【作者】 王芳；

【导师】 侯立中；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2004， 博士

【摘要】 细胞免疫是机体清除体内变异细胞的主要途径，其中细胞毒性T淋巴细胞(CTL)发挥关键作用。CTL的激活是通过识别抗原递呈细胞（APC）表面 MHCⅠ类分子递呈的抗原肽，被递呈的抗原一般为9～11个氨基酸的肽段。现已证实，合成肽不需要APC的加工、处理就能直接与APC表面的MHCⅠ类分子结合，与天然的内源性肽在激活免疫系统方面具有同等效力。肿瘤抗原肽的鉴定使直接应用明确的抗原肽作为肿瘤疫苗成为可能。但是合成肽疫苗分子较小，免疫原性较低，在体内半衰期短，容易被酶类降解,导致其诱导的特异性CTL存在时间短，效价低，不易达到使肿瘤消退所需的阈值。为此本研究主要从以下两方面着手突破这种低应答状态：①在不影响合成多肽疫苗与MHC-Ⅰ类分子结合的前提下，在合成多肽中掺入D型氨基酸，使其局部的旋光性发生改变。合成的D型抗原肽对蛋白酶的降解具有抗性，其稳定性提高，体内的半衰期延长，免疫原性增强。②联合应用免疫佐剂GM-CSF，活化巨噬细胞和其它免疫反应细胞，以期获得较强的细胞免疫应答反应。一．HER2/neu多肽疫苗序列的选择、设计与固相合成1．多肽氨基酸序列的选择与设计：应用SYFPETTHI基序法预测系统进行<WP=101>CTL表位初步预测，从抗原肽预测结果中选取两种区段P42-50 (HLYQGCQVV)、P782-790 ( VSRLLGICL)。因为MHC-Ⅰ类分子不同等位基因结合多肽的氨基酸残基变化主要局限在氨基末端，因此，在不改变多肽与MHC-Ⅰ类分子结合的前提下，改变多肽抗原的羧基端氨基酸，将P42-50的羧基端残基L-Leu替换为D-Leu , P782-790的羧基端残基L-Ser替换为D-Ser，合成了具有局部手性特征的多肽D-P42-50，D-P782-790，以期增强多肽在体内稳定性的同时提高其免疫原性。2．多肽合成：采用Fmoc固相多肽合成策略，合成上述四种多肽，L-P42-50，，D-P42-50，L-P782-790和D-P782-790。3．多肽的纯化及鉴定：利用中压反相液相层析对粗肽进行分析及纯化，L-P42-50、D-P42-50的纯度分别为99.2%及98.9% ，L-P782-790、D-P782-790的纯度分别为99.1%和99.0% 。利用激光解吸电离飞行时间质谱测定各色谱峰的分子量，经真空旋转蒸发及冻干获得纯品多肽。结果L-P42-50、D-P42-50总产率分别为63%及45%，，L-P782-790、D-P782-790总产率分别为58%及46%，。二．合成多肽疫苗体内诱导CTL作用研究小鼠乳腺癌模型的建立及多肽疫苗免疫 选用雌性BALB/c小鼠接种D2F2细胞,分别为对照组、GM-CSF组、L-P42-50、L-P42-50+ GM-CSF组、D-P42-50、D-P42-50+ GM-CSF组、L-P782-790、L-P782-790+ GM-CSF组、D-P782-790、D-P782-790+ GM-CSF组。1．与对照组小鼠相比，L-P42-50+GM-CSF组、L-P782-790+GM-CSF组、D-P42-50+GM-CSF组和D-P782-790+GM-CSF组小鼠脾细胞重量增加差异显著（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01），对淋巴细胞增殖的促进作用差异显著（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01），分泌IFN-γ的T淋巴细胞数明显增多（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01），对D2F2乳腺癌细胞的特异杀伤作用差异显著（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01）。这四种多肽联合应用GM-CSF 都能促进小鼠T细胞增殖，促进T细胞分泌IFN-γ及激活CTL杀伤乳腺癌细胞，说明这两种预测的L型多肽在体内与MHC-Ⅰ类分子相结合产生复合物，与T细胞受体结合，激活CTL<WP=102>细胞，对乳腺癌细胞产生杀伤作用。2．D-P42-50+GM-CSF组和L-P42-50+GM-CSF组、D-P782-790+GM-CSF组L-P782-790+GM-CSF相比，小鼠脾细胞重量的增加差异显著（P﹤0.05, P﹤0.05），对淋巴细胞增殖的促进作用差异显著（P﹤0.05, P﹤0.05）,分泌γ-IFN的T淋巴细胞数增多（P﹤0.05, P﹤0.05）,诱导对D2F2乳腺癌细胞的特异杀伤作用也有显著差异（P﹤0.05, P﹤0.05）。D-P42-50和D-P782-790辅以GM-CSF可显著促进小鼠T细胞增殖，促进T细胞分泌IFN-γ及激活CTL杀伤乳腺癌细胞，说明这两种经改造的手性多肽与MHC-Ⅰ类分子结合的能力并没有发生改变，与L型多肽一样，在体内与MHC-Ⅰ类分子相结合产生复合物，与T细胞受体结合，激活CTL细胞，对乳腺癌细胞产生杀伤作用。D-P42-50和 D-P782-790多肽在局部上具有了手性特征，具有了不同于体内肿瘤细胞所表达的HER2/neu多肽的结构特征，在一定程度上增加了免疫原性，从而使其更能够有效地诱导CTL。本研究的淋巴细胞增殖实验和CTL杀伤活性测定结果也肯定了这一实验设计的可行性和有效性。三．合成多肽疫苗体内抑瘤实验研究BALB/c小鼠接种小鼠乳腺癌细胞D2F2后，进行多肽疫苗接种。接种肿瘤细胞42天后处死小鼠，取肿瘤组织进行光镜及电镜观察。1．小鼠肿瘤组织体积和重量：与对照组小鼠相比，L-P42-50+GM-CSF组、L-P782-790+GM-CSF组、D-P42-50+GM-CSF组和D-P782-790+GM-CSF组的肿瘤重量和体积明显低于对照组，有显著性差异（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01），而且D-P42-50+GM-CSF组和D-P782-790+GM-CSF组的肿瘤重量和体积与L-P42-50+GM-CSF组和L-P782-790+GM-CSF组相比，有显著差异（P﹤0.05, P﹤0.05）。NaCl组、GM-CSF 组、L-P42-50 组、D-P42-50组、L-P782-790组和 D-P782-790组小鼠与对照组相比无显?更多还原

【Abstract】 Cell immunogy is the major to eliminate the tumor cells in the body,and cototo-xic T cells(CTL) is the most immportant.CTL is activated by the compound of MHC-Ⅰmolecuar and antigen peptide which is presented by antigen presentation cells(APC).The length of the antigen peptide is most 9～11 amino acids.It is has been proved that synthesized peptide can combine with MHC-Ⅰmolecuar on the surface of APC directly without the processing of APC,have the same effect as the natual endogenous peptide.The identification of the tumor antigen peptide can apply the peptide as the tumor vaccine.But peptide molecular is small ,the immunogenicity of peptide is low,half-life time of peptide is short and the peptide is hydrolized by enzyme easily.All these can influence the existing time and valence of CTL.Two methods were used to improve these situation : ①Change regional optical rotation by turn some L-peptide to D-peptide without altering the combination of peptide and MHC-Ⅰmolecuar. The synthesized D-peptide can be stable by resisting the hydrolization of the protein enzyme and increase the half-life time thus increade immunogenicity of peptide. ②Immunity ajuvant GM-CSF is used to activate the macrophage and other immunity cells and get the more powerful cell immunity.1.The selection,designation and synthesis of the HER2/neu peptide:(1) Design of the amino acid sequence of peptide: SYFPETTHI sequence <WP=105>analysis system was used to predict MHC-Ⅰmolecular restricted CTL epi-position of HER2/neu protein ,and select two region P42-50 (HLYQGCQVV) and P782-790 ( VSRLLGICL).Because the change of combination region of the peptide with the different allel gene of MHC-Ⅰmolecular is locolized the N-terminal amino acid of the peptide, so we change the C-terminal amino acid without changing the combination of the peptide and the MHC-Ⅰmolecular.We substitute D-Leu for L-Leu for the P42-50, substitute D-Ser for L-Ser for P782-790,and get two chiral peptide vaccine D-P42-50,D-P782-790.(2) Peptide synthesis: Peptides L-P42-50, D-P42-50,L-P782-790,D-P782-790 were synthesized with solid phase peptide synthesis method and Fmoc-strategy. Each amino acid was fully protected during the peptide synthesis procedure.(3) The purification and identification of peptide:The peptide was purifed by middle pressure reverse liquid chromatography.And the purity of L-P42-50, D-P42-50 is 99.2%,98.9%, the purity of L-P782-790, D-P782-790 is 99.1%,99.0%. The fraction of interested was confirmed by measuring the molecular weight of each fraction with EMS. Then the crude peptide was purified on preparative RP-HPLC. The purified peptide was received through the collection of interested fraction and the disposal of vacuum rotation evaporation and ice freeze. The product of pure peptide L-P42-50, D-P42-50 is 63% and 45% ,the product of pure peptide L-P782-790,D-P782-790is 58% and 46% .2. Study on induction of specific CTL in vivo by peptide vaccine immunization. The construct of mouse breast tumor model and vaccinated with peptide vaccine:BALB/c mice were inoculated with mouse breast tumor cell line D2F2(which was transfected with full cDNA of human HER2/neu gene), then vaccinated with peptide vaccine and divided for ten group:Nacl group;GM-CSF group; L-P42-50 group;L-P42-50+ GM-CSF group;D-P42-50 group; D-P42-50+ GM-CSF group;L-P782-790 <WP=106>group; L-P782-790 + GM-CSF group; D-P782-790 group; D-P782-790 + GM-CSF group.(1) Compared with the control group; L-P42-50+ GM-CSF group; D-P42-50+ GM-CSF group; L-P782-790+ GM-CSF group and D-P782-790 + GM-CSF group have significant difference on the increase of the weight of spleen（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01）;promoting the proliferation of T lymphocytes（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01）;the secreting γ-IFN of T cells（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01）; and HER2/neu specific CTL（P﹤0.05, P﹤0.05, P﹤0.01, P﹤0.01）. The results indicated all these four peptides can form compound with MHC-Ⅰmolecular,bind to the T cell receptor,activated special CTL.(2) Compared with L更多还原