构建重组肿瘤基因疫苗对肿瘤进展及生存率影响的实验结局(英)

目的:利用P815肿瘤动物模型观察IL-12对肿瘤动物模型抗肿瘤作用的效应。方法:将小鼠肥大细胞瘤P815特异抗原基因P1A克隆到真核表达质粒pCI-neo中;用P815细胞对DBA/2小鼠右腹侧皮下注射,构建P815小鼠肿瘤模型;以重组基因疫苗单独或与鼠IL-12真核表达质粒一起肌肉注射,观察肿瘤的消长、特异细胞毒T淋巴细胞激活和抗体的生成情况。结果:重组基因疫苗在体外有很好的表达,注射后细胞毒性T淋巴细胞(CTL)的杀伤效率为40%,IL-12共注射的CTL杀伤效率达到60%。免疫后,30%小鼠的肿瘤出现消退;同IL-12共注射则有50%的小鼠的肿瘤出现消退。两种情况下都不能检测到任何特异抗体的产生。结论:长期存在的IL-12可以持续刺激机体细胞免疫,使肿瘤的生存环境持续恶化,从而达到治疗肿瘤的目的。     

构建重组肿瘤基因疫苗对肿瘤进展及生存率影响的实验结局

目的：利用P815肿瘤动物模型观察IL-12对肿瘤动物模型抗肿瘤作用的效应。方法：将小鼠肥大细胞瘤P815特异抗原基因P1A克隆到真核表达质粒pCI—neo中；用P815细胞对DBA／2小鼠右腹侧皮下注射，构建P815小鼠肿瘤模型；以重组基因疫苗单独或与鼠IL-12真核表达质粒一起肌肉注射，观察肿瘤的消长、特异细胞毒T淋巴细胞激活和抗体的生成情况。结果：重组基因疫苗在体外有很好的表达，注射后细胞毒性T淋巴细胞(CTL)的杀伤效率为40％，IL-12共注射的CTL杀伤效率达到60％。免疫后，30％小鼠的肿瘤出现消退；同IL-12共注射则有50％的小鼠的肿瘤出现消退。两种情况下都不能检测到任何特异抗体的产生。结论：长期存在的IL-12可以持续刺激机体细胞免疫，使肿瘤的生存环境持续恶化，从而达到治疗肿瘤的目的。     

基于人/鼠嵌合模型的人黑色素瘤mage-3基因疫苗的免疫学作用

目的 利用人 /鼠嵌合模型评价人黑色素瘤基因 mage-3基因疫苗的免疫效果. 方法 建立并鉴定 Trimera动物模型,并用重组质粒 mage-3/pCI neo作为基因疫苗对其进行免疫.检测免疫动物脾细胞 CTL的杀伤活性和血清中 mage-3特异性抗体. 结果 构建的 Trimera模型脾脏和腹腔中出现了大量的人淋巴细胞.接种后, Trimera模型体内出现了效价为 1 256的 mage-3特异抗体.免疫动物 CTL体外杀伤实验显示,注射基因疫苗的 Trimera小鼠脾脏淋巴细胞对靶细胞 LB373的最大杀伤率为 50%左右,而对照小鼠只有 10%以下的杀伤水平. 结论 mage-3是一种理想的肿瘤疫苗,在人源化动物模型中能激发出特异的细胞和体液免疫,对共享 mage-3抗原的各种肿瘤细胞的临床治疗提供了实验依据.     

基于人／鼠嵌合模型的人黑色素瘤mage-3基因疫苗的免疫学作用

目的：利用人／鼠嵌合模型评价人黑色素瘤基因mage-3基因疫苗的免疫效果。方法：建立并鉴定Trimera动物模型，并用重组质粒mage-3／pCI-neo作为基因疫苗对其进行免疫。检测免疫动物脾细胞CTL的杀伤活性和血清中mage-3特异性抗体。结果：构建的Trimera模型脾脏和腹腔中出现了大量的人淋巴细胞。接种后，Trimera模型体内出现了效价为1：256的mage-3特异抗体。免疫动物CTL体外杀伤实验显示，注射基因疫苗的Trimera小鼠脾脏淋巴细胞对靶细胞LB373的最大杀伤率为50％左右。而对照小鼠只有10％以下的杀伤水平。结论：mage-3是一种理想的肿瘤疫苗，在人源化动物模型中能激发出特异的细胞和体液免疫，对共享mage-3抗原的各种肿瘤细胞的临床治疗提供了实验依据。     

丙肝核心抗原-CD40L胞外段真核表达载体的构建及其免疫原性分析

目的构建丙肝核心抗原-CD40L胞外段融合蛋白真核表达载体,并探讨其免疫原性。方法应用基因重组技术在原有载体的基础上构建真核表达载体pcDNA3.1-core-CD40L并加以鉴定。该表达质粒肌肉内注射免疫小鼠,ELISA法检测小鼠外周血中抗丙肝核心抗体,流式细胞仪检测小鼠外周血T淋巴细胞亚型,real time PCR法检测外周血单个核细胞内细胞因子,CTL杀伤实验检测小鼠脾淋巴细胞的杀伤活性。结果构建了真核表达载体pcD-NA3.1-core-CD40L,该载体能诱发小鼠产生高滴度的抗丙肝核心抗原的抗体,流式和real time结果证实该质粒能诱使小鼠T淋巴细胞由T0期向Th1和Th2转换,并以Th1为主,CTL杀伤结果显示该表达质粒能诱使小鼠产生高水平的细胞杀伤能力。结论成功构建了真核表达质粒pcDNA3.1-core-CD40L,该质粒能够在小鼠体内正确表达,并能诱发高水平的细胞杀伤活性。     

复苏因子E-DNA疫苗对BALB/C小鼠免疫效应的研究

目的:探索复苏因子E(RpfE)-DNA疫苗的小鼠免疫效果.方法:利用真核表达栽体PCDNA3.1构建PCDNA3.1-RpfE重组质粒.将SPF级BALB/C小鼠随机分为5组,即空质粒组、RpfE质粒组、BCG组、生理盐水组、空白对照组,于0、3、6周先后3次免疫小鼠,在第3次免疫后3周,心脏取血ELISA方法检测血清RpfE IgG抗体效价、干扰素γ(IFNγ)水平;并行脾淋巴细胞培养,以RpfE蛋白刺激淋巴细胞,CCK-8法检测淋巴细胞增殖指数、ELISA方法检测上清诱导IFNγ和白介素12(IL-12)产生水平,乳酸脱氢酶试验检测小鼠脾细胞毒性T淋巴(CTL)杀伤效应.结果:(1)RpfE质粒组小鼠血清中RpfE抗体水平明显高于其他各组(P<0.001);RpfE质粒组IFNγ水平明显高于空质粒组、生理盐水组、空白对照组和BCG组(P<0101或P<0.05).(2)RpfE质粒组具有较强的刺激淋巴细胞增殖作用,CCK-8实验表明其增殖指数高于其他各组(P<0.05);上清ELISA检测表明IFNγ和IL-12产生水平也高于其他各组(P<0.05).(3)RpfE质粒组脾淋巴细胞特异性CTL杀伤性与其他各组比较有统计学意义(P<0.05).结论:RpfE-DNA疫苗可诱导小鼠的免疫应答,具有良好的体液和细胞免疫原性.     

嗜肺军团菌momp基因疫苗的制备及其免疫原性研究

目的构建真核重组质粒GFP-momp作为核酸疫苗,已构建的原核重组质粒pET-momp表达的蛋白作为蛋白疫苗,二者联合免疫BALB/c雌性小鼠,观察其免疫原性。方法 (1)以嗜肺军团菌DNA作为模板,扩增得到momp基因,并克隆至pEGFP-C1载体获得重组质粒GFP-momp。鉴定正确后,将其转染到NIH3T3细胞,利用荧光显微镜观察GFP-momp的表达。(2)选取BALB/c雌性小鼠60只,平均分为4组,即磷酸盐缓冲液(PBS)组(A组)、空质粒pEGFP-C1组(B组)、DNA疫苗组(C组)、联合疫苗组(D组)。以重组质粒GFP-momp作为DNA疫苗,重组质粒GFP-momp和MOMP蛋白作为联合疫苗,第1天A组肌肉注射PBS 50μL,B组注射pEGFP-C1 50μg,C、D组各注射GFP-momp50μg;第14天各组以相同剂量追加免疫1次;第21天A、B、C组用相同剂量再追加免疫1次,D组注射10μg纯化的MOMP蛋白。借助对各试验组小鼠的抗体水平、脾淋巴细胞增殖活性、细胞毒性T淋巴细胞(CTL)杀伤活性等指标的动态检测,来评价DNA疫苗与蛋白疫苗的免疫原性。结果扩增出831bp的momp基因,构建重组质粒GFP-momp,转染入NIH3T3细胞并在细胞内表达出绿色荧光蛋白。在淋巴细胞增殖试验中:A组和B组比较差异无统计学意义(P0.05);C组和D组淋巴细胞增殖活性均高于B组,差异有统计学意义(P0.05);C组高于D组,差异有统计学意义(P0.05)。间接ELISA测定血清中抗原特异性抗体水平结果显示,A、B两组比较差异无统计学意义(P0.05);C、D两组均高于B组,差异有统计学意义(P0.05);D组抗体滴度高于C组,差异有统计学意义(P0.05)。CTL杀伤活性试验结果显示,A、B两组比较差异无统计学意义(P0.05);C、D两组均高于B组,差异有统计学意义(P0.05);D组杀伤活性高于C组,差异有统计学意义(P0.05)。结论成功构建momp基因的真核表达载体并在NIH3T3细胞中得到表达,并进一步得出嗜肺军团菌momp基因诱导产生的DNA疫苗和DNA-蛋白疫苗均能产生细胞免疫和体液免疫,具有免疫原性。     

bcr-abl融合基因真核表达载体诱导小鼠特异性免疫应答

目的在小鼠体内外研究bcr-abl融合基因真核表达载体诱导的特异性免疫应答,探索肿瘤免疫治疗的新途径。方法构建表达bcr-abl融合基因cDNA片段的真核细胞质粒pVbcr-abl,将 pVbcr-abl质粒用纳米颗粒聚乙烯亚胺包裹后给BALB／c小鼠肌内注射,检测小鼠血清中bcr-abl特异性抗体水平。小鼠免疫后20 d皮下接种具有相同遗传背景的SP2／0／ber-abl细胞(H-2~d),观察小鼠生存情况、移植肿瘤的生长情况和肿瘤细胞浸润情况。利用免疫组织化学方法观察肿瘤组织中T淋巴细胞浸润情况;流式细胞术分析免疫小鼠脾脏T细胞亚群的变化;LDH释放法检测脾脏细胞毒性T淋巴细胞(CTL)活性。结果成功构建真核表达载体pVbcr-abl,bcr-abl融合基因cDNA片段在真核细胞中可得到高效表达。pVbcr-abl免疫BALB／c小鼠能激活机体免疫系统,诱导产生特异性抗体和特异性CTL活性,并形成特异性免疫保护力。免疫小鼠的移植肿瘤形成时间、表面出现破溃时间、生长速度明显降低,荷瘤生存时间明显延长。免疫小鼠移植肿瘤组织中有大量CD3~+T细胞浸润。免疫小鼠的脾细胞杀伤SP2／0／bcr-abl细胞的细胞毒活性明显升高。小鼠脾脏T细胞亚群发生改变,CIM~+／ CD8~+细胞比值升高到1．54±0．29。结论pVbcr-abl真核表达载体除能在小鼠体内诱导产生特异性抗体以外,还能诱导高水平特异性CTL活性,直接杀伤肿瘤细胞,抑制肿瘤细胞的生长速度。     

高强度聚焦超声制备肿瘤疫苗对小鼠抗肿瘤免疫增强作用的研究

目的 探讨高强度聚焦超声(HIFU)制备的肿瘤疫苗对小鼠抗肿瘤免疫的增强作用.方法 每组小鼠20只,分别以2种声强的HIFU瘤苗、高温瘤苗及生理盐水注射,接种H22细胞后观察小鼠肿瘤发生率及生存期;检测免疫小鼠淋巴细胞的增殖及小鼠细胞毒性淋巴细胞(CTL)对肿瘤的杀伤;观察不同声强对HIFU瘤苗效能的影响.结果 HIFU瘤苗组小鼠肿瘤发生率减低,生存期延长;淋巴细胞在同源肿瘤刺激下增殖明显,CTL对肿瘤细胞有特异性的杀伤,且比高温瘤苗组有更高的增殖率和杀伤率(P＜0.05);显著提高声强后制备的HIFU瘤苗效能降低.结论 一定参数下制备的HIFU瘤苗明显增强了小鼠抗同源肿瘤的免疫力.     

弓形虫SAG1、GRA2单基因疫苗及SAG1-GRA2复合基因疫苗的免疫效果观察

目的 检测SAG1和GRA2基因编码的单基因及复合基因疫苗诱导小鼠的免疫应答,评价其抗弓形虫感染的保护性免疫效果.方法 大量制备pcDNA3.1-SAG1、pcDNA3.1-GRA2、pcDNA3.1-SAG1-GRA2真核表达重组质粒,肌内注射BALB/c小鼠,分别于2周、4周后加强免疫一次.另设立pcDNA3.1及PBS对照组.于3次免疫后4周作免疫指标测定(包括ELISA法测定小鼠血清IgG抗体及细胞因子IFN-γ、IL-4,MTT法测定小鼠淋巴细胞增殖能力及NK细胞杀伤活性),并观察弓形虫速殖子攻击感染后小鼠的生存时间.结果 SAG1-GRA2组小鼠血清IgG抗体、IFN-γ、淋巴细胞增殖能力及NK细胞杀伤活性均高于SAG1及GRA2组(P＜0.05);各组均未检测到IL-4;复合基因组小鼠生存时间较单基因疫苗组延长(P＜0.01).结论 弓形虫SAG1-GRA2复合基因疫苗较SAG1、GRA2单基因疫苗具有更好的免疫保护性.     

Intravenous cytokine gene delivery by lipid-DNA complexes controls the growth of established lung metastases

Local expression of cytokine genes by ex vivo transfection or intratumoral gene delivery can control the growth of cutaneous tumors. However, control of tumor metastases by conventional nonviral gene therapy approaches is more difficult. Intravenous injection of lipid-DNA complexes containing noncoding plasmid DNA can significantly inhibit the growth of early metastatic lung tumors. Therefore, we hypothesized that delivery of a cytokine gene by lipid-plasmid DNA complexes could induce even greater antitumor activity in mice with established lung metastases. The effectiveness of treatment with lipid-DNA complexes containing the IL-2 or IL-12 gene was compared with the effectiveness of treatment with complexes containing noncoding (empty vector) DNA. Treatment effects were evaluated in mice with either early (day 3) or late (day 6) established lung tumors. Lung tumor burdens and local intrapulmonary immune responses were assessed. Treatment with either noncoding plasmid DNA or with the IL-2 or IL-12 gene significantly inhibited the growth of early tumors. However, only treatment with the IL-2 or IL-12 gene induced a significant reduction in lung tumor burden in mice with more advanced metastases. Furthermore, the reduction in tumor burden was substantially greater than that achieved by treatment with recombinant cytokines. Treatment with the IL-2 or IL-12 gene was accompanied by increased numbers of NK cells and CD8+ T cells within lung tissues, increased cytotoxic activity, and increased local production of IFN-gamma by lung tissues, compared with treatment with noncoding DNA. Thus, cytokine gene delivery to the lungs by means of intravenously administered lipid-DNA complexes may be an effective method of controlling lung tumor metastases.     

Antitumor activity of lipopolysaccharide in tumor-bearing mice pretreated with BCG

The antitumor activity of lipopolysaccharide (LPS) was investigated in BCG-treated mice. C3H/He mice and CDF1 mice were injected with BCG and then were inoculated with syngeneic mouse hepatoma MH134 and mastocytoma P815 respectively. Hemorrhagic necrosis and retarded growth of tumor were observed after an intravenous (i.v.) injection of LPS, when tumor cells had been inoculated subcutaneously (s.c.). However an intraperitoneal (i.p.) injection of BCG plus LPS did not increase the mean survival time of mice that had been inoculated with tumor cells i.p. Sera from mice that had been treated with BCG plus LPS i.v. were cytotoxic for cultured tumor cells. These results seemed to indicate that growth-inhibitory effects of LPS on tumors inoculated s.c. were mediated by a humoral factor.     

IL-12 plasmid-enhanced DNA vaccination against carcinoembryonic antigen (CEA) studied in immune-gene knockout mice

Intramuscular (i.m.) injection of a plasmid encoding human carcinoembryonic antigen (CEA) elicited immunity against transplanted syngeneic (C57BL/6) CEA-positive Lewis lung carcinoma (CEA/LLC) cells, but tumors still appeared in all mice. In wild-type mice, coinjection of an IL-12 plasmid markedly enhanced anti-CEA humoral, T-helper-1 and cytotoxic T lymphocyte (CTL) responses, and resistance to a CEA/LLC tumor challenge such that 80% of mice remained tumor free. Injection of the IL- 12 plasmid alone was not protective. To analyze immune requirements, we immunized gene knockout (KO) mice of C57BL/6 background, deficient in either CD3, CD4, CD8, interferon gamma (IFNgamma), perforin or Fas ligand (FasL). Only CD3+ mice expressing both CD4 and CD8, which appear equally important, as well as IFNgamma and perforin, could fully resist a tumor challenge. IL-12 stimulated CTL activity, which was strictly CD3/CD8/perforin-dependent. FasL-KO mice had normal CTL activity and tumor resistance, indicating that only the perforin lytic pathway was involved. CD4-KO and IFNgamma-KO mice still generated CTLs. CEA-stimulated IFNgamma production occurred in both CD4- or CD8-KO mice and in both cases was augmented by IL-12. In IFNgamma-KO mice, IL-12 still enhanced anti-CEA antibody production but only moderately restored impaired DTH and tumor resistance. We conclude that the immune requirements for tumor rejection are stringent, involving multiple mechanisms which are all enhanced by IL-12.     

Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. I. Rejection by syngeneic mice

We have reported that it is possible to obtain variants that are incapable of forming progressive tumour in syngeneic mice (tum-) by mutagenesis and cloning of a teratocarcinoma and a Lewis lung carcinoma cell line. These observations were extended to the ascitic P815 mastocytoma of mouse strain DB/2. After a treatment with the mutagen N- methyl-N"-nitro-N-nitrosoguanidine, we obtained a high frequency (14%) of tum- clones. A colony assay indicated that after a period of rapid multiplication extending to approximately 10 d after injection, the P815 tum- cells were rejected by a process that was usually completed by day 15. No rejection was observed in sublethally irradiated animals. The immunological nature of the rejection of the P815 variants was further inferred because, upon rejection, the mice acquired a radioresistant specific protection that could be transferred adoptively with spleen cells. Cross-immunization patterns demonstrated the presence of singular antigenic specificities on three of the five variants that were examined. In addition, a common antigen was found on all the tum- variants and the original cells that were capable of forming progressive tumors in syngeneic mice (tum+). Mice injected with tum- cells were significantly protected against a tum+ challenge, even though no significant protection was generated by irradiated tum+ cells. A study of the T lymphocyte-mediated cytolysis against the P815 variants described here is presented in the accompanying report (9).     

Antisense IL-10 abrogates the inhibitory effects of IL-10 production by transfected tumor cells

Interleukin-10 (IL-10) is a pleiotropic cytokine that has a variety of downregulatory effects on immunologic and inflammatory processes. Ectopic tumor expression of IL-10 inhibited tumor growth, and local administration of antisense IL-10 significantly reversed the effects of IL-10 transfection in P815 mastocytoma. Tissue inhibitors of metalloproteinase (TIMPs) have been associated with decreased tumorigenesis and reduced metastasis, and TIMPs were increased in the region surrounding P815/IL-10 tumors and reduced in antisense IL-10-treated mice. In addition, the antisense IL-10 group had the largest tumor volume and poorest survival when compared with the P815/IL-10 control or sense groups. In summary, our data suggest that, in a mouse model, antisense IL-10 has substantive effects in reducing IL-10 translation and inhibiting IL-10-mediated TIMP upregulation, and, by doing so, allows IL-10-transfected mastocytoma to grow unchecked. Thus, ectopic tumor expression of IL-10 inhibits tumor growth, and antisense IL-10 administration in vivo reverses this protective effect.     

Tumor immunogenicity determines the effect of B7 costimulation on T cell-mediated tumor immunity

A costimulatory signal through B7 to its counter-receptor CD28 on T cells enhances T cell activation. We have generated recombinant retroviruses containing cDNA for murine B7 and transduced a panel of murine tumor lines with varying immunogenicity to study the effect of B7 costimulation on antitumor immunity. In contrast to the progressive outgrowth of all wild-type (B7-) tumors in unimmunized syngeneic mice, four immunogenic tumors, lymphoma RMA, EL4, mastocytoma P815, and melanoma E6B2, regressed completely when transduced with the B7 gene. In contrast, four nonimmunogenic tumors, sarcomas MCA101, MCA102, and Ag104, and melanoma B16, remained tumorigenic after transduction of the B7 gene. Immunization with B7-transduced immunogenic tumors enhanced protective immunity and increased specific cytotoxic T lymphocyte (CTL) activity against the respective wild-type tumors as compared to immunization with nontransduced or mock-transduced tumors. Moreover, cocultivation of CTL with B7-transduced EL4 cells augmented the specificity of tumor-reactive CTL in long-term cultures. Treatment by injection of B7-transduced tumor cells cured 60% of mice with established wild-type EL4 lymphoma. In contrast, immunization with nonimmunogenic tumors transduced with B7 did not provide protective immunity and did not increase specific CTL activity. Our results show that tumor immunogenicity is critical to the outcome of costimulation of T cell-mediated tumor immunity by B7.     

Regulation of T-helper-1 versus T-helper-2 activity and enhancement of tumor immunity by combined DNA-based vaccination and nonviral cytokine gene transfer

Intramuscular (i.m.) injections of a plasmid encoding human carcinoembryonic antigen (CEA) elicited both humoral and cellular immune responses in mice, but only partial inhibition of the growth of transplanted syngeneic CEA-positive P815 tumor cells (CEA/P815). Coinjection of the CEA vector with a vector encoding either interferon-gamma (IFN gamma) or IL-12 promoted IgG2a isotype anti-CEA antibody production, anti-CEA/P815 CTL activity and greater resistance to CEA/P815 tumor challenge. As well, CEA/P815-stimulated IFN gamma secretion in vitro was increased, but IL-4 diminished, consistent with a T-helper type 1 (Th1) response. In contrast, coinjection of the CEA vector with an IL-4 vector increased IgG1 production, but reduced CTL activity and resistance to tumor challenge. The latter treatment inhibited CEA/P815-dependent IFN gamma production but enhanced IL-4 secretion, consistent with a Th type 2 (Th2) response. Antitumor immunity was enhanced when the CEA and IL-12 plasmids were coinjected at the same muscle site, but not at separate sites despite increased serum IL-12 levels. Though the tumor cells expressed neomycin phosphotransferase, mice immunized with vectors encoding that protein (without CEA) were not protected against tumor growth, and produced no CTLs except for low levels when coinjected with an IL-12 vector. Thus, we show that immunity elicited by DNA vaccination against CEA can be biased to a protective type (high Th1 and CTL activity) or nonprotective type (high Th2 and low CTL activity) by i.m. coinjection of cytokine-expressing plasmids. IL-12 appears to act locally, but not systemically, through an adjuvant effect.     

Long-lasting anti-metastatic efficiency of interleukin 12-encoding plasmid DNA

Intramuscular injection of plasmid DNA encoding both subunits of the cytokine interleukin 12 (IL-12) exhibits strong antimetastatic activity against lung metastases induced by the malignant melanoma cell line B16-F10. The protective effect of IL-12 DNA is long-lasting, since administration of tumor cells 9 days after IL-12 DNA treatment prevented metastasis formation. No effects were observed with empty plasmid controls, DNA encoding the melanoma-associated antigen pmel17/gp100, the granulocyte-macrophage colony-stimulating factor GM-CSF, B7.1, or CpG-containing oligodeoxynucleotides. IL-12 DNA is required during early phases of metastasis formation and is ineffective when administered later. Its efficiency is dose dependent. The cytotoxic T cell response contributes to the antimetastatic effect as evidenced by genetically modified CD8- or perforin knockout mice. Depletion of natural killer (NK) cells by antibodies completely abrogated the effect. In contrast, the IL-12-induced antimetastatic effect was not mediated by interferon gamma (IFN-gamma) or tumor necrosis factor alpha (TNF-alpha) as shown with IFN-gamma receptor and TNF-alpha knockout mice, respectively. Toxic side effects by IL-12 were low. Our results suggest that plasmid DNA encoding IL-12 might have potential value as gene medicine against the initiation of metastasis formation.     

人B细胞淋巴瘤独特型DNA疫苗诱导抗肿瘤免疫反应的实验研究

本研究目的在于探讨含人B细胞淋巴瘤免疫球蛋白重链可变区基因片段的DNA疫苗诱导小鼠抗肿瘤免疫反应的情况，为人B细胞淋巴瘤疫苗的应用提供基础实验研究资料。本研究通过PCR方法获得人B细胞淋巴瘤细胞系Namalwa膜表面免疫球蛋白重链可变区(VH)基因片段，同时克隆小鼠单核细胞趋化蛋白-3(MCP-3)作为免疫佐剂分子，进一步以重组PCR的方法获得MCP-3和、VH基因的融合基因片段，构建DNA疫苗重组质粒。在体外以瞬时转染的方法证实以融合基因片段作为抗原基因的DNA疫苗质粒能够在真核细胞中正确表达。DNA疫苗质粒大量提取后免疫小鼠。用流式细胞术检测小鼠抗体生成情况，并用LDH释放法测定CTL活性以检测抗独特型细胞免疫。结果表明，从接种疫苗第8周开始小鼠体内特异性抗独特型抗体明显升高，并且抗体滴度可维持高水平至少至第20周。在DNA疫苗免疫组中5只免疫小鼠有3只产生抗体。所诱导的抗体只特异性识别肿瘤细胞表面独特型抗原，而不识别人A549对照细胞。用乳酸脱氢酶释放法未测到明显CTL反应的产生。结论：以MCP-3与VH的融合作为抗原基因构建的DNA疫苗能够诱导小鼠体内产生抗淋巴瘤细胞的特异性抗独特型抗体，为DNA疫苗临床用于人B细胞淋巴瘤治疗提供了初步实验支持。     

The gene coding for a major tumor rejection antigen of tumor P815 is identical to the normal gene of syngeneic DBA/2 mice

We showed previously that mouse mastocytoma P815 expresses several distinct antigens that are recognized by cytolytic T lymphocytes (CTL) of syngeneic DBA/2 mice. Antigens P815A and P815B are usually lost jointly and are targets for immune rejection responses in vivo. We used a cosmid library and a CTL stimulation assay to obtain transfectants expressing tumor rejection antigen P815A. From these transfectants we retrieved gene P1A which transferred the expression of both P815A and B. This gene is unrelated to three previously isolated genes coding for tum-antigens. It encodes a putative protein of 224 amino acids which contains two highly acidic domains showing homology with similar regions of nuclear proteins. The P1A gene expressed by tumor P815 is completely identical to the gene present in normal DBA/2 cells. Expression of the gene was tested by Northern blots. Cells from liver, spleen, and a number of mast cell lines were negative, but mast cell line L138.8A produced a high level of P1A message and was lysed by CTL directed against antigens P815A and B. We conclude that major tumor rejection antigens of P815 are encoded by a gene showing little or no expression in most normal cells of adult mice.