【作者】 蔡力汀；

【导师】 曾庆仁；

【作者基本信息】 中南大学 ， 病原生物学， 2006， 博士

【摘要】 第一篇日本血吸虫童虫虫源性细胞免疫小鼠诱生的保护性效果【目的】在前期研究中发现，用日本血吸虫(Sj)童虫的虫源性活细胞可诱导小鼠产生较明显的抗攻击感染保护性免疫。本研究目的是为了进一步确认其免疫效果；比较童虫细胞和成虫细胞，死细胞与活细胞诱导宿主所产生的保护性效果差异及其免疫应答特点；探索最佳的免疫次数。【方法】S．j尾蚴感染小鼠获得虫体。胰酶消化法制备虫源性细胞。台盼蓝染色检测细胞活率。活细胞经-20℃冻融后定为死细胞。PCR扩增鉴定血吸虫虫源性细胞的种属特异性并检测细胞的纯度。本研究设计了两个实验方案。在实验一中，53只昆明小鼠被分成4组，即对照组10只、18天童虫活细胞(LLC)免疫组18只、18天童虫死细胞(DLC)免疫组13只和42天成虫死细胞(DAC)免疫组11只。每两周分别用各免疫原对小鼠作皮下注射，共4次。于末次免疫后1周用30±1 S．j尾蚴对小鼠进行攻击感染。感染后第42天剖杀冲虫，观察结果。根据实验1结果，LLC是诱导小鼠产生最佳保护性效果的免疫原，因此，在实验二中选择了LLC对昆明小鼠作不同次数免疫，比较所诱导产生的保护性免疫，探索最佳免疫次数。28只小鼠随机分为4组：免疫1次组(V1)、2次组(V2)和3次组(V3)及对照组(Co)。于末次免疫后第2周用30±1 S．j尾蚴进行攻击感染。感染后第45天解剖小鼠冲虫。保护性效果的观察指标包括：计数虫荷及卵荷；测量虫体长度和肝组织内虫卵肉芽肿面积；对虫卵肉芽肿进行组织病理学观察。免疫学观察指标包括：ELISA检测免疫前后不同时点鼠血清中特异性总抗体(IgG+A+M)和IgG亚类抗体水平的动态变化；用SDS-PAGE及Western-blot对虫体与细胞的成分差异及免疫特性差异进行比较分析。【结果】Sj童虫源性细胞形态和大小不一，活率在80％以上。经PCR鉴定，细胞具有日本血吸虫种属特异性且无鼠源性宿主成分。免疫组与对照组的保护性指标比较显示：在实验一中，LLC，DLC和DAC组的减虫率分别为65.1％，54.5％和-2.4％，减卵率分别为76.5％，66.9％和-10.3％；在实验二中，用LLC免疫鼠2次和3次可分别获得64.5％和62.1％的减虫率及66.2％和66.1％的减卵率；此外，在实验2中，攻击后45天从V2和V3组小鼠体内所获得的虫体长度明显短于Co组的虫体；LLC免疫鼠肝表面虫卵结节明显少于DLC和DAC免疫鼠及对照鼠的；V3小鼠肝虫卵肉芽肿显著小于其他3组的。免疫学观察指标显示：LLC，DLC和DAC均能诱导小鼠产生抗童虫或成虫细胞抗原的IgG+A+M，抗体滴度与免疫次数呈正相关；LLC组小鼠血清中IgG2a／IgG1的比值明显高于DAC及对照组；SDS-PAGE与Western-blot的结果显示，18天童虫细胞蛋白区带与全虫蛋白有明显差别，18天童虫细胞制备物具有较强的抗原性，但与感染血清未见明显识别条带，童虫细胞中分子量约为38kDa组分仅被细胞抗原免疫血清所识别。【结论】本研究表明：来源于18天童虫的活细胞可诱导昆明鼠产生较理想的免疫保护效果；免疫2次或3次的保护性效果无显著差异；IgG亚类绝对水平增高及IgG2a／IgG1比值增高间接反映了Th1型免疫反应与保护性免疫呈正相关；用于免疫接种的细胞源于虫体实质成分，其中38kDa组分可能是一种优势抗原分子，但对其性质与功能有待于进一步研究。第二篇日本血吸虫童虫培养细胞免疫小鼠诱导的保护性效果【目的】前文研究证明，日本血吸虫(Sj)童虫源性细胞免疫小鼠可获得较为满意的抗攻击感染效果。基于本课题组在Sj细胞体外传代培养技术突破，本研究拟用Sj体外培养细胞为免疫原，观察其诱导昆明小鼠所产生的免疫保护性效果以及免疫应答类型，以探索研发此类疫苗的可能性。【方法】获取Sj尾蚴感染小鼠后不同发育期虫体，在无菌条件下制备虫源性细胞，用PRIM-1640-40Sj特定培基进行体外原代和传代培养。用台盼蓝染色鉴定细胞活率。PCR扩增鉴定传代培养细胞的种属特异性并检测细胞的纯度。通过两组实验观察传代培养细胞所诱导的保护性效果及免疫反应类型。在实验一中，3组小鼠每组8只，按1次／两周间隔经腹腔分别注射生理盐水(NSCo)，18天童虫培养细胞(JC-18)及12天童虫培养细胞(JC-12)，共进行3次免疫。末次接种后第2周用40±2尾／鼠的Sj尾蚴攻击感染。感染后第42天剖杀冲虫，观察保护性效果。在实验二中，根据实验1结果(发现JC-12的保护效果优于JC-18)，进一步比较了体外培养的12天童虫细胞(JC-12)和30天成虫细胞(AC-30)所诱导的免疫保护性差异。在本实验中，设计了两个接种途径：腹膜内注射(ip)和腹股沟皮下注射(sc)。JCip，JCse，ACsc和ACip 4个免疫组，每组10只，按1次／两周的接种间隔共免疫3次。10只对照组小鼠(Co)皮下注射D’hanks组。各组小鼠于末次免疫后第4周用30±1尾／鼠Sj尾蚴进行攻击感染，感染后第40天剖杀冲虫，观察了所诱导的保护性效果及免疫应答类型。用SDS-PAGE和Western-blot对成虫(AWA)，童虫(JWA)和童虫培养细胞(JCA)的蛋白组分进行了分析。用JC-12和AC-30免疫鼠及对照鼠的脾细胞和血清分别对预感染Sj尾蚴(50尾±5／鼠)后第5天的小鼠作腹腔注射1次(血清：0.2ml／鼠，脾细胞：4.5×10~6／鼠)，每组6只鼠。被动免疫后第35天剖杀冲虫，观察了所诱导的保护性效果及免疫反应类型。用免疫鼠脾细胞与Sj尾蚴于体外共同培养，观察24h，48h和72h的虫体死亡率。检测经相应抗原刺激后的免疫鼠和非免疫鼠脾细胞分泌的LI-4和IFN-γ水平。【结果】经体外传代培养的JC-12，JC-18和AC-30的细胞形态呈大小不等的圆形或椭圆形，可见成对细胞分裂相。与对照组比较，在实验一中，JC-12组和JC-18组小鼠分别获得了36.52％和26.92％的减虫率及47.07％和35.53％的减卵率。两类细胞均诱导宿主产生高水平抗18天童虫抗原的IgG抗体。在实验二中，JCip，JCsc小鼠分别获得了41.17％和56.87％的减虫率及54.13％和66.87％的减卵率；而ACsc和ACip的减虫率分别为-2.70％和-16.08％，肝卵减少率分别为-0.27％和-2.30％。两组JC免疫鼠的肝表面卵结节及肝卵成熟率明显降低。来自于JCsc小鼠体内的虫体长度明显短于来自于AC免疫鼠和对照组鼠。ELISA结果显示：JC免疫诱导产生相对较高水平的特异性IgE，IgG和IgG2a抗体，并在其经抗原刺激的脾细胞和血清中测出高水平的IFN-γ。AC-30免疫鼠尽管诱导产生了高水平的特异性抗体及IL-4，但并没有诱导产生明显的保护性效果。被动转移实验中，仅发现JC免疫鼠血清获得部分保护性效果。体外杀伤实验结果显示，JC和AC免疫鼠脾细胞均具有部分杀尾蚴作用，但以JC组杀伤率较高。SDS-PAGE分析结果显示，体外传代培养的Jc-12与JWA在成分上有较大差异。【结论】用体外培养的童虫细胞免疫昆明小鼠可诱导产生抗血吸虫攻击感染的保护性效果，但JC-18所诱生的免疫效果不如JC-12，提示可能越幼期的虫体细胞诱生的保护性效果越好。与前文比较，18天童虫虫源性细胞免疫所获得效果优于18天童虫培养细胞，其原因可能与实验批间差异或与细胞经培养后某些成分被丢失有关。实验1和实验2的免疫效果有明显差异，这可能与免疫源制备的稳定性或实验批间差异有关。在有效免疫中，显示出以Th1型为主的免疫应答机制，但结合体外试验结果可发现，高水平的保护性效果可能与Th1型和Th2型协同免疫所发挥的作用相关。成虫培养细胞不能诱生抗攻击感染作用的原因以及早期童虫细胞免疫接种的最佳方案还有待于进一步探索。第三篇日本血吸虫细胞型免疫原模拟表位的筛选及鉴定【目的】根据前述研究中证明S.j童虫细胞可在小鼠动物模型中诱导产生有效的抗感染免疫。本研究为了从分子水平了解此类免疫原的保护性机制，采用噬菌体展示技术对Sj童虫细胞抗原模拟肽表位进行了免疫筛选与鉴定。【方法】S.j12天童虫细胞经体外培养至4代后，用其活细胞免疫接种昆明小鼠，收集经保护性实验证明有效的攻击感染前免疫血清，用饱和硫酸胺沉淀法初纯化IgG抗体(抗SjJC-12抗体)后对噬菌体随机12肽库进行免疫筛选。经过4轮免疫淘洗，阳性克隆得到16250倍有效富集。从第4轮筛选结果中随机挑选20个克隆，经ELISA检测鉴定出15个阳性克隆；通过DNA测序和生物信息学分析出12个不同序列的阳性克隆。将此12个克隆分别免疫小鼠制备抗血清。用ELISA鉴定12个克隆的免疫原性以及是否为血吸虫细胞的免疫模拟表位。用8个含精氨酸残基的克隆和4个不含精氨酸残基的克隆分别对昆明鼠进行联合免疫(1次／10d，共3次)，于末次免疫后两周用24尾／鼠S.j尾蚴进行攻击感染，观察保护性效果。【结果】用抗SjJC-12IgG免疫筛选随机12肽库获得12个不同序列阳性克隆。经生物信息学分析与基因比对，仅发现与比目鱼的一种分子有38％的同源，提示这些多肽可能是JC-12抗原决定簇空间构象的模拟表位。ELISA检测显示：JC-12抗血清与12个克隆中11个发生反应，其中的clone-6不与JC-12免疫血清及JWA-12发生免疫反应，同时也不能诱导宿主产生抗体应答，提示该克隆所含序列与JC-12的抗原决定簇表位无关；12个克隆免疫鼠血清中有2个可诱导宿主产生强烈的抗体应答且可识别JC-12细胞抗原，其免疫血清也可识别JC-12细胞抗原及AWA-12可溶性抗原，提示这两个克隆为JC-12抗原模拟表位，具有进一步研究价值；两组多克隆联合免疫鼠血清均可识别JC-12抗原。含精氨酸残基和不含精氨酸残基的噬菌体克隆分别对小鼠作联合免疫，抗攻击感染实验正在观察中。【结论】用抗JC-12抗体对噬菌体随机12肽库进行免疫筛选得到12个不同序列的克隆(含精氨酸残基的8个)；经鉴定显示，有11个免疫宿主产生抗体应答，用JWA检出10个克隆免疫血清呈阳性反应，其中两个克隆具有进一步研究的价值。更多还原

【Abstract】 Section OneSchistosoma japonicum: Protective Immunity Induced bySchistosomulum-derived Cells in Mouse ModelWe have previously reported that immunization with intact live cellsresourced from Schistosomulum japonicum (S.j) was capable of inducingproduction of partial immunoprotection against challenge infection by S.jcercariae in Kunming strain murine model. In present work, two schemes ofimmune protective experiment were designed with Kunming strain of mice asexperimental model to further validate the immune protective effect inducedby this type of vaccine and explore the optimal immunization protocolincluding the number of inoculations and parasite stages from whichimmunogenic cells derived. In experiment 1 (EXPⅠ), a four timesvaccination program was formulated to compare the protective efficacyamong three antigens including LLC (live cells resourced from18-daypost-infection larval worms), DLC (dead cells derived from 18-daypost-infection larval worms) and DAC (dead cells derived from 42-daypost-infection adult worms). In experiment 2 (EXPⅡ), LLC was selected tovaccinate animals once (V1), twice (V2) and three times (V3) fordetermination of an optimal inoculation number as it was proved to be thebest immunogen in EXPⅠ. Challenge infection with 30±1 S.j cercariae wascarried out one week for EXPⅠand two weeks for EXPⅡafter the lastvaccination. At the day 42 for EXPⅠand day 45 for EXPⅡ, mice weresacrificed and perfused. Afterward, the worm burden and liver egg burdenwere counted, the development status of worm bodies observed and the sizesof egg granulomas in liver tissues measured. PCR showed that there was nocontaminate of host tissue ingredient in both crude cells. Synchronously,primary dynamic levels of specific total antibody classes and IgG subclasseswere observed by ELISAs. Ingredients and characters of effectiveimmunogen were initially identified by using SDS-PAGE and Western-blottechniques, respectively. By parallel comparison with control group, the results from experimental groups revealed that the reduction rate of wormburden and liver egg burden in EXPⅠwere 65.1％and 76.5％for LLC group,54.5％and 66.9％for DLC, -2.4％and -10.3％for DAC. In EXPⅡ, 64.5％and 62.1％worm burden reduction rate and 66.2％and 66.1％liver eggreduction rate were obtained from the mice immunized with LLC twice andthree times, respectively. Moreover, in mice of EXPⅡ, the development ofworms in the day 45 post infection was stunted. The best exterior of livers inEXPⅠwas from LLC group of mice and followed by DLC group, controlgroup and DAC group while the best outward appearance of livers in EXPⅡwas from mice vaccinated twice and three times followed by that vaccinatedonce and controls. The areas of egg granulomas in liver slices from LLCgroup of mice were significantly smaller than that from other three groups.Compared with DAC and control groups, the levels of specific antibodies andthe ratio of IgG2a/IgG1 were elevated in LLC group. Despite theelectrophoresed polypeptide bands of the hepatic stage juvenile worm cellson acrylamide resolving gel were far less than that of worm bodies, butpossessed potent immunogenicity and antigenicity. Furthermore, the bands of18-day larval warm cell preparations were notrecognized by infected micesera. Our results demonstrated that live cells from 18-day old hepatic stage ofS.j could induce production of the higher level of protective efficacy than thatfrom 42-day old adult worms in murine-S.j challenge model. There was nosignificant difference in protective effects and immune response typebetween vaccination for twice and three times animals. In addition, aTh1-biased immune response was indirectly reflected in high protectivegroups as evidenced by an elevated absolute level of IgG subclasses and theratio of IgG2a/IgG1.Section TwoSchistosoma Japonicum: Cultured Schistosomules Cells Induced aPartial Protection Against Challege Infection In Mouse ModelSchistosomiasis is believed to rank the second behind malaria in global importance. A vaccine would contribute to reduction of schistosomiasismorbidity and mortality through induced immuno-modulatory responsesleading to reduced worm burdens, reduced egg production(anti-fecundity)and reduced viability of eggs(anti-embryonation). A novel cell-type vaccinederived from schistosomulues was explored in our laboratory. To furtherdevelop this cell-type vaccine, this research observed through twoexperiments immune protective effects against challenge infection in Kunminstrain mice model induced by m vitro passage cultured 4th generations of livecells from 12-day and 18-day juvenile worms or 30-day adult worms.Immunogens above were abbreviated as JC-12, JC-18 and AC-30,respectively. After identification of without contamination of host constituentin sub-cultured cells with PCR, the mice were vaccinated intraperitoneallywith JC-12 and JC-18, respectively, in experiment I(EXPⅠ). Anotherexperiment (EXPⅡ) was designed according to the result of EXP I and newindexes. The mice were immunized with AC-30 and JC-12 without adjuvantby different injection routes, respectively. In both experiments, animals werechallenged with 40±2 or 30±1 Schistosomajaponicum (Sj) cercariae after thelast booster dose and sacrificed at day 40 post challenge infection for formerexperiment and day 42 post challenge infection for latter experiment.Parasitological and immunological results were reported here. PCR showedthat no contamination of host constituent was found in sub-cultured cells. Themice immunized with JC-12 recovered significant fewer worms and livereggs than those immunized with JC-18 (p＜0.05) or injected with normalsaline (NS) (p＜0.01) in EXPⅠand those immunized with AC-30 (p＜0.01) orinjected with D’hanks (p＜0.01) in EXPⅡ. Parasitological results obtainedfrom animals immunized with AC-30 were not significant different fromthose from controls. Photomicrographs (×100) of hepatic granulomas in EXPⅠshowed a mixed inflammatory reaction. The immunological studies haveshown: 1, High levels of IgG antibodies against soluble adult and juvenileworm antigens were observed in animals immunized with either culturedcells from juvenile or adult worms; 2, A Th1-type immune response tojuvenile antigen was suggested by ratio of IgG1/IgG2a＜1, coinciding withelevated level of interferon-γ, (IFN-γ) production in animals immunized withJC-12; in contrast, a dominant Th2-type immune response, with elevated IgG1 response to adult antigens and coinciding with detectable interleukin 4(IL-4) production, was generated in animals with AC-30 vaccination; 3,Anti-adult worm antigen (anti-AWA) IgE antibodies and anti-juvenile wormantigen (anti-JWA) IgE in sera from animals immunized with JC-12 or withAC-30 were significantly increased; 4, Passive transfer of sera from bothJC-12 and AC-30 immunized mice conferred partial protection to recipientmice; 5, Splenocytes from JC-12 and AC-30 immunized mice induced/nvitro killing effect on S. japonicum cercariae. Totally, identification ofantibody isotypes and cytokine correlates of partial immunity to subsequentinfection in our experiments suggests a great potential for live cultured cellvaccines as a novel approach against schistosomiasis. However, consistenthigh resistance was not demonstrated in EXP I with other batches of JC-12and JC-18, though a significant protection and IgG against 12-day old larvalworm crude soluble antigen was induced by JC-12 and JC-18, respectively,suggesting that the stability of immunogens remains to be further probed into.Section ThreeScreen and Characterization of Mimotopes of SchistosomulumJaponicum Cell-type ImmunogenIn previous study, our group proved that immunization of Kunmingstrain of murine model with sub-cultured live cells from 12-day oldSchistosoma japonicum(Sj)juvenile(JC-12) induced a partial protectionagainst the parasite challenge infection. In an attempt to further clarify theinduced immunoprotection mechanism on molecular level, isolate andidentify the peptides mimicking epitopes on JC-12 and test their protectivepotentiality against S.j, IgG were prepared by purification from polyclonalimmunosera against JC-12 to screen a 12-mer random phage peptide library.Four rounds of bioparming were performed and resulted in an effectiveenrichment. Of 20 randomly picked clones from the fourth elute, fifteenwerefound to be positive by evaluating the binding to anti-JC-12 sera by a reverse ELISA. Amino acid sequences were deduced by sequencing. Twelveclones were found to possess distinct sequences. Bioinformatics analysisamong 12 different clones was carried out with aid of several sorftwares. Bycomparing each peptide sequence with others, the highest 41.6% of identitywas found between Clone-5 and Clone-7. Independent phage clone and twogroups of mixed phage clone, according to whether containing Arginineresidue in peptide sequence or not, were served for immunization of mice,respectively. Induced immune response (antibody levels) and specialty of theresponse were evaluated by a direct ELSA. All, but one, animals developed aimmune response against these phagotops. Clone-10 could induce thestrongest antibody response, and the most intensely reacted with anti-JC-12sera. However, as to react with soluble 12-day old worm antigen,anti-Clone-7 immunosera revealed the strongest response profile. From theELISA results we concluded that Clone 10 and Clone 7 may be twointeresting mimotopes related to JC-12. Both combinatorial immunizationgroups were all induced to product IgG antibody, especially the group witharginine residue in sequences. The protective experiment is being under way,and the results will be reported soon afterward.更多还原