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。     

用基因组DNA制备独特型抗淋巴瘤核酸疫苗的实验研究

本研究观察从人B细胞淋巴瘤细胞系基因组DNA及RNA分别构建的表达载体作为核酸疫苗诱导小鼠产生抗肿瘤免疫反应的情况。分别提取人B淋巴瘤细胞系Namalwa细胞的基因组DNA及RNA，将免疫球蛋白重链可变区（IgHV)基因片段克隆到pcDNA3.0真核表达载体中作为独特型核酸疫苗，其中DNA 核酸疫苗用LipofectAMINE转染COS细胞，多面手用RT－PCR观察转录及剪接的情况。两种不同来源的核酸疫苗分别肌内注射免疫小鼠后，用间接免疫荧光法检测抗独特型抗体的生成。结果发现，基因组DNA来源的核酸疫苗在COS细胞中能成功地转录，转录产物大小为477bp，其中86bp的内含子区域被剪接；两种疫苗免疫动物后均可诱导针对Namalwa细胞特异的抗独特型抗体，并于第6周达高峰。结论提示，淋巴瘤基因组DNA和RNA来源的IgHV基因片段均可制备核酸疫苗，能诱发小鼠产生抗淋巴瘤免疫反应。     

独特型Ig/TCR DNA疫苗抗淋巴系统肿瘤的研究进展

利用淋巴系肿瘤细胞Ig/TCR基因重排的独特型特点,设计独特型Ig/TCR DNA疫苗,可诱导机体产生抗独特型特异性免疫应答,在小鼠中已显示了其抗肿瘤效应,并已进入人体研究阶段,具有较好的应用前景.     

TCR独特型DNA疫苗诱导抗淋巴系统肿瘤免疫反应的实验研究

本研究观察ＴＣＲ独特型ＤＮＡ疫苗诱导ＢＡＬＢ／ｃ小鼠抗肿瘤免疫反应的情况。用ＣＥＭ淋巴瘤细胞系和ＢＡＬＢ／ｃ小鼠作模型,ＲＴ－ＰＣＲ扩增ＣＥＭ系特异性重排的ＴＣＲ Ｖβ基因,克隆到真核表达载体ｐｃＤＮＡ３中作为ＤＮＡ疫苗。用肌内注射法免疫小鼠,间接免疫荧光法检测小鼠抗体生成和用ＭＴＴ法测定ＣＴＬ活性以检测抗独特型的细胞免疫。结果发现,接种ＤＮＡ疫苗后第４周开始小鼠血清中即可检测到特异性抗独特型抗体     

融合型B细胞淋巴瘤独特型DNA疫苗表达质粒的构建

已有研究证实,B细胞淋巴瘤病人自身免疫球蛋白独特型可视为肿瘤特异性抗原用于独特型疫苗。为探究带有细胞因子的融合型独特型肿瘤疫苗是否会提高免疫效果,制备小鼠B细胞型淋巴瘤细胞的独特型单链可变区片段,与免疫佐荆单核细胞趋化因子MCP3融合,同时融合报告基因EGFP,构建融合型独特型淋巴瘤DNA疫苗。用RT—PCR法扩增BALB／c小鼠源B细胞淋巴瘤细胞株A20的IgVH和IgVL基因,重组PCR法将编码（Gly4Ser）3的核苷酸片段连接两基因,制备scFv片段。用相同PCR方法,选用1段编码NDAQAPKS连接肽连接趋化因子MCP3基因与scFv片段,获得MCP3-scFv融合基因片段。将scFv和MCF3-scFv融合基因片段分别插入真核表达质粒pTARGET,并在融合基因的下游插入报告基因EGFP,构建真核表达质粒pTARGET／scFv—EGFP和pTARGET／MCP3-scFv—EGFP。结果表明,成功扩增A20细胞的IgVH和IgVL基因片段及scFv—EGFP、MCP3-scFv—EGFP融合基因片段;酶切鉴定表明,成功制备重组真核表达质粒pTARGET／scFv—EGFP和pTARGET／MCP3-scFv—EGFP。结论：成功构建带有鼠源scFv片段、趋化因子MCP3和EGFP融合的独特型抗B细胞淋巴瘤疫苗表达质粒pTARGET／MCP3-scFv—EGFP和pTARGET／scFv—EGFP。所构建的重组表达质粒为下一步的抗B细胞淋巴瘤基因疫苗的体内动物实验奠定了基础。     

独特型Ig／TCRDNA疫苗抗淋巴系统肿瘤的研究进展

利用淋巴系肿瘤细胞Ig／TCR基因重排的独特型特点，设计独特型Ig／TCRDNA疫苗，可诱导机体产生抗独特型特异性免疫应答，在小鼠中已显示了其抗肿瘤效应，并已进入人体研究阶段，具有较好的应用前景。     

B细胞性非霍奇金淋巴瘤疫苗的研究进展

B细胞淋巴瘤肿瘤细胞来源于单-B细胞克隆,其细胞表面免疫球蛋白的独特型位点是唯一的,具有高度的特异性和均一性.B细胞表面的免疫球蛋白为独特型即肿瘤特异性抗原,使独特型淋巴瘤疫苗的研制成为可能.独特型蛋白疫苗的Ⅰ/Ⅱ期临床试验已显示了其治疗B细胞非霍奇金淋巴瘤的良好疗效,Ⅲ期临床试验目前已经完成.另外新型疫苗如DNA疫苗、树突细胞疫苗和脂质体疫苗正在研究阶段.笔者对独特型疫苗及新型疫苗的研究进展进行综述.     

Dsg3特异性的CD4~+ T细胞能够辅助B细胞产生天疱疮抗体

目的探讨CD4+T细胞在天疱疮致病性抗体产生中的作用。方法实验方面采用真核表达系统表达重组的天疱疮抗原桥粒芯糖蛋白3(Dsg3)胞外段功能区,联合不同佐剂(CFA或Alum)免疫C57BL/6小鼠,用免疫磁珠细胞分选(MACS)法分选出免疫后小鼠的CD4+T细胞,过继转移到T细胞受体β链(T cell receptor beta chain,TCRβ)缺陷小鼠,于转移后1、2、3、4周检测TCRβ缺陷小鼠体内CD4+T细胞的恢复,B细胞的增殖、活化,及特异性抗体的产生。结果过继转移后的TCRβ缺陷小鼠外周血中有CD4+T细胞的出现(比例有5%左右);CD4+T细胞进入缺陷小鼠脾脏中的淋巴小结,和B细胞相互作用;同时该小鼠血清中出现了特异性抗Dsg3抗体。结论 CD4+T细胞参与天疱疮致病性抗体的产生,在疾病中发挥极其重要的作用。     

免疫网络学说对ABO血型反定型试剂研制的理论指导意义

1974年，Jerne根据现代免疫学对抗体分子独特型的认识，在Burnet“克隆选择学说”的基础上提出了著名的“免疫网络学说”，认为任何抗体分子和淋巴细胞的抗原受体上均存在独特型，独特型可被体内另外一些淋巴细胞识别而诱发产生抗独特型抗体。以独特型与抗独特型的相互识别为基础，免疫系统内构成“网络”联系，在免疫调节中起重要作用。Jerne的网络学说强调了免疫系统是各个细胞克隆之间相互联系、相互制约所构成的对立统一整体，它对免疫学理论研究以及在生物学和医学领域中的实际应用都具有重大意义。用抗原免疫动物可产生抗体（Abl），用Abl免疫动物可产生抗独特型抗体（Ab2），Ab2免疫动物后可产生抗抗独特型抗体（Ab3）……Ab2的结合位点表现外来抗原或自身抗原决定簇的内在构象，即“内影像”，Ab3具有Ab1的“内影像”，因此，Ab2具有模拟抗原功能的潜能。     

布氏菌病双价DNA疫苗免疫保护效果的研究

【目的】构建布氏杆菌pcDNA3．1（＋）-L7／L12-BCSP31双价DNA疫苗，观察其免疫保护效果。【方法】分别克隆L7／L12、BCSP31基因，构建pcDNA3．1（＋）-L7／L12-BCSP31双价DNA疫苗。转染COS-7细胞，免疫细胞化学检测目的蛋白的表达。DNA疫苗免疫Balb／c小鼠，观察体液免疫和细胞免疫效果。布氏杆菌强毒株腹腔攻毒，观察双价DNA疫苗的免疫保护效果。【结果】构建的pcDNA3．1（＋）-L7／L12-BCSP31双价DNA疫苗在COS-7细胞有目的蛋白的表达。双价DNA疫苗免疫小鼠，ELISA检测双价DNA疫苗产生了高水平的特异性IgG抗体，且抗体亚型以IgG2a为主。FCM检测双价DNA疫苗的CD4^＋／CD8^＋比值明显下降。ELISPOT检测到疫苗免疫后小鼠分泌IFN-γ的T细胞增多。攻毒实验证明双价DNA疫苗能够产生有效的保护效果；部分数据及攻毒结果表明双价DNA疫苗的效果优于单价。【结论】研制的pcDNA3．1（＋）-L7／L12-BCSP31双价DNA疫苗免疫Balb／c小鼠能产生有效的免疫保护效果，且双价DNA疫苗效果优于单价。     

结核分枝杆菌Rv1009结构域多肽的免疫学特性研究

目的 研究结核分枝杆菌Rv1009结构域多肽的免疫学特性.方法 用原核表达的Rv1009结构域多肽免疫BALB/c小鼠3次.每次间隔2周.用ELISA法检测免疫小鼠血清中特异性抗体滴度.分离免疫小鼠的脾淋巴细胞,体外用抗原再刺激后,用MTT比色法检测免疫小鼠脾淋巴细胞的增殖指数.ELISA方法检测淋巴细胞悬液中γ干扰素(IFN-γ)、白细胞介素(IL)-10和IL-12的产生水平.另一部分免疫的小鼠经尾静脉感染MTB毒株H37Rv,4周后,计数脾脏细菌负荷数.结果 Rv1009结构域多肽免疫小鼠血清特异性抗体滴度为1:12 800.淋巴细胞增殖指数为2.40±0.18,明显高于生理盐水对照组的0.90±0.21.ELISA方法检测Rv1009结构域多肽免疫组IFN-γ、IL-10和IL-12水平为(1432±30)ng/L、(503±11)ng/L和(311±11)ng/L,显著高于生理盐水对照组的(256±20)ng/L、(76±6)ng/L和(56±8)ng/L(P<0.01).与生理盐水免疫组(细菌负荷6.64±0.13)相比较,Rv1009结构域多肽免疫组小鼠,对攻击感染后抗MTB在脾脏中增殖有显著作用(细菌负荷为4.86±0.14,P<0.05),但不及BCG免疫组的3.81±0.16.结论 Rv1009结构域多肽有可能作为新型结核疫苗的候选组分.     

Mechanisms of idiotype suppression. I. In vitro generation of idiotype- specific suppressor T cells by anti-idiotype antibodies and specific antigen

Normal BALB/c spleen cells are unresponsive in vitro to the phosphorylcholine (PC) determinant in the presence of anti-idiotype antibodies specific for the TEPC-15 myeloma protein (T15) which carries an idiotypic determinant indistinguishable from that of most anti-PC antibodies in BALB/c mice. The possibility that idiotype-specific suppressor cells may be generated during the culture period was examined by coculturing the cells with untreated syngeneic spleen cells. Cells that had been preincubated with anti-T15 idiotype (anti-T15id) antibodies and a PC-containing antigen, R36a for 3 d, were capable of specifically suppressing the anti-PC response of fresh normal spleen cells, indicating that idiotype-specific suppressor cells were generated during the culture period. The presence of specific antigen also appeared to be necessary because anti-T15id antibodies and a control antigen, DNP-Lys-Ficoll, were not capable of generating such suppressor cells. Suppressor cells were induced only in the population of spleen cells nonadherent to nylon wool and the suppressive activity was abrogated by treatment with anti-Thy 1.2 serum and complement. These results indicate that anti-idiotype antibodies and specific antigen can generate idiotype-specific suppressor T cells in vitro. These in vitro results may reflect in vivo mechanisms of idiotype suppression.     

Induction of antiphosphorylcholine antibody formation by anti-idiotypic antibodies

Anti-idiotypic antibodies have been used to mimic antigen in the mouse antiphosphorylcholine response in order to investigate the induction of precursors of antibody-forming cells. We have shown that interaction of anti-idiotype antibody with receptor antibody molecules induces the formation of antibodies that are specific for phosphorylcholine and carry the idiotypic determinants. This induction is dependent on the recognition of carrier determinants on the anti-idiotype antibody by helper T cells. We conclude that receptor antibody molecules on the surface of the precursors of antibody-forming cells deliver the antigenic signal for the induction of these cells.     

IL-2、IFN-γ和GM-CSF基因佐剂对pcDNA3/MDC-VP1 DNA疫苗免疫作用的影响

目的:观察IL-2、IFN-γ和GM-CSF重组质粒对pcDNA3/MDC-VP1 DNA疫苗免疫的免疫增强效果。方法:将50只4～6周龄雄性BALB/c小鼠随机分成pcDNA3组、pcDNA3/MDC-VP1组、pcDNA3/MDC-VP1+pcDNA3/hIL-2组、pcDNA3/MDC-VP1+pcDNA3/m IFN-γ组、pcDNA3/MDC-VP1+pcDNA3/mGM-CSF组,每组10只。均每3周接种1次,共3次。每次接种的第20d内眦静脉采血,用微量中和试验(固定病毒-稀释血清法)检测血清中和抗体效价。第3次免疫后3周,每组取3只小鼠脾脏制备淋巴细胞悬液,检测淋巴细胞增殖活性与特异性细胞毒性T淋巴细胞(CTL)杀伤活性。结果:各组血清中和抗体滴度随免疫次数增加而提高(P0.05);第3次免疫后pcDNA3/MDC-VP1+pcDNA3/mGM-CSF组的血清中和抗体滴度、脾脏淋巴细胞增殖活性和特异性CTL杀伤活性均高于其他组(P0.05)。结论:三种细胞因子基因佐剂均能增强pcDNA3/MDC-VP1的免疫效果,GM-CSF基因佐剂的免疫效果优于IL-2和IFN-γ。     

Antibodies elicited by pneumococcal antigens bear an anti-DNA--associated idiotype.

There is evidence in both murine and human lupus that the production of anti-DNA antibodies may be triggered by environmental antigens. To explore this further, we studied the serum of 10 nonautoimmune individuals immunized with a polyvalent pneumococcal polysaccharide vaccine. All 10 patients showed a rise in the titer of antipneumococcal antibodies bearing an anti-DNA-associated idiotype. The antipneumococcal response was specific as no idiotypic antitetanus antibodies were detected. Furthermore, no anti-DNA antibodies were present in postvaccination sera. The molecular analysis of antipneumococcal and anti-DNA antibodies bearing a common idiotype will help elucidate how foreign antigen might lead to the production of anti-DNA antibodies in susceptible individuals.     

丙型肝炎病毒包膜区基因DNA免疫的研究

目的:构建丙型肝炎病毒包膜区基因(E1、E2)真核表达载体,命名为PCI-neo-E1和E2。方法:将真核重组体转染NIH-3T3细胞中,利用RT-PCR方法鉴定相应的基因片段,显示获得了相应的稳定转染的细胞克隆。提取转染细胞蛋白,进行WesternBlot分析。结果:HCVE1、E2基因在真核细胞中获得有效表达。将所构建的PCI-neo-E1、E2免疫Balb/C小鼠,获得特异的抗体反应。结论:通过HCV包膜区DNA免疫的研究表明能激发特异的免疫反应,为进一步进行HCV疫苗研究奠定基础。     

Idiotypy of clonal responses to influenza virus hemagglutinin

Anti-idiotype antisera were raised in syngeneic (BALB/c mice) and homologous (A/J mice) systems to study the cross-reactive idiotypes among monoclonal antibodies to PR8 and B/Lee virus HA and the expression of these idiotypes during primary and secondary antiviral responses of BALB/c mice. Extensive idiotypic cross-reactivity was demonstrated among monoclonal antibodies specific for distinct antigenic determinants on PR8 hemagglutinin (HA). The study of idiotypy of monoclonal antibodies against the same or overlapping antigenic determinants on B/Lee HA showed that these monoclonal antibodies may bear (a) a true individual idiotype not shared by other monoclonal antibodies, (b) idiotypes shared by few monoclonal antibodies, and (c) true cross-reactive idiotypes shared by all of these monoclonal antibodies. In contrast, no cross-reactive idiotypes were detectable among monoclonal antibodies to B/Lee HA and monoclonal antibodies to PR8 HA. Furthermore, we have shown that the anti-idiotype antibodies we used recognize determinants on monoclonal antibodies closely associated with antigenic binding sites. Finally, studies of the idiotypes expressed during primary and secondary antiviral HA responses of mice immunized with B/Lee virus revealed persistence of some idiotypes during both primary and secondary responses, whereas others were only expressed in the primary or secondary response.     

Antigen- and receptor-driven regulatory mechanisms. IV. Idiotype-bearing I-J+ suppressor T cell factors induce second-order suppressor T cells which express anti-idiotypic receptors

Administration of azobenzenearsonate (ABA)-coupled syngeneic spleen cells intravenously to A/J mice leads to the generation of suppressor T cells (Ts1) which exhibit specific binding to ABA-bovine serum albumin (BSA)-coated dishes. These Ts1 share idiotypic determinants with the major cross-reactive idiotype (CRI) of the anti-ABA antibodies of A/J mice, and also produce a soluble suppressor factor (TsF) bearing CRI and I-J subregion-coded determinants. Injection of this TsF into naive A/J mice elicits a second set of specific suppressor cells (Ts2) which are not lysed by anti-CRI antibody plus C, and which do not bind to ABA- BSA-coated dishes. However, in contrast with Ts1, these Ts2 do bind to plates bearing CRI+ anti-ABA immunoglobulin. Thus, Ts2 exhibit anti- idiotypic specificity. These data indicate that antigen elicits the production of a soluble T cell product bearing both variable portion of the Ig heavy chain (VH) and I-J subregion-coded determinants which serves to communicate between T cell subsets to establish an idiotype- anti-idiotype regulatory pathway.