本文選題：日本血吸蟲 + pEGFP-Sj26GST��； 參考：《華中科技大學(xué)》2011年博士論文

【摘要】：日本血吸蟲病是一種廣泛流行并嚴(yán)重影響人畜健康的寄生蟲病。我國目前對(duì)血吸蟲病的防治重點(diǎn)在于滅螺及人畜同步化療。但是由于吡喹酮僅對(duì)侵入皮膚3 h的童蟲和成蟲有效,且目前在很多國家已經(jīng)出現(xiàn)對(duì)吡喹酮敏感性降低的報(bào)道。因此,安全有效的疫苗對(duì)血吸蟲病的防治具有重要的作用。 日本血吸蟲疫苗的研究經(jīng)歷了死疫苗、減毒活疫苗、基因工程疫苗和核酸疫苗等過程。其中核酸疫苗中最常用的是DNA疫苗,因其具有制備簡單、保存及運(yùn)輸方便和能誘導(dǎo)廣泛和持久的體液免疫和細(xì)胞免疫的優(yōu)點(diǎn)而備受研究者青睞。目前血吸蟲DNA疫苗在血吸蟲研究中占據(jù)著主導(dǎo)地位。但是截至目前為止,單價(jià)DNA疫苗的免疫保護(hù)效果并不令人滿意,WHO推薦的40%或以上的保護(hù)力仍然沒有達(dá)到。究其原因在于,血吸蟲作為一種多細(xì)胞生物,基因組非常復(fù)雜,且在與宿主長期進(jìn)化的過程中產(chǎn)生了多種免疫逃避機(jī)制。 新近證實(shí),調(diào)節(jié)性T細(xì)胞(Tregs)與寄生蟲逃避宿主免疫攻擊關(guān)系非常密切。在很多感染性疾病中,尤其是寄生蟲感染時(shí)Tregs的水平顯著升高,從而成為了寄生蟲一個(gè)逃生的“窗口”幫助其逃避宿主的免疫攻擊。在瘧疾感染的小鼠模型中,消耗Tregs有利于機(jī)體清除病原體并降低小鼠出現(xiàn)致死性感染的可能。因此探究血吸蟲感染中Tregs的變化,并針對(duì)該變化采取相應(yīng)的治療方案必將有助于血吸蟲疫苗的研究。 西咪替丁(CIM)作為H2受體阻滯劑在臨床上一直用于治療胃酸引起的胃腸功能紊亂并有確定的療效。除此之外,CIM在臨床上還廣泛的用于治療皰疹病毒,HIV病毒等感染性疾病引起的免疫功能下降,以及阻抑腫瘤的發(fā)生及生長。研究表明,其作用機(jī)制主要在于抑制抑制性T細(xì)胞的功能從而發(fā)揮免疫增強(qiáng)作用。有報(bào)道稱,直接將CIM和毗喹酮合用可顯著提高吡喹酮的殺蟲效果。同時(shí)也有很多將CIM作為一種免疫調(diào)節(jié)劑來增強(qiáng)疫苗的免疫原性的報(bào)道。 因此,本研究選用CIM和日本血吸蟲26KD谷胱甘肽S-轉(zhuǎn)移酶(Sj26)DNA疫苗合用,一方面觀察CIM和日本血吸蟲pEGFP-Sj26GST DNA疫苗聯(lián)合使用的免疫保護(hù)作用；另一方面觀察CIM和日本血吸蟲pEGFP-Sj26GST DNA疫苗聯(lián)合使用后宿主體內(nèi)Tregs的變化,并探討其免疫機(jī)制。 本課題分為以下三個(gè)部分： 一、西咪替丁對(duì)日本血吸蟲感染小鼠免疫應(yīng)答的影響研究 目的：探討不同劑量的C1M對(duì)血吸蟲感染小鼠免疫應(yīng)答的影響。 方法：32只6-8周齡的BALB/c雌性小鼠隨機(jī)分成3組,即50 mg/kg CIM組(CIM50)、25 mg/kg CIM組(CIM25)及感染對(duì)照組(Control)。CIM50組采用50mg/kg的CIM皮下注射三次,每次間隔兩周；CIM25組采用25 mg/kg的CIM皮下注射三次,每次間隔兩周,即第1、14、28天分別用藥一次；Control為單純感染組。第42天時(shí)各組小鼠攻擊感染日本血吸蟲尾蚴40條/鼠。感染后第6周殺鼠取脾淋巴細(xì)胞計(jì)數(shù),檢測(cè)脾淋巴細(xì)胞中CD4+CD25+FoxP3+Tregs百分比及IL-2、IFN-γ、IL-4和IL-5水平。 結(jié)果：和感染對(duì)照組相比,使用25mg/kg CIM組調(diào)節(jié)性T細(xì)胞的比例顯著降低(P0.01)且小鼠脾細(xì)胞培養(yǎng)上清中IL-2、IFN-γ的水平均較對(duì)照組升高(P0.05),而IL-4、IL-5的水平雖較對(duì)照組升高,但無顯著性差異(P0.05)。而和感染對(duì)照組相比,使用50mg/kg CIM組在調(diào)節(jié)性T細(xì)胞的比例及小鼠脾細(xì)胞培養(yǎng)上清中細(xì)胞因子的水平上均與對(duì)照組無顯著性差異(P0.05)。 結(jié)論：CIM可作為免疫調(diào)節(jié)劑,增強(qiáng)小鼠的免疫功能,從而提高小鼠對(duì)血吸蟲感染的免疫保護(hù)作用,但是該作用與劑量有關(guān)。 二、西咪替丁和日本血吸蟲pEGFP-Sj26GST DNA疫苗聯(lián)合使用的免疫保護(hù)作用研究 目的：觀察CIM對(duì)pEGFP-Sj26GST DNA疫苗免疫保護(hù)作用的影響。 方法：從本室保存的DH5a大腸桿菌體內(nèi)提取已構(gòu)建好的pEGFP-Sj26GST重組質(zhì)粒,進(jìn)行鑒定。50只BALB/c小鼠隨機(jī)分為5組,即感染對(duì)照組、CIM單用組、pEGFP-N3空載體對(duì)照組、pEGFP-Sj26GST DNA疫苗組及CIM和pEGFP-Sj26GST DNA疫苗合用組。其中疫苗組每只小鼠在第1、14及28天時(shí)分別經(jīng)股四頭肌注射100μgpEGFP-Sj26GST DNA疫苗或pEGFP-N3空載體,CIM組每只小鼠從首次免疫前兩天開始每天皮下注射25 mg/kg的CIM直至感染前。末次免疫后2周,各組小鼠均經(jīng)腹部皮膚感染日本血吸蟲尾蚴40條/鼠。感染后第6周殺鼠沖蟲,計(jì)數(shù)每只小鼠的蟲荷及肝內(nèi)蟲卵數(shù)。計(jì)算減蟲率和肝組織中每條雌蟲的減卵率。取出小鼠肝組織用福爾馬林固定,脫水、石蠟切片、HE染色,檢測(cè)各組小鼠肝組織內(nèi)蟲卵肉芽腫的變化。 結(jié)果：將大量提取出的pEGFP-Sj26GST重組質(zhì)粒及pEGFP-N3空載體質(zhì)粒在核酸分析儀上檢測(cè)發(fā)現(xiàn)濃度達(dá)到3mg/ml且純度較高。動(dòng)物保護(hù)性試驗(yàn)結(jié)果顯示,CIM和pEGFP-Sj26GST DNA疫苗聯(lián)合使用后減蟲率高達(dá)79%,顯著高于pEGFP-Sj26GST疫苗單獨(dú)使用組(27%)及其他各組。肝臟內(nèi)蟲卵計(jì)數(shù)發(fā)現(xiàn)其肝減卵率與pEGFP-Sj26GSTDNA疫苗單用組相比,聯(lián)合使用CIM和pEGFP-Sj26GST DNA疫苗也有顯著的降低(22.48%vs 68.35%)。小鼠肝組織HE染色顯示,和感染對(duì)照組相比,CIM和PEGFP-Sj26GST DNA疫苗合用組的蟲卵肉芽腫數(shù)目(16.25±2.87 vs 4.5±1.76)顯著減少。顯微鏡下可見,感染對(duì)照組的肝肉芽腫體積較大,而pEGFP-Sj26GST疫苗組及與CIM合用組的肝肉芽腫體積則明顯縮小。 結(jié)論：CIM可作為佐劑增強(qiáng)血吸蟲pEGFP-Sj26GST DNA疫苗的免疫保護(hù)效果。且CIM和疫苗合用可以顯著減少血吸蟲感染宿主肝內(nèi)蟲卵肉芽腫的數(shù)目。 三、西咪替丁增強(qiáng)日本血吸蟲pEGFP-Sj26GST DNA疫苗的免疫保護(hù)作用的機(jī)制研究 目的：觀察CIM對(duì)宿主體內(nèi)CD4+CD25+Tregs及相關(guān)細(xì)胞因子的影響,探討CIM增強(qiáng)pEGFP-Sj26GST DNA疫苗免疫保護(hù)作用的機(jī)制。 方法：80只雌性BALB/c小鼠隨機(jī)分成五組,即感染對(duì)照組、CIM組、pEGFP-N3空載體對(duì)照組、pEGFP-Sj26GST DNA疫苗組及CIM和pEGFP-Sj26GST DNA疫苗合用組。其中疫苗組每只小鼠在第1、14及28天時(shí)分別經(jīng)股四頭肌注射100μgpEGFP-Sj26GST DNA疫苗或pEGFP-N3空載體,即每只小鼠免疫三次,每次間隔2周。CIM組每只小鼠從首次免疫前兩天開始每天皮下注射25mg/kg的CIM直至感染前。各組取6只小鼠在末次免疫后一周剖殺,取眼眶血,靜置后離心取上清,檢測(cè)其血清中特異性GST抗體的含量。將剩余的50只小鼠在末次免疫后2周均經(jīng)腹部皮膚感染日本血吸蟲尾蚴40條/鼠。感染后第6周,剖殺小鼠,無菌取脾,制備單個(gè)脾細(xì)胞懸液,流式細(xì)胞儀檢測(cè)脾淋巴細(xì)胞中CD4+CD25+Tregs百分比。將無菌取出的脾細(xì)胞懸液在體外用絲裂原ConA刺激后培養(yǎng)48小時(shí),MTT法檢測(cè)在體外各組脾細(xì)胞對(duì)絲裂原的增值比率。體外培養(yǎng)72小時(shí)離心收集各組脾細(xì)胞上清,夾心ELISA法分別檢測(cè)脾細(xì)胞上清中Th1細(xì)胞因子γ-干擾素(IFN-γ)、白細(xì)胞介素-2(IL-2)和Th2細(xì)胞因子白細(xì)胞介素-4(IL-4)、白細(xì)胞介素-5(IL-5)和白細(xì)胞介素-10(IL-10)等細(xì)胞因子含量。 結(jié)果： 1)使用pEGFP-Sj26GST DNA疫苗后小鼠血清中特異性的抗Sj26GST抗體的水平明顯高于空載體組及單用CIM組(p0.05),且將pEGFP-Sj26GST DNA疫苗和CIM合用后小鼠血清中特異性的抗Sj26GST抗體的水平亦顯著高于單獨(dú)使用pEGFP-Sj26GST DNA疫苗組(p0.05)。 2)和感染對(duì)照組相比,單用pEGFP-Sj26GST DNA疫苗可以降低其脾淋巴細(xì)胞中CD4+CD25+Foxp3+T細(xì)胞的比例(p0.05),而將CIM和pEGFP-Sj26GST DNA疫苗合用則可以顯著降低其脾淋巴細(xì)胞中CD4+CD25+Foxp3+ T細(xì)胞的比例(p0.01)。 3)和感染對(duì)照組相比,單用CIM后脾淋巴細(xì)胞對(duì)ConA的增殖反應(yīng)明顯提高,而合用CIM及pEGFP-Sj26GST DNA疫苗后,脾淋巴細(xì)胞對(duì)ConA的增殖反應(yīng)則較單獨(dú)使用pEGFP-Sj26GST DNA疫苗組亦有顯著提高(p0.05)。 4)CIM和pEGFP-Sj26GST DNA疫苗合用組小鼠脾細(xì)胞培養(yǎng)上清中IL-12、IFN-γ的水平均較感染對(duì)照組高(P0.05),IL-10的含量較對(duì)照組降低(P0.05),而IL-4、IL-5的水平雖較對(duì)照組升高,但無顯著性差異(P0.05)。 結(jié)論： 1)提取出的pEGFP-Sj26GST DNA疫苗具有較強(qiáng)的免疫原性,將CIM與疫苗合用后能誘導(dǎo)機(jī)體產(chǎn)生更高水平的特異性抗體。 2)CIM和疫苗合用可以降低血吸蟲感染宿主脾淋巴細(xì)胞中CD4+CD25+ Tregs的百分比。 3)CIM可以輔助DNA疫苗在體外增強(qiáng)T淋巴細(xì)胞對(duì)絲裂原的反應(yīng),增強(qiáng)T細(xì)胞的功能。 4)CIM和疫苗合用可以增強(qiáng)機(jī)體的Thl型免疫反應(yīng),提高疫苗的保護(hù)性免疫效果。 課題的特色和創(chuàng)新點(diǎn)： 1)證明了CIM可作為一種免疫調(diào)節(jié)劑增強(qiáng)機(jī)體的Thl型免疫應(yīng)答。 2)首次將CIM與日本血吸蟲的DNA疫苗合用,并證實(shí)能有效提高抗日本血吸蟲感染的免疫保護(hù)作用。 3)證明CIM增強(qiáng)疫苗保護(hù)作用的機(jī)制與其下調(diào)宿主CD4+CD25+Tregs的水平有關(guān)。
[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 - 緯 , IL - 4 and IL - 5 levels in spleen lymphocytes were detected .


Results : Compared with the control group , the proportion of IL - 2 and IFN - 緯 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 鹵 2.87 vs 4.5 鹵 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 - 緯 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 .
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 薛德聯(lián);西咪替丁治療哮喘持續(xù)狀態(tài)23例療效觀察[J];臨床薈萃;1992年06期

2 楊新榮;甲氰咪呱臨床應(yīng)用新進(jìn)展[J];臨床薈萃;1993年13期

3 陳曉燕;西咪替丁與維生素k_1聯(lián)用治療喘憋性肺炎的臨床觀察[J];臨床薈萃;1996年08期

4 曾明輝;西咪替丁臨床應(yīng)用新進(jìn)展[J];臨床薈萃;1996年12期

5 聞勤生,張國治,孔憲濤,李石;甲氰脒胍對(duì)肝硬化患者白細(xì)胞介素2活性影響的研究[J];臨床肝膽病雜志;1993年03期

6 吳紹熙,郭寧如;替硝唑加甲氰脒胍治療尋常痤瘡的開放研究[J];臨床皮膚科雜志;1999年01期

7 酆之月,鄒淑萍;幾種老藥的臨床新用途[J];山東醫(yī)藥工業(yè);1998年01期

8 劉瑞梓,趙懷玉,賈繼東,許世穩(wěn);西米替丁對(duì)常見胃病患者體外淋巴細(xì)胞活性影響的研究[J];上海免疫學(xué)雜志;1990年06期

9 張莉,陳建平;日本血吸蟲疫苗候選分子的研究進(jìn)展[J];寄生蟲病與感染性疾病;2005年01期

10 李柳哲,石佑恩,寧長修,鄧偉文,韓家俊;日本血吸蟲混合DNA疫苗免疫效果觀察[J];中國血吸蟲病防治雜志;2001年04期

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