西咪替丁对日本血吸虫DNA疫苗免疫保护作用的影响及其机制研究

发布时间：2018-07-09 22:07

本文选题：日本血吸虫 + pEGFP-Sj26GST　；参考：《华中科技大学》2011年博士论文

【摘要】：日本血吸虫病是一种广泛流行并严重影响人畜健康的寄生虫病。我国目前对血吸虫病的防治重点在于灭螺及人畜同步化疗。但是由于吡喹酮仅对侵入皮肤3 h的童虫和成虫有效,且目前在很多国家已经出现对吡喹酮敏感性降低的报道。因此,安全有效的疫苗对血吸虫病的防治具有重要的作用。日本血吸虫疫苗的研究经历了死疫苗、减毒活疫苗、基因工程疫苗和核酸疫苗等过程。其中核酸疫苗中最常用的是DNA疫苗,因其具有制备简单、保存及运输方便和能诱导广泛和持久的体液免疫和细胞免疫的优点而备受研究者青睐。目前血吸虫DNA疫苗在血吸虫研究中占据着主导地位。但是截至目前为止,单价DNA疫苗的免疫保护效果并不令人满意,WHO推荐的40%或以上的保护力仍然没有达到。究其原因在于,血吸虫作为一种多细胞生物,基因组非常复杂,且在与宿主长期进化的过程中产生了多种免疫逃避机制。新近证实,调节性T细胞(Tregs)与寄生虫逃避宿主免疫攻击关系非常密切。在很多感染性疾病中,尤其是寄生虫感染时Tregs的水平显著升高,从而成为了寄生虫一个逃生的“窗口”帮助其逃避宿主的免疫攻击。在疟疾感染的小鼠模型中,消耗Tregs有利于机体清除病原体并降低小鼠出现致死性感染的可能。因此探究血吸虫感染中Tregs的变化,并针对该变化采取相应的治疗方案必将有助于血吸虫疫苗的研究。西咪替丁(CIM)作为H2受体阻滞剂在临床上一直用于治疗胃酸引起的胃肠功能紊乱并有确定的疗效。除此之外,CIM在临床上还广泛的用于治疗疱疹病毒,HIV病毒等感染性疾病引起的免疫功能下降,以及阻抑肿瘤的发生及生长。研究表明,其作用机制主要在于抑制抑制性T细胞的功能从而发挥免疫增强作用。有报道称,直接将CIM和毗喹酮合用可显著提高吡喹酮的杀虫效果。同时也有很多将CIM作为一种免疫调节剂来增强疫苗的免疫原性的报道。因此,本研究选用CIM和日本血吸虫26KD谷胱甘肽S-转移酶(Sj26)DNA疫苗合用,一方面观察CIM和日本血吸虫pEGFP-Sj26GST DNA疫苗联合使用的免疫保护作用；另一方面观察CIM和日本血吸虫pEGFP-Sj26GST DNA疫苗联合使用后宿主体内Tregs的变化,并探讨其免疫机制。本课题分为以下三个部分：一、西咪替丁对日本血吸虫感染小鼠免疫应答的影响研究目的：探讨不同剂量的C1M对血吸虫感染小鼠免疫应答的影响。方法：32只6-8周龄的BALB/c雌性小鼠随机分成3组,即50 mg/kg CIM组(CIM50)、25 mg/kg CIM组(CIM25)及感染对照组(Control)。CIM50组采用50mg/kg的CIM皮下注射三次,每次间隔两周；CIM25组采用25 mg/kg的CIM皮下注射三次,每次间隔两周,即第1、14、28天分别用药一次；Control为单纯感染组。第42天时各组小鼠攻击感染日本血吸虫尾蚴40条/鼠。感染后第6周杀鼠取脾淋巴细胞计数,检测脾淋巴细胞中CD4+CD25+FoxP3+Tregs百分比及IL-2、IFN-γ、IL-4和IL-5水平。结果：和感染对照组相比,使用25mg/kg CIM组调节性T细胞的比例显著降低(P0.01)且小鼠脾细胞培养上清中IL-2、IFN-γ的水平均较对照组升高(P0.05),而IL-4、IL-5的水平虽较对照组升高,但无显著性差异(P0.05)。而和感染对照组相比,使用50mg/kg CIM组在调节性T细胞的比例及小鼠脾细胞培养上清中细胞因子的水平上均与对照组无显著性差异(P0.05)。结论：CIM可作为免疫调节剂,增强小鼠的免疫功能,从而提高小鼠对血吸虫感染的免疫保护作用,但是该作用与剂量有关。二、西咪替丁和日本血吸虫pEGFP-Sj26GST DNA疫苗联合使用的免疫保护作用研究目的：观察CIM对pEGFP-Sj26GST DNA疫苗免疫保护作用的影响。方法：从本室保存的DH5a大肠杆菌体内提取已构建好的pEGFP-Sj26GST重组质粒,进行鉴定。50只BALB/c小鼠随机分为5组,即感染对照组、CIM单用组、pEGFP-N3空载体对照组、pEGFP-Sj26GST DNA疫苗组及CIM和pEGFP-Sj26GST DNA疫苗合用组。其中疫苗组每只小鼠在第1、14及28天时分别经股四头肌注射100μgpEGFP-Sj26GST DNA疫苗或pEGFP-N3空载体,CIM组每只小鼠从首次免疫前两天开始每天皮下注射25 mg/kg的CIM直至感染前。末次免疫后2周,各组小鼠均经腹部皮肤感染日本血吸虫尾蚴40条/鼠。感染后第6周杀鼠冲虫,计数每只小鼠的虫荷及肝内虫卵数。计算减虫率和肝组织中每条雌虫的减卵率。取出小鼠肝组织用福尔马林固定,脱水、石蜡切片、HE染色,检测各组小鼠肝组织内虫卵肉芽肿的变化。结果：将大量提取出的pEGFP-Sj26GST重组质粒及pEGFP-N3空载体质粒在核酸分析仪上检测发现浓度达到3mg/ml且纯度较高。动物保护性试验结果显示,CIM和pEGFP-Sj26GST DNA疫苗联合使用后减虫率高达79%,显著高于pEGFP-Sj26GST疫苗单独使用组(27%)及其他各组。肝脏内虫卵计数发现其肝减卵率与pEGFP-Sj26GSTDNA疫苗单用组相比,联合使用CIM和pEGFP-Sj26GST DNA疫苗也有显著的降低(22.48%vs 68.35%)。小鼠肝组织HE染色显示,和感染对照组相比,CIM和PEGFP-Sj26GST DNA疫苗合用组的虫卵肉芽肿数目(16.25±2.87 vs 4.5±1.76)显著减少。显微镜下可见,感染对照组的肝肉芽肿体积较大,而pEGFP-Sj26GST疫苗组及与CIM合用组的肝肉芽肿体积则明显缩小。结论：CIM可作为佐剂增强血吸虫pEGFP-Sj26GST DNA疫苗的免疫保护效果。且CIM和疫苗合用可以显著减少血吸虫感染宿主肝内虫卵肉芽肿的数目。三、西咪替丁增强日本血吸虫pEGFP-Sj26GST DNA疫苗的免疫保护作用的机制研究目的：观察CIM对宿主体内CD4+CD25+Tregs及相关细胞因子的影响,探讨CIM增强pEGFP-Sj26GST DNA疫苗免疫保护作用的机制。方法：80只雌性BALB/c小鼠随机分成五组,即感染对照组、CIM组、pEGFP-N3空载体对照组、pEGFP-Sj26GST DNA疫苗组及CIM和pEGFP-Sj26GST DNA疫苗合用组。其中疫苗组每只小鼠在第1、14及28天时分别经股四头肌注射100μgpEGFP-Sj26GST DNA疫苗或pEGFP-N3空载体,即每只小鼠免疫三次,每次间隔2周。CIM组每只小鼠从首次免疫前两天开始每天皮下注射25mg/kg的CIM直至感染前。各组取6只小鼠在末次免疫后一周剖杀,取眼眶血,静置后离心取上清,检测其血清中特异性GST抗体的含量。将剩余的50只小鼠在末次免疫后2周均经腹部皮肤感染日本血吸虫尾蚴40条/鼠。感染后第6周,剖杀小鼠,无菌取脾,制备单个脾细胞悬液,流式细胞仪检测脾淋巴细胞中CD4+CD25+Tregs百分比。将无菌取出的脾细胞悬液在体外用丝裂原ConA刺激后培养48小时,MTT法检测在体外各组脾细胞对丝裂原的增值比率。体外培养72小时离心收集各组脾细胞上清,夹心ELISA法分别检测脾细胞上清中Th1细胞因子γ-干扰素(IFN-γ)、白细胞介素-2(IL-2)和Th2细胞因子白细胞介素-4(IL-4)、白细胞介素-5(IL-5)和白细胞介素-10(IL-10)等细胞因子含量。结果： 1)使用pEGFP-Sj26GST DNA疫苗后小鼠血清中特异性的抗Sj26GST抗体的水平明显高于空载体组及单用CIM组(p0.05),且将pEGFP-Sj26GST DNA疫苗和CIM合用后小鼠血清中特异性的抗Sj26GST抗体的水平亦显著高于单独使用pEGFP-Sj26GST DNA疫苗组(p0.05)。 2)和感染对照组相比,单用pEGFP-Sj26GST DNA疫苗可以降低其脾淋巴细胞中CD4+CD25+Foxp3+T细胞的比例(p0.05),而将CIM和pEGFP-Sj26GST DNA疫苗合用则可以显著降低其脾淋巴细胞中CD4+CD25+Foxp3+ T细胞的比例(p0.01)。 3)和感染对照组相比,单用CIM后脾淋巴细胞对ConA的增殖反应明显提高,而合用CIM及pEGFP-Sj26GST DNA疫苗后,脾淋巴细胞对ConA的增殖反应则较单独使用pEGFP-Sj26GST DNA疫苗组亦有显著提高(p0.05)。 4)CIM和pEGFP-Sj26GST DNA疫苗合用组小鼠脾细胞培养上清中IL-12、IFN-γ的水平均较感染对照组高(P0.05),IL-10的含量较对照组降低(P0.05),而IL-4、IL-5的水平虽较对照组升高,但无显著性差异(P0.05)。结论： 1)提取出的pEGFP-Sj26GST DNA疫苗具有较强的免疫原性,将CIM与疫苗合用后能诱导机体产生更高水平的特异性抗体。 2)CIM和疫苗合用可以降低血吸虫感染宿主脾淋巴细胞中CD4+CD25+ Tregs的百分比。 3)CIM可以辅助DNA疫苗在体外增强T淋巴细胞对丝裂原的反应,增强T细胞的功能。 4)CIM和疫苗合用可以增强机体的Thl型免疫反应,提高疫苗的保护性免疫效果。课题的特色和创新点： 1)证明了CIM可作为一种免疫调节剂增强机体的Thl型免疫应答。 2)首次将CIM与日本血吸虫的DNA疫苗合用,并证实能有效提高抗日本血吸虫感染的免疫保护作用。 3)证明CIM增强疫苗保护作用的机制与其下调宿主CD4+CD25+Tregs的水平有关。
[Abstract]:Schistosoma japonicum is a kind of parasitic disease which is widely epidemic and seriously affects the health of human and livestock .

DNA vaccine is most commonly used in the research of Schistosoma japonicum because it has the advantages of simple preparation , easy preservation and transportation , and can induce wide and lasting humoral immunity and cellular immunity .

In many infectious diseases , especially parasite infection , the level of Terence is significantly increased , thus becoming a " window " to escape the host . In the model of mice infected with malaria , the consumption of Tofu is beneficial to the organism to remove pathogens and reduce the possibility of lethal infection in mice . Therefore , it is possible to study the change of Terence in the infection of Schistosoma japonicum and to take corresponding treatment protocols for this change , which will contribute to the research of schistosomiasis vaccine .

Simitedin ( CIM ) , as a H2 receptor blocker , has been used in clinic to treat gastrointestinal disorders induced by gastric acid . In addition , CIM has been widely used in the treatment of infectious diseases such as herpes virus , HIV virus , and its growth .

Therefore , the use of the DNA vaccine of the 26KD glutathione S - transferase ( Sj26 ) of CIM and Schistosoma japonicum was selected in this study . On the one hand , the protective effects of CIM and Sj26GST DNA vaccine of Schistosoma japonicum were observed .
On the other hand , we observed the changes of T lymphocyte in host body after combined use of DNA vaccine of CIM and Sj26GST , and discussed its immune mechanism .

The subject is divided into the following three parts :

Study on the Effect of Imitedin on Immune Response of Mice Infected with Schistosoma japonicum

Objective : To study the effect of different doses of C1M on immune responses of mice infected with Schistosoma japonicum .

Methods : Thirty - two BALB / c female mice 6 - 8 weeks old were randomly divided into 3 groups , 50 mg / kg CIM group ( CIM50 ) , 25 mg / kg CIM group ( CIM25 ) and control group ( Control ) .
The CIM25 group was subcutaneously injected with CIM at 25 mg / kg for two weeks , i.e . 1 , 14 and 28 days , respectively ;
Control was a simple infection group . At the 42 th day , the mice were infected with 40 / mouse cercariae of Schistosoma japonicum . After infection , the mice were killed at 6 weeks after infection . The percentage of CD4 + CD25 + FoxP3 + Tlymphocyte and IL - 2 , IFN - 纬 , IL - 4 and IL - 5 levels in spleen lymphocytes were detected .

Results : Compared with the control group , the proportion of IL - 2 and IFN - 纬 was significantly lower than that in the control group ( P0.05 ) , but the level of IL - 4 and IL - 5 was higher than that in the control group ( P0.05 ) .

Conclusion : CIM can be used as an immune regulator to enhance the immune function of mice , so as to improve the immune protective effect of mice against Schistosoma japonicum infection , but the effect is related to the dose .

Study on the Protective Effects of Imitedin and Sj26GST DNA Vaccine of Schistosoma japonicum

Objective : To observe the effect of CIM on the immune protective effect of DNA vaccine ( Sj26GST ) DNA vaccine .

Methods : Twenty - five BALB / c mice were randomly divided into five groups : the infected control group , the CIM single - use group , the eukaryotic expression vector control group , and the fusion group of the CIM - Sj26GST DNA vaccine .

Results : The results showed that the recombinant plasmid and the eukaryotic expression vector of the eukaryotic expression vector , which were extracted from the eukaryotic expression vector , were 3 mg / ml and 3 mg / ml , respectively . The results of the protective tests showed that the reduction rate was 79 % higher than that in the single - use group ( 27 % ) and the other groups . The results showed that the DNA vaccine of CIM and the Sj26GST DNA vaccine was significantly lower ( 22.48 % vs 68.35 % ) than that in the single - use group . Compared with the control group , the number of eggs granuloma ( 16.25 卤 2.87 vs 4.5 卤 1.76 ) in the combined group of CIM and PEGFP - Sj26GST DNA vaccine was significantly reduced compared with the control group .

Conclusion : CIM can be used as adjuvant to enhance the immune protective effect of Sj26GST DNA vaccine .

Study on the mechanism of enhancing the immune protective effect of simitedin on the DNA vaccine of Schistosoma japonicum

Objective : To observe the effect of CIM on the CD4 + CD25 + T cell and related cytokines in the host , and to explore the mechanism of CIM to enhance the immune protection function of DNA vaccine .

Methods : 80 female BALB / c mice were randomly divided into five groups : the control group , the CIM group , the empty vector control group , the expression CIM group and the eukaryotic expression vector of Sj26GST DNA vaccine .

Results :

1 ) The level of anti Sj26GST antibody was significantly higher in mouse serum than empty vector group and CIM group ( p < 0.05 ) .

2 ) Compared with the infected control group , the ratio of CD4 + CD25 + Foxp3 + T cells in the spleen lymphocytes of spleen lymphocytes could be decreased by the single use of the recombinant plasmid ( p0.01 ) , but the ratio of CD4 + CD25 + Foxp3 + T cells in the spleen lymphocytes could be significantly reduced by the combination of the CIM ( p0.01 ) and the Sj26GST DNA vaccine .

3 ) Compared with the control group , the proliferative response of spleen lymphocytes to Con A was increased significantly after CIM , while the proliferation response of spleen lymphocytes to Con A was significantly increased compared with the control group ( p < 0.05 ) .

4 ) The levels of IL - 12 and IFN - 纬 were higher than those in the control group ( P0.05 ) , but the levels of IL - 4 and IL - 5 were higher than those in the control group ( P0.05 ) .

Conclusion :

( 1 ) The DNA vaccine has stronger immunogenicity , and can induce the organism to generate higher specific antibody after the CIM is combined with the vaccine .

2 ) The combination of CIM and vaccine can reduce the percentage of CD4 + CD25 + T lymphocyte in the host spleen lymphocytes infected with Schistosoma japonicum .

3 ) CIM can enhance T lymphocyte response to mitogen in vitro and enhance T cell function in vitro .

4 ) the combination of CIM and vaccine can enhance the Thl type immune response of the organism and improve the protective immunity effect of the vaccine .

Characteristics and innovation points of the subject :

1 ) It is proved that CIM can be used as an immune regulator to enhance the Thl - type immune response of the organism .

2 ) CIM is combined with DNA vaccine of Schistosoma japonicum for the first time , and the protective effect of anti - Japanese infection can be effectively improved .

3 ) It is shown that the mechanism of CIM - enhanced vaccine protection is related to its downregulation of the level of CD4 + CD25 + TVB .
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392

【参考文献】