【摘要】： 血管生成是胚胎發(fā)育、傷口愈合、炎癥中的生理反應(yīng),但是在實(shí)體瘤、風(fēng)濕性關(guān)節(jié)炎、糖尿病性視網(wǎng)膜炎等疾病中卻屬于病理過程。眾多的證據(jù)表明,實(shí)體瘤的生長(zhǎng)和轉(zhuǎn)移依賴于血管生成,腫瘤血管生成也因此成為腫瘤學(xué)研究的熱點(diǎn)領(lǐng)域之一。目前,部分化學(xué)合成的抗血管生成的藥物已進(jìn)入臨床試驗(yàn),并證明抗血管生成是有效的腫瘤治療方法。由于抗血管生成是一個(gè)長(zhǎng)期的治療過程,持續(xù)給藥不僅麻煩而且給患者帶來很重的經(jīng)濟(jì)負(fù)擔(dān)。主動(dòng)免疫治療在少數(shù)幾次給藥的情況下,便可以獲得長(zhǎng)期的保護(hù)性效果,不但方便而且便宜,所以,抗腫瘤血管生成疫苗的運(yùn)用前景非常具有吸引力。到目前為止,主動(dòng)免疫治療在動(dòng)物實(shí)驗(yàn)中取得了一定的療效,但還存在一些缺點(diǎn),比如靶抗原的特異性不高等等。2000年Croix發(fā)現(xiàn)多條在結(jié)腸癌血管內(nèi)皮中特異性高表達(dá)的基因,命名為腫瘤內(nèi)皮標(biāo)志物(tumor endothelial markers,TEMs)。隨后的研究提示提示TEM8是目前特異性最高的免疫治療候選靶抗原,但TEM8是否為抗腫瘤治療的有效靶抗原并沒有得到證明。本課題的目的就在于評(píng)估基于TEM8的抗腫瘤免疫治療的有效性和毒副作用,從而評(píng)價(jià)是否有必要對(duì)TEM8進(jìn)行更多的免疫治療方面的研究。 我們首先構(gòu)建編碼人TEM8-Ⅰ基因的真核表達(dá)質(zhì)粒pTEM8-Ⅰ和編碼人TEM8-Ⅰ、小鼠GM-CSF基因的雙表達(dá)質(zhì)粒pTEM8/mGM-CSF。經(jīng)限制性酶切和測(cè)序鑒定后轉(zhuǎn)化營(yíng)養(yǎng)缺陷型小鼠沙門氏菌株SL7207,制備基于TEM8的口服疫苗。以編碼綠色熒光蛋白的真核表達(dá)質(zhì)粒pEGFP-N1轉(zhuǎn)化SL7207后制備示蹤疫苗,口服免疫C57BL/6小鼠,3天后,在Peyer’s結(jié)和脾細(xì)胞中均可發(fā)現(xiàn)表達(dá)綠色熒光蛋白的抗原提呈細(xì)胞,確認(rèn)了這種疫苗傳遞抗原的有效性和最佳條件。隨后,我們以pTEM8-Ⅰ和pTEM8/mGM-CSF免疫C57BL/6J小鼠,取脾,以羊抗小鼠TEM8特異性抗體作免疫組化,檢測(cè)到TEM8蛋白在脾內(nèi)APC的細(xì)胞膜和細(xì)胞漿中表達(dá),證實(shí)了所構(gòu)建的疫苗能夠完成質(zhì)粒DNA由細(xì)菌到體內(nèi)APC的傳遞,以及在APC內(nèi)表達(dá)目的抗原的工作。 在證實(shí)上述載體的抗原傳遞能力之后,我們研究了疫苗的抗腫瘤能力。主要包括保護(hù)性免疫和治療性免疫兩種方案,在B16黑色素瘤和CT26結(jié)腸癌模型中完成。在保護(hù)性免疫組,給小鼠口服免疫3次后,皮下接種腫瘤細(xì)胞建立皮下腫瘤模型或者經(jīng)尾靜脈注射腫瘤細(xì)胞建立肺轉(zhuǎn)移瘤模型;在治療性免疫組,在第一次口服免疫后3天接種腫瘤細(xì)胞,按免疫方案繼續(xù)給小鼠口服免疫2次。實(shí)驗(yàn)結(jié)果顯示,以TEM8為靶抗原的抗腫瘤血管生成疫苗,對(duì)這兩種腫瘤都有明顯的抑制作用,其中以CT26結(jié)腸癌皮下模型最明顯;TEM8-Ⅰ和pTEM8/mGM-CSF免疫組的腫瘤體積、肺轉(zhuǎn)移瘤的數(shù)目與對(duì)照組有明顯差異(p0.05),小鼠生存期延長(zhǎng)。腫瘤組織內(nèi)血管內(nèi)皮細(xì)胞采用抗CD31單克隆抗體作免疫組織化學(xué)染色,結(jié)果顯示TEM8疫苗免疫組微血管密度(MVD)較對(duì)照組明顯降低;藻酸鹽體內(nèi)實(shí)驗(yàn)顯示TEM8疫苗免疫組腫瘤血管生成明顯少于對(duì)照組,FITC-Dextran攝取率也低于對(duì)照組。以pTEM8/mGM-CSF免疫后,取脾細(xì)胞以標(biāo)準(zhǔn)51Cr釋放試驗(yàn)檢測(cè)TEM8特異性CTL,顯示免疫組小鼠脾細(xì)胞對(duì)CT26無殺傷作用,但可以殺傷對(duì)轉(zhuǎn)染了TEM8基因的CT26細(xì)胞。以羊抗鼠TEM8抗體A16作免疫組織化學(xué)檢測(cè)證實(shí)CT26結(jié)腸癌組織中只有血管內(nèi)皮細(xì)胞表達(dá)TEM8蛋白,進(jìn)一步說明TEM8疫苗免疫后,是通過殺傷腫瘤血管內(nèi)皮細(xì)胞取得抗腫瘤效果。 確認(rèn)了疫苗的抗腫瘤作用之后,我們進(jìn)一步研究了疫苗的抗腫瘤機(jī)理和毒副作用。分別利用CD4或者CD8基因敲除小鼠作保護(hù)性免疫試驗(yàn),結(jié)果發(fā)現(xiàn)CD8缺陷時(shí),小鼠不能獲得疫苗免疫后的保護(hù)性效果;而在CD4缺陷時(shí),實(shí)驗(yàn)組和對(duì)照組小鼠腫瘤體積仍然有顯著性差異(p0.05),提示CD8+T細(xì)胞在抗腫瘤中取主要作用。給小鼠口服免疫pTEM8/mGM-CSF后觀察體重、主要內(nèi)臟、一般情況等,未發(fā)現(xiàn)明顯差別。皮膚致傷實(shí)驗(yàn)結(jié)果顯示pTEM8/mGM-CSF免疫小鼠和對(duì)照組皮膚傷口的愈合時(shí)間和組織學(xué)特征均無顯著差別,提示該疫苗免疫后無明顯副作用。 綜合以上研究,可以得出以下結(jié)論:①以減毒沙門氏菌SL7207為載體的口服DNA疫苗可以打破自身耐受,誘導(dǎo)針對(duì)自身抗原的免疫反應(yīng);②以TEM8為靶抗原的疫苗可通過抗血管生成發(fā)揮抗腫瘤作用;③CD8+T細(xì)胞在以減毒沙門氏菌為載體的口服DNA疫苗所誘導(dǎo)的抗腫瘤免疫應(yīng)答中取主要作用。
[Abstract]:Angiogenesis is a physiological response in embryonic development, wound healing and inflammation, but it is a pathological process in solid tumors, rheumatic arthritis, and diabetic retinitis. Numerous evidence suggests that growth and metastasis of solid tumors depend on angiogenesis and tumor angiogenesis is also one of the hot spots in oncology research. At present, some of the chemically synthesized anti-angiogenic drugs have entered clinical trials and demonstrate that anti-angiogenic is an effective method of tumor therapy. Because anti-angiogenesis is a long-term treatment process, continuous administration is not only a problem but also a heavy economic burden for patients. Active immune therapy can obtain a long-term protective effect in a few times of administration, and is not only convenient and cheap, so the application prospect of the anti-tumor angiogenesis vaccine is very attractive. So far, active immunotherapy has achieved a certain therapeutic effect in animal experiments, but there are some disadvantages, such as the high specificity of the target antigen and so on. TEMs). The subsequent study suggests that TEM8 is the most specific candidate target antigen with the highest specificity, but whether TEM8 is the effective target antigen for anti-tumor therapy is not shown. The purpose of this study is to evaluate the effectiveness and toxic and side effects of TEM8-based anti-tumor immunotherapy, so as to evaluate the need for more immunotherapy for TEM8. We first construct the eukaryotic expression plasmid pTEM8-I of the human TEM8-I gene and the double-expression plasmid pTEM8/ mGM-C of the human TEM8-I and the mouse GM-CSF gene. SF. transformed auxotrophic Salmonella strain SL7207 after restriction enzyme digestion and sequencing to prepare a TEM8-based oral administration The vaccine was prepared by transforming SL7207 with the eukaryotic expression plasmid pEGFP-N1 of the green fluorescent protein. After 3 days, the antigen-presenting cells expressing the green fluorescent protein can be found in the Peyer's junction and the spleen cell, and the effectiveness and the optimum of the vaccine delivery antigen are confirmed. Conditions. Subsequently, we immunized C57BL/ 6J mice with pTEM8-I and pTEM8/ mGM-CSF, and the spleen and the mouse TEM8 specific antibody were used as immunohistochemistry to detect the expression of the TEM8 protein in the cell membrane and the cytoplasm of the APC in the spleen, and it was confirmed that the constructed vaccine was able to complete the plasmid DNA from the bacteria to the in vivo APC. And the expression of the target antigen in the APC. Work. After confirming the antigen delivery ability of the above-mentioned vector, we studied the vaccine. Anti-tumor ability. It mainly includes both protective and therapeutic immunization protocols, in B16 melanoma and CT26 colon cancer The model was completed. In the protective immune group, after three times of oral immunization of the mice, the tumor cells were inoculated subcutaneously to establish a subcutaneous tumor model or a lung metastasis tumor model was established by the tail vein injection of the tumor cells; in the therapeutic immunization group,3 days after the first oral immunization, Tumor cells, continued to the mouse port according to the immune protocol The results of the experiment show that the anti-tumor angiogenesis vaccine with TEM8 as the target antigen has an obvious inhibitory effect on the two tumors, in which the tumor volume of the CT26 colon cancer subdermal model is the most obvious; the number of the lung metastases in the TEM8-I and pTEM8/ mGM-CSF immune groups is significantly different from that of the control group (p 0.05), mouse The results showed that the microvessel density (MVD) in the immune group of the TEM8 vaccine was significantly lower than that in the control group. In less than the control group, the FITC-Dextran uptake rate was also After immunization with pTEM8/ mGM-CSF, the spleen cells were taken to test the TEM8-specific CTL with the standard 51Cr release test. T26 cells. The expression of TEM8 protein in the vascular endothelial cells in the CT26 colon cancer tissue was confirmed by the immunohistochemical study of the goat anti-mouse TEM8 antibody A16, and it was further demonstrated that the TEM8 vaccine was immunized with the anti-tumor vascular endothelial cells. The anti-tumor effect of the vaccine is confirmed, and the anti-swelling of the vaccine is further studied after the anti-tumor effect of the vaccine is confirmed. The mechanism of the tumor and the toxic and side effects of the mice were tested by using the CD4 or CD8 gene knockout mice. The results showed that the mice were unable to obtain the protective effect after the vaccine immunization, and the volume of the tumor in the experimental group and the control group was still significant in the case of the CD4 defect. Heteropoly (p0.05), suggesting that CD8 + T cells are resistant to The main role of the tumor is to observe body weight, main internal organs, general conditions, etc. after oral administration of pTEM8/ mGM-CSF to mice. No significant difference was found. The results of skin injury showed no significant difference in the healing time and the histological characteristics of the skin wound in the mice and the control group. The following conclusions can be obtained by combining the above studies: the oral DNA vaccine which is the carrier of the attenuated Salmonella SL7207 can break the self-tolerance and induce the immune response to the autoantigen; and the vaccine with the TEM8 as the target antigen can be used as a target antigen. Anti-tumor effect of blood vessel production; anti-swelling induced by oral DNA vaccine with attenuated Salmonella as carrie
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392;R730.5

【共引文獻(xiàn)】

相關(guān)期刊論文 前9條

1 樊慧婷;林洪生;李杰;祁鑫;裴迎霞;;Lewis肺癌荷瘤小鼠CD4~+CD25~+Foxp3~+調(diào)節(jié)性T細(xì)胞及髓樣抑制細(xì)胞與腫瘤生長(zhǎng)的關(guān)系[J];現(xiàn)代免疫學(xué);2009年05期

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2 覃林花;趨化因子與共刺激分子聯(lián)合基因治療肝癌的實(shí)驗(yàn)研究[D];第二軍醫(yī)大學(xué);2001年

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4 劉春雨;基因重組BCG的研究——phIFN-α-2B和pYL-GFP穿梭質(zhì)粒的構(gòu)建及其在BCG中的表達(dá)[D];天津醫(yī)科大學(xué);2003年

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6 李林;黑色素瘤抗原MAGE-A10基因克隆、表達(dá)和分布的研究[D];第四軍醫(yī)大學(xué);2004年

7 陳玉丙;樹突狀細(xì)胞抗原負(fù)載及MAGE-3 DNA瘤苗研制[D];吉林大學(xué);2004年

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10 雷曉;胃癌Treg對(duì)DC免疫功能的抑制作用及其機(jī)理研究[D];第三軍醫(yī)大學(xué);2005年

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1 楊靜;MAGE-A1/A3蛋白在膠質(zhì)瘤的表達(dá)及意義[D];廣西醫(yī)科大學(xué);2011年

2 丁春艷;腫瘤抗原MAGE-A9、-A10、-A12在乳腺癌組織中的表達(dá)及對(duì)乳腺癌細(xì)胞增殖的影響[D];河北醫(yī)科大學(xué);2011年

3 范鐘婷;精子相關(guān)抗原-9在外陰上皮內(nèi)瘤變及外陰鱗狀細(xì)胞癌組織中的表達(dá)及意義[D];山東大學(xué);2011年

4 方皓舒;腫瘤損傷釋放物質(zhì)對(duì)腫瘤生長(zhǎng)影響的研究[D];華中科技大學(xué);2009年

5 徐小培;恩度對(duì)C57BL/6小鼠肺轉(zhuǎn)移性黑色素瘤免疫狀態(tài)的影響[D];華中科技大學(xué);2010年

6 陳欣;LPS、腫瘤細(xì)胞壞死釋放物質(zhì)對(duì)小鼠黑色素瘤B16細(xì)胞侵襲、轉(zhuǎn)移能力的影響[D];華中科技大學(xué);2010年

7 鄧學(xué)峰;一種植物蛋白的腫瘤抗原活性的研究[D];河北醫(yī)科大學(xué);2002年

8 毛愉燕;上皮性卵巢癌腫瘤浸潤(rùn)性樹突狀細(xì)胞的密度和激活狀態(tài)及其與VEGF的相關(guān)性[D];浙江大學(xué);2003年

9 周宇;肝片吸蟲保護(hù)性抗原基因在卡介苗中的表達(dá)[D];四川大學(xué);2003年

10 岳君秋;MAGE-A1在肺癌中的表達(dá)與腫瘤細(xì)胞增殖的相關(guān)性研究[D];武漢大學(xué);2004年

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