysine,[K]n),它們可通過電性中和作用與富含負電荷(磷酸根基團)的質(zhì)粒DNA發(fā)生聚合、壓縮,從而將直徑約數(shù)百納米、松散的質(zhì)粒DNA聚縮成為幾十納米的致密顆粒,使之易于被真核細胞攝入從而達到基因轉(zhuǎn)染的目的。構(gòu)建“肽-DNA復合疫苗”,這種疫苗可以打破自身耐受,操作方便,是比較理想的選擇之一。 本課題的目的就在于評估基于TEM-8的分子疫苗經(jīng)過陽離子穿膜肽改造后的抗腫瘤免疫治療的有效性和毒副作用,從而評價是否有必要對TEM-8進行更多的免疫治療方面的研究。 方法: 1.我們首先構(gòu)建陽離子融合肽[K]16-tat49-57,融合肽經(jīng)反相高壓液相色譜鑒定其純度,用質(zhì)譜鑒定其分子量,在特定NaCl濃度下,將其與富含負電荷的pTEM-8真核表達質(zhì)粒結(jié)合構(gòu)建成新型顆粒性疫苗,用透射電鏡掃描顯示其顆粒大小,用westernblot檢測TEM-8蛋白的表達; 2.構(gòu)建CT-26結(jié)腸癌模型,我們以基于TEM-8的新型顆粒性疫苗免疫Balb/c小鼠,研究疫苗的抗瘤能力。免疫小鼠每周一次,連續(xù)三次。觀察小鼠的生存時間和腫瘤體積變化情況,用免疫組化觀察腫瘤組織血管生長情況,并計算腫瘤微血管密度; 3.確認了疫苗的抗腫瘤作用之后,我們進一步研究了疫苗的抗腫瘤機理和毒副作用; 結(jié)果: 1.對合成的多肽進行高效液相色譜鑒定其純度達到了96.57%用質(zhì)譜分析其分子量為3390.44 Da,并成功構(gòu)建了顆粒性疫苗,透射電鏡及軟件分析,其顆粒大小均勻,直徑小于25nm,符合疫苗要求,并對其轉(zhuǎn)染和免疫印跡檢測分子量為63KD符合預期結(jié)果; 2.實驗結(jié)果顯示,基于TEM-8的顆粒性疫苗免疫組的腫瘤體積與對照組有明顯差異(p0.05),荷瘤小鼠生存期延長。腫瘤組織內(nèi)血管內(nèi)皮細胞采用抗CD31單克隆抗體作免疫組織化學染色,結(jié)果顯示TEM-8疫苗免疫組微血管密度(MVD)較對照組明顯降低; 3.在激發(fā)CTL的51Cr釋放實驗證明,經(jīng)過疫苗免疫的小鼠能夠激發(fā)CTL免疫反應,ELISPOT檢測顯示在體內(nèi)疫苗能有效激發(fā)抗原特異性CTL效應,分別利用抗CD4+或者抗CD8+單抗作保護性免疫試驗,結(jié)果發(fā)現(xiàn)抗CD8+時,小鼠不能獲得疫苗免疫后的保護性效果;而在抗CD4+時,實驗組和對照組小鼠腫瘤體積仍然有顯著性差異(p0.05),提示CD8+T細胞在抗腫瘤中起主要作用; 結(jié)論: 1.成功構(gòu)建基于TEM-8的新型顆粒性疫苗,同時能夠有效介導基因的表達; 2.基于TEM-8的顆粒性疫苗能有效抑制腫瘤的生長速度、提高荷瘤動物的生存率; 3.CD8+T細胞在以DNA疫苗所誘導的抗腫瘤免疫應答中起主要作用;
[Abstract]:Background and purpose:
Generation, growth and metastasis of solid tumors depends on angiogenesis therefore, anti angiogenesis has become an effective means of anti-tumor therapy. At present, the drug for passive immunization of VEGF as a target of anti angiogenesis has made some progress in clinical, anti angiogenesis therapy has also been followed further, in order to activate cytotoxic T lymphocytes (cytotoxic T, lymphocyte, CTL) plays an important role in the comprehensive treatment of active immunotherapy based on malignant tumor. Compared with other traditional methods of treatment, active immunotherapy can induce tumor specific immune response itself, not only can identify and eliminate tumor cells. But also can induce immune memory, prevent the recurrence of the tumor. At the same time can reduce the continuous administration of patients brought trouble and serious economic burden. Therefore, anti tumor angiogenesis A vaccine application is very attractive. So far, the molecular vaccine anti angiogenic therapy, target antigen is mainly VEGFR-2, VEGF, FGFR, MMP-2 molecules, but these molecules not only in tumor tissue was abundant, and its expression level was also higher in some normal tissues, so the molecular vaccine similar target design has the potential danger to themselves.
2000 Croix found 9 high specific expression in vascular endothelial cells of colon cancer gene, named as tumor endothelial marker 1~9 (tumor endothelial markers, TEMs). By in situ hybridization detected in adult mice, TEM-8 mRNA expression or trace expression in normal tissues, and its expression in tumor in the endothelium is very abundant in the human body, TEM-8mRNA and TEM-8 protein in almost all colon cancer, esophageal cancer, bladder cancer, high expression of lung cancer cases, and in the same case paracancerous tissues and normal tissues but not expression in the corpus luteum and wound healing in the process of new blood vessels in no detection of TEM-8 protein. These results suggest that TEM-8 is one of the most ideal current immunotherapy candidate targets. Confirmed by TEM-8 single molecular vaccine immunogenicity low molecular vaccine in the later study, and After binding with other antigens, it can effectively stimulate the immune response. Later scholars introduced DC vaccine in clinic, which can effectively improve the immunogenicity of TEM-8, and has obvious anti-tumor effect. However, there are some characteristics such as low transport efficiency, inconvenient operation and high cost.
Cationic peptides DNA transporter (cationic peptides DNA delivery systems) is one of the rapid progress of the field. The class DNA transporter for some rich in positively charged amino acid polypeptide, such as polylysine (ploylysine, [K]n), which can be through the electrical neutralization and rich negative charge (phosphate). DNA polymerization, compression, which will be about hundreds of nanometer diameter, plasmid DNA loose condensation become dense particles of tens of nanometers, which is easy to eukaryotic cells so as to achieve the purpose of intake of gene transfection. Construction of peptide -DNA complex vaccine, this vaccine can break self tolerance, convenient operation, is one of the choice the ideal.
The purpose of this project is to evaluate the effectiveness and side effects of TEM-8 based molecular vaccine for anti-tumor immunotherapy after cationic transmembrane peptide modification, so as to evaluate whether it is necessary to carry out more immunotherapy for TEM-8.
Method:
We constructed 1. cationic fusion peptide [K]16-tat49-57 fusion peptide by reversed phase high pressure liquid chromatography. The purity and identification of the molecular weight by mass spectrometry, in particular NaCl concentration, with the rich negative charge pTEM-8 eukaryotic expression plasmid constructed with a novel particle vaccine, using transmission electron microscope scanning showed that the particle size, use to detect the expression of TEM-8 protein westernblot;
2. to construct the CT-26 model of colon cancer, we use a novel particle vaccine Balb/c mice were immunized with TEM-8 based vaccine antitumor ability of immunized mice. Once a week, three times in a row. The survival time and tumor volume changes were observed in mice, using immunohistochemical observation of tumor angiogenesis, and tumor microvessel calculation density;
3. after confirming the antitumor effect of the vaccine, we further studied the antitumor mechanism and side effects of the vaccine.
Result:
1. of the synthesized peptide by HPLC. The purity reached 96.57% by mass spectrometry analysis of the molecular weight of 3390.44 Da, and successfully constructed the particle vaccine, TEM analysis and software, the uniform particle size, diameter less than 25nm, meet the requirements of molecular detection and vaccine, the transfection and immunoblotting was 63KD in accordance with the expected results;
2.. Experimental results show that the TEM-8 based particle vaccine immune group of tumor size and the control group had significant difference (P0.05), prolong the survival time of tumor bearing mice. Vascular endothelial cells in tumor tissue using anti CD31 monoclonal antibodies for immunohistochemical staining in the immunized group TEM-8 vaccine microvessel density (MVD) decreased significantly compared with the control group;
3. excitation at CTL 51Cr release experiments show that after the vaccine can stimulate the immune response of CTL mice, ELISPOT assay can effectively stimulate antigen-specific CTL effect in vivo vaccine, respectively using anti CD4+ or anti CD8+ monoclonal antibody for protective immunity test results anti CD8+, protective effect in mice can not get vaccinated after; and in anti CD4+, the experimental group and the control group the tumor volume still has significant difference (P0.05), suggesting that CD8+T cells play a major role in anti tumor;
Conclusion:
1. the new granular vaccine based on TEM-8 was successfully constructed, and the gene expression could be effectively mediated.
2. TEM-8 based granular vaccine can effectively inhibit the growth rate of tumor and improve the survival rate of tumor bearing animals.
3.CD8+T cells play a major role in the anti-tumor immune response induced by DNA vaccine.

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R392

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相關(guān)期刊論文 前1條

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本文編號：1670682