謝榮華，舒衡平

黃芪對(duì)弓形蟲wx2b2a表位疫苗免疫小鼠后抗急性弓形蟲感染的免疫調(diào)節(jié)作用

謝榮華1，舒衡平2

目的 探討黃芪對(duì)弓形蟲wx2b2a表位疫苗免疫小鼠后抗急性弓形蟲感染的免疫保護(hù)作用。方法 將小鼠隨機(jī)分成pcDNA3-W2b2a組、pcDNA3-W2b2a +黃芪治療組、pcDNA3 +黃芪治療組、黃芪治療組、pcDNA3及生理鹽水對(duì)照組。將弓形蟲pcDNA3-W2b2a肌注免疫pcDNA3-W2b2a組、pcDNA3-W2b2a +黃芪治療組小鼠3次,末次免疫后4周，每組小鼠腹腔注射102個(gè)速殖子，感染后2 d起，pcDNA3-W2b2a +黃芪治療組、pcDNA3 +黃芪治療組及黃芪治療組小鼠給予黃芪75 mg/d灌胃治療，連續(xù)用藥7 d，用ELISA法測(cè)定免疫前、末次免疫后4周、感染后6 d小鼠血清IgG水平及免疫前、末次免疫后2周、感染后4、6、8 d小鼠IFN-γ、IL-18水平，并觀察弓形蟲感染后小鼠的生存時(shí)間。結(jié)果 pcDNA3-W2b2a免疫小鼠后血清IgG抗體水平均高于其它組(P<0.05)，末次免疫后4周,感染后第6 d pcDNA3-W2b2a、pcDNA3-W2b2a +黃芪治療組小鼠特異性IgG抗體水平無(wú)顯著性差異(P>0.05)；末次免疫后2周、感染后4 d、6 d、8 d pcDNA3-W2b2a+黃芪治療組小鼠血清IFN-γ水平分別高于其它組(P<0.05)；感染后各組小鼠血清IL-18水平持續(xù)上升,但感染后8 d，黃芪治療后小鼠血清IL-18水平顯著低于pcDNA3和生理鹽水對(duì)照組(P<0.05)；小鼠RH株速殖子感染后，pcDNA3-W2b2a +黃芪治療組小鼠存活時(shí)間明顯長(zhǎng)于pcDNA3-W2b2a組、pcDNA3 +黃芪治療組及黃芪治療組 (P<0.05)。結(jié)論 黃芪可增強(qiáng)弓形蟲wx2b2a表位疫苗免疫小鼠后抗急性弓形蟲感染的免疫調(diào)節(jié)作用。

弓形蟲；表位疫苗；黃芪；免疫調(diào)節(jié)

弓形蟲是一種機(jī)會(huì)性致病原蟲,引起人獸共患弓形蟲病, 免疫功能正常者感染弓形蟲絕大多數(shù)無(wú)癥狀，或者癥狀很輕,免疫功能抑制或缺陷者(如器官移植、惡性腫瘤及艾滋病人) 感染是導(dǎo)致其死亡的原因之一。目前常采用乙胺嘧啶和磺胺嘧啶聯(lián)合治療弓形蟲病,但其毒副作用發(fā)生率高,且不能根治[1]。表位疫苗是利用基因重組技術(shù)將一個(gè)或者多個(gè)抗原表位的編碼基因連接在一起，在宿主細(xì)胞內(nèi)利用真核啟動(dòng)子來(lái)持續(xù)高效表達(dá)多個(gè)多肽類免疫原，以提高疫苗的免疫效果，其安全性好，誘發(fā)的免疫應(yīng)答針對(duì)性強(qiáng)。本研究組在前期已構(gòu)建了弓形蟲新基因wx2b2a 表位疫苗質(zhì)粒,能夠誘導(dǎo)小鼠產(chǎn)生一定的抗弓形蟲感染保護(hù)性免疫[2]，但效果也不甚理想。人體感染弓形蟲后體內(nèi)免疫應(yīng)答失衡,而中藥黃芪具有雙向免疫應(yīng)答的調(diào)節(jié)作用[3]，本研究探討黃芪能否增強(qiáng)弓形蟲wx2b2a表位疫苗免疫小鼠后抗急性弓形蟲感染的免疫保護(hù)作用。

1 材料與方法

1.1 菌株與質(zhì)粒 弓形蟲RH株為本室傳代保種、pcDNA3、pcDNA3-W2b2a為本室構(gòu)建。

1.2 實(shí)驗(yàn)動(dòng)物 6周齡昆明小鼠，雌雄各半，18～20 g，購(gòu)自南華大學(xué)實(shí)驗(yàn)動(dòng)物中心。

1.3 主要試劑與藥物 黃芪顆粒，四川百利藥業(yè)有限責(zé)任公司，國(guó)藥準(zhǔn)字Z20003380；HRP標(biāo)記的羊抗小鼠IgG購(gòu)自北京博大泰克公司；小鼠IFN-γ ELISA試劑盒，深圳晶美生物有限公司；小鼠IL-18 ELISA檢測(cè)試劑盒, 日本MBL公司。

1.4 動(dòng)物分組及實(shí)驗(yàn)方案 昆明小鼠90只隨機(jī)分成6組，雌雄各半，分別為Ⅰ組：pcDNA3-W2b2a質(zhì)粒組、Ⅱ組:pcDNA3-W2b2a質(zhì)粒 +黃芪治療組、Ⅲ組: pcDNA3 +黃芪治療組、Ⅳ組：黃芪治療組、V組：pcDNA3、VI組：生理鹽水組。將pcDNA3-W2b2a質(zhì)粒100 μL分別免疫Ⅰ組和Ⅱ組小鼠，從鼠左后腿脛后肌下端，以平行于肌肉縱軸的方向進(jìn)針，共免疫3次，每次間隔2周；Ⅲ組、V組小鼠注射pcDNA3，Ⅳ組、VI組小鼠注射100 μL生理鹽水。末次免疫后4周，每鼠腹腔注射0.2 mL含102的弓形蟲RH株速殖子懸液。小鼠感染后2 d起, Ⅱ組、Ⅲ組及Ⅳ組小鼠給予黃芪灌胃治療, 給藥劑量每只鼠75 mg/d, 灌藥量0.4 mL/鼠, 其它組給予相同量的蒸餾水灌胃。灌胃前2 h禁水禁食。連續(xù)給藥7 d。

1.5 ELISA 法測(cè)定小鼠血清IgG 分別于免疫前、末次免疫后4周及感染后6 d小鼠斷尾法取血,分離收集小鼠血清。將每次所取血清樣品做1∶100稀釋，用間接ELISA法檢測(cè)免疫小鼠IgG抗體。

1.6 IFN-γ及IL-18 測(cè)定 免疫前、末次免疫后第2周及感染后4、6、8 d小鼠斷尾法取血，分離收集小鼠血清，ELISA 方法測(cè)定小鼠血清IFN-γ及IL-18濃度。按試劑盒說(shuō)明書操作。

2 結(jié) 果

2.1 小鼠血清特異性IgG抗體水平 pcDNA3-W2b2a免疫小鼠后血清IgG抗體水平均高于其它組(P<0.05)；末次免疫后4周,感染后6 d pcDNA3-W2b2a、pcDNA3-W2b2a +黃芪治療組小鼠特異性IgG抗體水平差異無(wú)統(tǒng)計(jì)學(xué)意義(P>0.05)(圖1)。

2.2 小鼠血清IFN-γ水平 末次免疫后2周、感染后4 d、6 d、8 d pcDNA3-W2b2a +黃芪治療組小鼠血清IFN-γ水平分別高于其它組(P<0.05)，而pcDNA3-W2b2a組、pcDNA3 +黃芪治療組、黃芪治療組小鼠血清IFN-γ水平分別高于pcDNA3組和生理鹽水對(duì)照組；感染后8 d各組小鼠血清IFN-γ水平較感染后6 d有所下降(P<0.05)(圖2)。

圖1 ELISA檢測(cè)小鼠血清特異性IgG抗體結(jié)果

圖2 ELISA檢測(cè)小鼠血清IFN-γ結(jié)果

2.3 小鼠血清IL-18水平 末次免疫后2周、感染后4 d、6 d、8 d各組小鼠血清IL-18水平呈上升趨勢(shì)；感染后8 d，黃芪治療后小鼠血清IL-18低于pcDNA3和生理鹽水對(duì)照組(P<0.05)(圖3)。

圖3 ELISA檢測(cè)小鼠血清IL-18結(jié)果

2.4 小鼠感染后的存活時(shí)間 末次免疫后4周，每鼠腹腔注射0.2 mL含102的弓形蟲RH株速殖子，實(shí)驗(yàn)小鼠均死亡。pcDNA3-W2b2a +黃芪治療組小鼠存活時(shí)間長(zhǎng)于其它組(P<0.05 )，pcDNA3-W2b2a、pcDNA3 +黃芪治療組及黃芪治療組小鼠存活時(shí)間長(zhǎng)于pcDNA3 和生理鹽組(P<0.05 )(表1)。

表1 速殖子攻擊后各組小鼠存活時(shí)間±s)

注：﹡與pcDNA3-W2b2a、pcDNA3+黃芪治療組、黃芪治療組比較，P<0.05；#與pcDNA3和生理鹽水組比較，P<0.05。

Note: ﹡As compared with the pcDNA3-W2b2a group, pcDNA3 +Astragalustreatment group ,Astragalustreatment group,P<0.05；# As compared with the pcDNA3 group, physiological sale group,P<0.05.

3 討 論

為增強(qiáng)弓形蟲疫苗的免疫效果，用其制成多表位疫苗，能誘導(dǎo)機(jī)體產(chǎn)生較高水平的保護(hù)性免疫應(yīng)答。魏慶寬[4]構(gòu)建pcDNA3-ROP2-p30-HSP70多基因核酸疫苗能誘發(fā)小鼠產(chǎn)生細(xì)胞免疫和體液免疫，實(shí)驗(yàn)組小鼠存活時(shí)間明顯延長(zhǎng)。范久波[5]等研究弓形蟲新基因wx、wx2表位疫苗能夠誘導(dǎo)小鼠產(chǎn)生抗弓形蟲感染保護(hù)性免疫。但單用弓形蟲疫苗免疫效果還不是十分理想，目前有學(xué)者研究中藥黃芪具有免疫調(diào)節(jié)作用及保護(hù)作用。黃芪可促進(jìn)中性粒細(xì)胞及巨噬細(xì)胞的吞噬功能和殺菌能力，提高免疫抑制小鼠的IL-2、TNF-a、IFN-γ的水平，并可促進(jìn)T淋巴細(xì)胞增殖[6]。邱宇安[7]等研究黃芪含藥血清對(duì)血管緊張素Ⅱ致內(nèi)皮細(xì)胞凋亡具有保護(hù)作用。黃芪注射液能提高重癥急性胰腺炎大鼠外周血GSH-PX、SOD 活性，降低ROS活性和MDA濃度[8]，對(duì)重癥急性胰腺炎有保護(hù)性作用。俞天虹[9]等研究大劑量黃芪組方的補(bǔ)陽(yáng)還五湯能顯著促進(jìn)腦缺血后室下區(qū)神經(jīng)干細(xì)胞增殖和神經(jīng)功能恢復(fù)。黃芪在寄生蟲病防治方面也具有一定的發(fā)展?jié)摿?，但其在直接抗寄生蟲病效果方面還有待進(jìn)一步研究，黃佩珺[10]等研究結(jié)果表明黃芪對(duì)低劑量速殖子感染的小鼠有保護(hù)作用，而對(duì)高劑量速殖子感染小鼠的保護(hù)作用不明顯。有研究發(fā)現(xiàn)，通過(guò)黃芪提取物(Astragalusmembranaceus extracts，AmE)治療弓形蟲感染小鼠，或接種疫苗的小鼠能夠顯著延長(zhǎng)存活時(shí)間，降低寄生蟲的數(shù)量，并增強(qiáng)Th1型細(xì)胞免疫反應(yīng)[11]。本研究結(jié)果顯示，pcDNA3-W2b2a可誘導(dǎo)小鼠產(chǎn)生高水平的IgG特異性抗體，但黃芪對(duì)此作用不明顯；pcDNA3-W2b2a可刺激小鼠產(chǎn)生高水平IFN-γ，感染后4、6 、8 d黃芪治療后小鼠血清IFN-γ水平分別高于非治療組，且pcDNA3-W2b2a +黃芪治療組小鼠血清IFN-γ水平最高，提示黃芪可以促進(jìn)pcDNA3-W2b2a刺激機(jī)體產(chǎn)生IFN-γ,增加宿主對(duì)速殖子的抗蟲能力，但感染后8 d小鼠血清IFN-γ水平較感染后6 d有所下降，提示小鼠可能處于代償性抗炎反應(yīng)(CARS)引起的免疫抑制階段,黃芪則通過(guò)誘生IFN-γ 改善宿主的免疫抑制狀態(tài)。張曉莉[12]等研究黃芪水煎劑及黃芪多糖可提高隱孢子蟲感染小鼠CD4、CD4/CD8 及IL-2、IL-4 和IFN-γ水平增強(qiáng)免疫功能促進(jìn)隱孢子蟲感染小鼠恢復(fù)。末次免疫后2周、感染后4、6、8 d各組小鼠血清IL-18水平呈持續(xù)上升；感染后8 d，pcDNA3-W2b2a +黃芪治療組小鼠血清IL-18顯著低于pcDNA3和生理鹽水對(duì)照組，提示黃芪可能抑制IL-18的表達(dá)。本實(shí)驗(yàn)提示黃芪對(duì)弓形蟲感染具有雙向調(diào)節(jié)作用，一方面,誘導(dǎo)IFN-γ早期生成，上調(diào)抗蟲免疫應(yīng)答；另一方面抑制IL-18生成，下調(diào)免疫應(yīng)答[13]。Mordue[14]等發(fā)現(xiàn)唯一可決定急性弓形蟲感染小鼠生存的因子是IL-18，中和IL-18的作用可延長(zhǎng)小鼠2 d的生存時(shí)間。小鼠感染試驗(yàn)表明，pcDNA3-W2b2a +黃芪治療組小鼠存活時(shí)間明顯長(zhǎng)于pcDNA3-W2b2a組和黃芪治療組。由此可見，黃芪可增強(qiáng)弓形蟲wx2b2a表位疫苗免疫小鼠后抗急性弓形蟲感染的免疫保護(hù)作用。

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Shu Heng-ping, Email: hengpingshu@xysm.net

Immunomodulatory effect ofAstragaluson immune mice from the epitope vaccineswx2b2a ofToxoplasmagondiiagainst acuteT.gondiiinfection

XIE Rong-hua1,SHU Heng-ping2

(1.HunanPolytechnicofEnvironmentandBiology,Hengyang421001,China; 2.XiangyaSchoolofMedicine,CentralSouthUniversity,Changsha410078,China)

We studied the immunomodulatory effect ofAstragaluson immune mice from the epitope vaccineswx2b2a ofToxoplasmagondii(T.gondii) against acuteT.gondiiinfection. The mice were randomly divided into Group pcDNA3-W2b2a, Group pcDNA3-W2b2a +Astragalustreatment, Group pcDNA3 +Astragalustreatment, GroupAstragalustreatment, and Group pcDNA3 and physiological saline. The mice in Group pcDNA3-W2b2a and Group pcDNA3-W2b2a +Astragalustreatment were intramuscularly immunized with pcDNA3-W2b2a ofT.gondiifor three times. Four weeks after the last immunization, the mice in each group were intraperitoneally injected with 102tachyzoites. Starting from 2 days after infection, the mice in Group pcDNA3-W2b2a +Astragalustreatment, Group pcDNA3 +Astragalustreatment, GroupAstragalustreatment were orally treated withAstragaluswith 75 mg/d for 7 days. ELISA method was taken to assay the serum levels of IgG before immunization, 4 weeks after the last immunization and 6 day post-infection (dpi) , and the levels of IFN-γ and IL-18 before immunization, 2 weeks after the last immunization and 4, 6 and 8 dpi, as well as to observe the survival time of the mice attacked byT.gondii. The serum levels of IgG of the mice which were immune with pcDNA3-W2b2a were higher than those in other groups (P<0.05). The levels of IgG antibodies in Group pcDNA3-W2b2a, Group pcDNA3-W2b2a +Astragalustreatment were almost the same in four weeks after the last immunization and 6 dpi (P>0.05). The levels of IFN-γ in Group pcDNA3-W2b2a +Astragalustreatment were higher than those in other groups (P<0.05) in 2 weeks after the last immunization and 4, 6, 8 dpi. The serum levels of IL-18 kept elevating after infection. But they are significantly lower than those in Group pcDNA3 and physiological saline after 8 days withAstragalustreatment (P<0.05). After infection with RH tachyzoites, the mice in Group pcDNA3-W2b2a +Astragalustreatment lived longer than those in Group pcDNA3-W2b2a, Group pcDNA3 +Astragalustreatment, and GroupAstragalustreatment (P<0.05).Astragaluscan strengthen the immunomodulatory effect on immune mice from the epitope vaccineswx2b2a ofT.gondiiagainst acuteT.gondiiinfection.

Toxoplasmagondii; epitope vaccine;Astragalus; immunomodulatory

10.3969/j.issn.1002-2694.2015.09.008

舒衡平，Email: hengpingshu@xysm.net

1.湖南環(huán)境生物職業(yè)技術(shù)學(xué)院，衡陽(yáng) 421001； 2.中南大學(xué)湘雅醫(yī)學(xué)院，長(zhǎng)沙 410078

Supported by grants from the Special Foundation of Forestry Science and Technology Innovation of Hunan (No. XLK201432), the Science and Technology Project of Hunan (No. 2014FJ3126), the Social Science Fund of Hengyang (No. 2014D100), the Science and Technology Development Project of Hengyang (No. 2014KJ27)

R382.5

A

1002-2694(2015)09-0822-04

2015-02-15；

2015-07-20

湖南省林業(yè)科技創(chuàng)新專項(xiàng)資金(No.XLK201432),湖南省科技計(jì)劃項(xiàng)目(No.2014FJ3126),衡陽(yáng)市社會(huì)科學(xué)基金(No.2014D100),衡陽(yáng)市科學(xué)技術(shù)發(fā)展計(jì)劃項(xiàng)目(No.2014KJ27)聯(lián)合資助