【摘要】：腫瘤的生長離不開營養(yǎng)的供給，因此，腫瘤新生血管的形成對于腫瘤的生長具有重要意義。在眾多血管生成有關的細胞因子中，VEGF是最重要的調節(jié)因子，很多腫瘤的生長過程都伴隨著VEGF的高表達，許多研究表明，抑制VEGF的生物學活性就能抑制腫瘤的生長。 目前，用VEGF抗體治療腫瘤取得了一定的臨床效果，但仍存在許多不足。臨床使用的VEGF抗體為人源化抗體，雖然人源化程度較高，但仍存在異源性的問題。由于臨床上用抗體治療腫瘤需要反復多次用藥，因此，抗體本身的異源性問題不容忽視。另外，抗體臨床用量較大，其生產(chǎn)成本較高，導致抗體藥物的價格較高，也一定程度上限制了其使用范圍。 用外源VEGF抗體治療腫瘤是一種被動免疫治療。如果能夠采用主動免疫治療，則可以避免VEGF抗體治療的一些不足，如用藥次數(shù)多、存在異源性等問題。但是VEGF是機體自身產(chǎn)生的蛋白質，有其正常的生理功能，機體對正常的自身蛋白存在免疫耐受，用自身VEGF蛋白難以誘導機體產(chǎn)生針對自身VEGF的抗體。 本文的目的就是要設計一種mVEGF疫苗，繞開或打破機體的免疫耐受，讓機體能夠產(chǎn)生識別mVEGF的中和性抗體，以抑制腫瘤新生血管的生成，最終實現(xiàn)抑制腫瘤生長的目的。 為了實現(xiàn)上述目的，我們確定了mVEGF疫苗設計的基本策略，即以mVEGF空間結構為依據(jù)，尋找mVEGF可能的抗原表位，將這些可能的抗原表位展示在一種簡單的蛋白質骨架之上，形成一種新蛋白，用這種新蛋白作為可能的mVEGF疫苗。 首先，我們選擇了抗體重鏈可變區(qū)作為抗原表位展示的骨架蛋白，由于抗體的抗原結合部位是由抗體的輕鏈和重鏈可變區(qū)共同組成，單獨的重鏈抗體可變區(qū)存在穩(wěn)定性差的缺點，經(jīng)過分析，我們將抗體重鏈可變區(qū)的部分氨基酸進行了突變，突變后的重鏈可變區(qū)蛋白可以很容易地實現(xiàn)原核表達，并可復性為穩(wěn)定的蛋白質。由于抗體的重鏈可變區(qū)的CDR3區(qū)的是高度可變的，能夠容忍不同長度序列的插入或置換，因此，CDR3區(qū)被我們選為抗原表位展示部位。 根據(jù)人VEGF165的空間結構，我們分析了小鼠VEGF164的可能的抗原表位，其中表位1（EYPDEIEYIFKP）、表位2（KSHEVIKFMDV）和表位3（IMRIKPHQSQH）有可能是mVEGF的中和性抗原表位。 我們用表位2的氨基酸序列替換抗體重鏈可變區(qū)的CDR3區(qū)氨基酸序列，形成一種新的蛋白質，命名為mFV2。將此序列轉換為其基因編碼序列，全基因合成其編碼序列，并克隆到pET-24a表達載體上，構建了表位2表達載體。將該載體轉化BL21（DE3）宿主菌，，通過IPTG誘導實現(xiàn)了mFV2的高表達，表達的mFV2蛋白主要以包涵體形式存在，包涵體經(jīng)過洗滌、純化、裂解后，用SephacrylS-100凝膠過濾柱進行了純化，獲得了高純度的mFV2蛋白，通過稀釋法成功復性了mFV2蛋白。 通過重疊PCR法將表位1和表位3編碼基因成功插入到抗體可變區(qū)載體蛋白的CDR3區(qū)中，同樣實現(xiàn)了mFV1和mFV3的表達、純化和復性。 在獲得了三種小鼠表位疫苗蛋白mFV1、mFV2和mFV3后，我們首先驗證了三種疫苗蛋白的免疫效果。用三種疫苗蛋白分別免疫雌性Balb/c小鼠，免疫5周后小鼠眼眶取血，獲得三種免疫血清，命名為pAb1、pAb2和pAb3。ELISA檢測表明，三種免疫血清的滴度均達到1：104，并且能夠特異性識別VEGF164，而不與無關蛋白反應，這表明,這三種疫苗蛋白的確能夠誘導機體產(chǎn)生特異性識別VEGF164的抗體。WesternBlot檢測顯示，免疫血清pAb1和pAb2能夠識別VEGF164，但pFV3則不能識別VEGF164，很可能表位3是一種空間表位，在WesternBlot中該表位不能保持其空間結構。另外，通過免疫熒光實驗證明，三種免疫血清都能識別B16小鼠黑色素瘤細胞中表達的VEGF164，B16細胞表達的VEGF164主要分布在胞漿中。為了驗證三種免疫血清是否具有VEGF164中和活性，我們分析了三種免疫 血清對人臍靜脈內(nèi)皮細胞（HUVEC）增殖、遷移和成管能力的影響。實驗結果表明，VEGF164能夠促進HUVEC的增殖、遷移及成管，而pAb1、pAb2、pAb3均表現(xiàn)出了一定的mVEGF抑制活性，其中pAb3表現(xiàn)出了理想的VEGF164抑制活性。上述體外實驗結果證明，三種免疫血清的確具有VEGF164中和活性，能夠 抑制VEGF164的生物學功能。在此基礎上，我們開展了mVEGF疫苗治療腫瘤的實驗研究。首先，我們采用預防接種的方式觀察三種mVEGF疫苗是否具有抑制H22 實體瘤生長的作用。實驗選用6-8周的雌性BALB/c小鼠，分為四組，分別是模型組、mFV1組、mFV2組和mFV3組。mFV1組、mFV2組和mFV3組分別用mFV1蛋白、mFV2蛋白和mFV3蛋白免疫三次，第5周通過ELISA檢測免疫效果，并開始接種H22腫瘤細胞，待腫瘤生長到可用手摸到時開始記錄腫瘤生長狀態(tài)，并最終處死小鼠，分離腫瘤，稱量腫瘤大小。實驗結果表明，三種mVEGF疫苗均表現(xiàn)出了一定的抑制H22腫瘤生長的能力，其中，mFV3疫苗的效果最理想，抑瘤率達到83.8%，mFV2次之，mFV1最差。為了更直觀地了解mVEGF疫苗的抑瘤機制，將H22腫瘤模型實驗中各組 的腫瘤組織進行了免疫組化檢測，結果顯示，疫苗治療組微血管密度比模型組低，其中mFV3免疫組腫瘤的微血管密度減少最為明顯，這就是為什么在三種疫苗蛋白中mFV3抑瘤作用最好的原因。 同樣地，利用S180腫瘤模型驗證了三種mVEGF疫苗的抑瘤效果。在S180腫瘤模型上，三種疫苗的抑瘤表現(xiàn)與在H22腫瘤模型上的表現(xiàn)一致，也是mFV3疫苗的效果最理想，抑瘤率為80.3%，mFV2次之，mFV1最差。 為了模擬臨床上疫苗使用的實際情況，我們又開展了治療性接種mVEGF疫苗對腫瘤生長影響及與化療藥物共用的效果觀察。實驗仍選用6-8周的雌性BALB/c小鼠，分為四組，分別是模型組、mFV3治療組、順鉑治療組和mFV3+順鉑組。實驗中接種H22腫瘤細胞與給藥同時進行，模型組僅注射生理鹽水，順鉑組注射順鉑2mg/kg，每周一次，連續(xù)用藥2次，mFV3+順鉑組為順鉑和mFV3聯(lián)合治療，小鼠接種腫瘤細胞后，注射順鉑（2mg/kg）并同時進行mFV3免疫，順鉑每周一次，連續(xù)用藥2次，第14天進行mFV3第二次免疫，于第28天處死小鼠，并剝離腫瘤組織，測量腫瘤大小。實驗結果為，單純mFV3免疫組腫瘤抑制率為27.3%,單純順鉑治療組腫瘤抑制率為35.7%，而mFV3免疫+順鉑治療組的腫瘤抑制率達到了66.2%。可見，mVEGF疫苗治療和化療之間并不沖突，二者有較好的協(xié)同效應，可聯(lián)合用于腫瘤的治療。 綜上所述，本文建立了一種基于抗體可變區(qū)的抗原表位展示系統(tǒng)，利用該系統(tǒng)構建了三種mVEGF疫苗，它們能夠有效誘導小鼠產(chǎn)生VEGF164中和抗體，最終抑制小鼠實體瘤的生長。
[Abstract]:The growth of the tumor is essential for the growth of the tumor, so the formation of the tumor-based new blood vessel is of great significance to the growth of the tumor. VEGF is the most important regulatory factor in many angiogenesis-related cytokines, and the growth of many tumors is associated with the high expression of VEGF, and many studies have shown that the inhibition of the biological activity of VEGF can inhibit the growth of the tumor. At present, the treatment of the tumor with the VEGF antibody has achieved a certain clinical effect, but there are still many problems that do not The VEGF antibody used in clinical use is a humanized antibody, although the humanizing degree is high, there is still a heterogenous question. in that clinical use of the antibody for the treatment of the tumor, repeated use of the medicament is required, therefore, the problem of the heterogenous problem of the antibody itself is not allowed to be In addition, the clinical dosage of the antibody is large, the production cost is high, the price of the antibody drug is high, and the use of the antibody is limited to a certain extent. Perimeter. The treatment of a tumor with an exogenous VEGF antibody is a passive if active immunotherapy can be used, some deficiency of VEGF antibody treatment can be avoided, However, VEGF is the protein produced by the body itself, has its normal physiological function, and the body is immune tolerance to the normal self-protein, and it is difficult to induce the body to produce the self-VEGF by using the self-VEGF protein. The purpose of the present invention is to design a mVEGF vaccine, to bypass or to break the immune tolerance of the body, to enable the body to generate neutralizing antibodies to recognize the mVEGF, to inhibit the generation of the tumor new blood vessels, and finally to realize the inhibition of the tumor. In order to achieve the above objects, we have determined the basic strategy of the design of mVEGF vaccine, i. e., based on the space structure of mVEGF, to find the potential epitope of mVEGF, to show these potential epitopes on a simple protein skeleton, to form a new protein that is possible with this new protein mVEGF vaccine. First, we selected the antibody heavy chain variable region as the skeleton protein shown by the epitope of the antigen, because the antigen binding site of the antibody is composed of the light chain and the heavy chain variable region of the antibody, the single heavy chain antibody variable region has stability The disadvantage of the difference is that, after the analysis, we have mutated some of the amino acids in the variable region of the antibody heavy chain, and the mutant heavy chain variable region protein can easily realize the prokaryotic expression and can Renaturation is a stable protein. Since the CDR3 region of the heavy chain variable region of the antibody is highly variable, insertion or displacement of the different length sequences can be tolerated, so that the CDR3 region is selected by us The potential epitope of the mouse VEGF164 is analyzed according to the spatial structure of the human VEGF165, wherein the epitope 1 (EYPDEIEYIFKP), the epitope 2 (KSHEVIKFMDV) and the epitope 3 (IMRIKPHQSQH) may be mV. The neutralizing antigen epitope of the EGF is formed by replacing the amino acid sequence of the CDR3 region of the heavy chain variable region of the antibody with the amino acid sequence of the epitope 2, The novel protein is named mFV2. The sequence is converted into its gene coding sequence, the whole gene is synthesized with its coding sequence and cloned into pET-24a expression carrier. and the expression vector of the epitope 2 is constructed on the body, the vector is transformed into the BL21 (DE3) host strain, the high expression of the mFV2 is realized through the induction of IPTG, the expressed mFV2 protein is mainly in the form of an inclusion body, the inclusion body is washed, purified and cracked, and purified by a Sephacryl S-100 gel filtration column, high-purity mFV2 protein is obtained, The mFV2 protein is successfully renatured by the release method. The epitope 1 and the epitope 3 encoding gene are successfully inserted into the CDR3 region of the antibody variable region vector protein by the overlapping PCR method, and the mFV is also realized. Expression, purification and renaturation of 1 and mFV3. After three mouse epitope vaccine proteins mFV1, mFV2 and mFV3 were obtained, I The three kinds of vaccine proteins were used to immunize the female Balb/ c mice, and the mice were immunized for 5 weeks to obtain three kinds of immune serum, named pAb1, pAb2 and pAb3. The ELISA test showed that the titer of the three kinds of immune serum was 1:104, and it was able to specifically recognize the VEGF. 164, without reacting with extraneous proteins, suggesting that these three vaccine proteins are indeed capable of inducing the body The antibodies that specifically identify the VEGF164 are produced. The Western Blot test shows that the immune serum pAb1 and pAb2 are able to recognize the VEGF164, but pFV3 does not recognize the VEGF164, and it is likely that the epitope 3 is a space epitope, in Western The epitope in Blot cannot maintain its spatial structure. In addition, by immunofluorescence experiments, all three kinds of immune serum can recognize the expression of VEGF164 and B16 cell in B16 mouse melanoma cells. The amount of VEGF164 is mainly distributed in the cytoplasm. In order to verify that the three kinds of immune serum have VEG In F164 and activity, we analyzed three kinds of immune serum on human umbilical vein endothelial cells ( The results showed that VEGF164 could promote the proliferation, migration and tube formation of HUVEC, while pAb1, pAb2, pAb3 showed a certain mVEGF inhibitory activity, of which pAb 3. The results of the above-mentioned in vitro test show that the three kinds of immunity Serum does have a neutralizing activity of VEGF164 Sex, can inhibit the biological function of VEGF164. On this basis, We carried out an experimental study of the treatment of tumors with the mVEGF vaccine. First of all, we use a prophylactic vaccination. The three types of mVE were observed. Whether the GF vaccine has the effect of inhibiting the growth of H22 solid tumor. In the experiment,6-8 weeks of female BALB/ c mice were selected and divided into four groups. In the model group, mFV1 group, mFV2 group and mFV3 group, mFV1 group, mFV2 group and mFV3 group were immunized three times with mFV1 protein, mFV2 protein and mFV3 protein, and the immune effect was detected by ELISA at week 5, and H22 tumor cells were inoculated, and when the tumor was grown to the available hand, start to record the tumor The results showed that the three mVEGF vaccines showed a certain ability to inhibit the growth of H22 tumor, and the effect of mFV3 vaccine was the most ideal. The tumor rate was 83.8%, mFV2 was the second, and mFV1 was the worst. In order to understand the mV more intuitively, The anti-tumor mechanism of the EGF vaccine was carried out by the immunohistochemical staining of the tumor tissues in the H22 tumor model. The results showed that the microvessel density ratio in the vaccine treatment group was higher than that of the model group. The decrease of the microvessel density in the mFV3 immune group is the most obvious, which is It is the best reason why mFV3 is the best in the three vaccine proteins. Similarly, The tumor-inhibiting effect of three kinds of mVEGF vaccines is verified by using the S180 tumor model. In the S180 tumor model, the tumor-inhibiting performance of the three vaccines is consistent with the expression on the H22 tumor model, and also the effect of the mFV3 vaccine The most ideal, the tumor-inhibiting rate is 80.3%, mFV2 is the second, and mFV1 is the worst. In order to simulate the actual situation of the clinical vaccine, we have also carried out the treatment The effect of the mVEGF vaccine on the growth of the tumor and the effect of the administration of the mVEGF vaccine on the growth of the tumor were observed. The 6-8 weeks of female BALB/ c mice were selected and divided into four groups. The model group, the mFV3 treatment group, the cisplatin treatment group, and the mFV3 + cisplatin group were respectively inoculated. In the experiment, the H22 tumor cells were inoculated with the administration, and the model group was injected with normal saline only, and the cisplatin group was injected with cisplatin for 2 mg/ kg once a week. In combination with cisplatin and mFV3, the FV3 + cisplatin group was treated with cisplatin and mFV3. After the mice were inoculated with tumor cells, cisplatin (2 mg/ kg) was injected and mFV3 immunization was performed at the same time. Cisplatin was administered once a week for 2 times, and mFV3 was performed on day 14. After the second immunization, the mice were sacrificed on day 28, and the tumor tissues were removed and the size of the tumor was measured. The experimental results were as follows: the tumor inhibition rate of the pure mFV3 group was 27.3%, and the tumor inhibition rate of the pure cisplatin group was 35.7%. %, and the tumor inhibition rate of mFV3 + cisplatin-treated group reached 66.2%. In conclusion, an antigen epitope display system based on an antibody variable region is established, and three mVEGF vaccines are constructed by using the system,
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R392

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