黃芪多糖、香菇多糖增強(qiáng)弓形蟲wx2b4a表位疫苗免疫小鼠的保護(hù)作用

謝榮華，范久波，舒衡平

黃芪多糖、香菇多糖增強(qiáng)弓形蟲wx2b4a表位疫苗免疫小鼠的保護(hù)作用

謝榮華1，范久波2，舒衡平3

目的 探討黃芪多糖(Astragalan)、香菇多糖(Lentinan)增強(qiáng)弓形蟲wx2b4a表位疫苗刺激機(jī)體產(chǎn)生免疫應(yīng)答和保護(hù)免疫的效果。方法 將黃芪多糖、香菇多糖分別與弓形蟲wx2b4a表位疫苗混合肌注免疫小鼠，ELISA法檢測(cè)小鼠血清中IgG抗體水平，pcDNA3-W2b4a刺激下培養(yǎng)各組小鼠脾臟淋巴細(xì)胞，ELISA法檢測(cè)IL-2、IFN-γ分泌水平，CCK-8法檢測(cè)免疫鼠脾細(xì)胞增殖活性，并觀察其受到弓形蟲攻擊感染后的生存時(shí)間。結(jié)果 各免疫組小鼠血清IgG抗體水平顯著高于對(duì)照組(P<0.05)，且pcDNA3-W2b4a+黃芪多糖組要高于pcDNA3-W2b4a+香菇多糖組(P<0.05)；pcDNA3-W2b4a+黃芪多糖組、pcDNA3-W2b4a+香菇多糖組小鼠脾細(xì)胞IL-2、IFN-γ水平高于pcDNA3-W2b4a組(P<0.05)；各免疫組T細(xì)胞增殖活性與對(duì)照組比較明顯增強(qiáng)(P<0.05)，且pcDNA3-W2b4a+香菇多糖組要高于pcDNA3-W2b4a+黃芪多糖組；小鼠攻擊試驗(yàn)表明，pcDNA3-W2b4a+黃芪多糖組、pcDNA3-W2b4a+香菇多糖組小鼠存活時(shí)間明顯長(zhǎng)于對(duì)照組和pcDNA3-W2b4a組(P<0.05)。結(jié)論 黃芪多糖、香菇多糖可增強(qiáng)弓形蟲wx2b4a表位疫苗產(chǎn)生免疫應(yīng)答，并顯示較好的免疫保護(hù)效應(yīng)。

弓形蟲；黃芪多糖；香菇多糖；表位疫苗；保護(hù)力

弓形蟲寄生引起的感染非常普遍，估計(jì)全世界約有1/3 的人感染,多數(shù)感染者無癥狀或癥狀輕微。但嚴(yán)重免疫缺陷的病人，如艾滋病人等，發(fā)生感染，后果就很嚴(yán)重。孕婦感染可致胎兒畸形、流產(chǎn)及死胎等。目前尚無理想的藥物及疫苗防治弓形蟲病,研究弓形蟲疫苗預(yù)防弓形蟲病是目前研究的熱點(diǎn)。本研究組在前期已構(gòu)建了弓形蟲新基因wx2b4a 表位疫苗質(zhì)粒[1],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]，但效果也不理想,非常需要用免疫佐劑來增強(qiáng)弓形蟲疫苗免疫原性,產(chǎn)生更有效和持久的保護(hù)性免疫，生物活性多糖是一類從植物、動(dòng)物、微生物等生物體中提取的, 具有多種生物活性的碳水化合物。研究表明, 許多生物活性多糖具有免疫調(diào)節(jié)作用,且與其它免疫增強(qiáng)劑相比, 多糖具有毒副作用小、多效性、雙向調(diào)節(jié)性及來源廣等特點(diǎn)[3],成為當(dāng)今研制新型免疫增強(qiáng)劑的發(fā)展方向之一。本研究探討黃芪多糖、香菇多糖能否增強(qiáng)弓形蟲wx2b4a表位疫苗刺激小鼠機(jī)體產(chǎn)生免疫應(yīng)答和保護(hù)免疫的效果。

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-W2b4a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，購自南華大學(xué)實(shí)驗(yàn)動(dòng)物中心。

1.3 主要試劑與藥物 HRP標(biāo)記的羊抗小鼠IgG購自北京博大泰克公司；DMEM-H 基礎(chǔ)培養(yǎng)基、青霉素鏈霉素和胎牛血清購自Gibco公司；IL-2、IFN-γ小鼠細(xì)胞因子檢測(cè)ELISA試劑盒購自Excell公司；細(xì)胞增殖-毒性檢測(cè)試劑盒(cck-8)購自上海美季生物技術(shù)有限公司；香菇多糖注射液(金陵藥業(yè)股份有限公司福州梅峰制藥廠，國藥準(zhǔn)字：H20030131)、注射用黃芪多糖(天津賽諾制藥有限公司，國藥準(zhǔn)字：220040086)

1.4 實(shí)驗(yàn)動(dòng)物分組及動(dòng)物免疫 昆明小鼠115只，隨機(jī)分為5組，雌雄各半，分別為Ⅰ組：pcDNA3-W2b4a質(zhì)粒100 μg，Ⅱ組:pcDNA3-W2b4a質(zhì)粒100 μg +黃芪多糖(50 mg/kg/d)，Ⅲ組：pcDNA3-W2b4a質(zhì)粒100 μg+香菇多糖(2.5 mg/kg/d)，Ⅳ組:pcDNA3 100 μg, V組:生理鹽水100 μL，將定量的黃芪多糖、香菇多糖分別與pcDNA3-W2b4a 質(zhì)粒混合, 加PBS溶液至100 μL，從小鼠左后腿肌肉注射,每次100 μL,共免疫3次,每次間隔2周, 生理鹽水組和pcDNA3組作對(duì)照。

1.5 ELISA 法測(cè)定小鼠血清IgG 分別于免疫前及末次免疫后第2、4 周, 斷尾法取血，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抗體水平。按試劑盒說明書操作。

1.6 小鼠脾細(xì)胞懸液制備 小鼠末次免疫后2周,在攻擊實(shí)驗(yàn)前,每組小鼠隨機(jī)處死8只，無菌取脾臟置于200目銅網(wǎng),研磨后用1640培養(yǎng)液沖洗,收集脾細(xì)胞,破紅細(xì)胞后,加入含有10%小牛血清的1640培養(yǎng)液吹打混勻,將脾細(xì)胞數(shù)調(diào)整至5×106/mL,用于細(xì)胞因子釋放檢測(cè)和脾細(xì)胞增殖實(shí)驗(yàn)。

1.7 細(xì)胞因子釋放檢測(cè) 加入含100 mL/L 胎牛血清和10 mL/L雙抗的DMEM-H培養(yǎng)基將每組脾細(xì)胞數(shù)調(diào)整為1×105/mL，每組分別取雙份100 μL加入24孔板中，加入10 μL pcDNA3-W2b4a質(zhì)粒，在飽和濕度為50 mL/L CO2培養(yǎng)箱中37 ℃培養(yǎng)72 h后收集培養(yǎng)液上清，采用小鼠細(xì)胞因子ELISA檢測(cè)試劑盒，嚴(yán)格按照說明書要求操作，檢測(cè)IL-2、IFN-γ的水平。

1.8 免疫鼠脾細(xì)胞增殖實(shí)驗(yàn)(CCK-8法) 將各組脾細(xì)胞懸液100 μL (2×104/mL)分別加入96 孔細(xì)胞培養(yǎng)板(每組設(shè)2個(gè)復(fù)孔和對(duì)照孔),各組分別加入ConA(終濃度為10 μg/mL), 對(duì)照孔不加入ConA,37 ℃,5%CO2培養(yǎng)48 h。于培養(yǎng)結(jié)束前4 h 各孔加入10 μL CCK-8試劑。培養(yǎng)結(jié)束后,吸去100 μL上清,測(cè)定時(shí)每孔加入二甲亞砜(DMSO)100 μL,振蕩溶解,用酶標(biāo)儀測(cè)定吸光度(A450)

1.9 攻擊感染實(shí)驗(yàn) 末次免疫后第4周，實(shí)驗(yàn)組和對(duì)照組小鼠經(jīng)腹腔注射弓形蟲RH株速殖子(500個(gè)/鼠)，觀察記錄小鼠的感染情況和死亡時(shí)間。

2 結(jié) 果

2.1 小鼠血清特異性IgG抗體水平 各免疫組小鼠特異性IgG抗體均高于對(duì)照組(P<0.05)，且均于末次免疫后第4周達(dá)到最高值；pcDNA3-W2b4a+黃芪多糖、pcDNA3-W2b4a+香菇多糖及pcDNA3-W2b4a組IgG抗體水平較對(duì)照組同期要高(P<0.05)，且末次免疫后第4周pcDNA3-W2b4a+黃芪多糖IgG抗體水平最高(A值為0.397±0.041)，而生理鹽水組IgG抗體水平最低(A值為0.121±0.057)(圖1)。

圖1 ELISA 法檢測(cè)小鼠血清IgG 的結(jié)果

2.2 細(xì)胞因子釋放檢測(cè) 以pcDNA3-W2b4a質(zhì)粒刺激免疫小鼠脾細(xì)胞檢測(cè)IL-2 、IFN-γ細(xì)胞因子的分泌(圖2)。各免疫組小鼠脾細(xì)胞IL-2 、IFN-γ水平高于對(duì)照組(P<0.05)；pcDNA3-W2b4a+黃芪多糖組、pcDNA3-W2b4a+香菇多糖組小鼠脾細(xì)胞IL-2 、IFN-γ水平高于pcDNA3-W2b4a組(P<0.05)(圖2)。

圖2 ELISA 法檢測(cè)小鼠脾細(xì)胞IL-2、IFN-γ 的結(jié)果

Fig.2 Detection of IL-2 and IFN-γ in splenoctes of mice by ELISA

2.3 免疫鼠脾細(xì)胞增殖實(shí)驗(yàn)(CCK-8法) 末次免疫后2周,ConA 刺激小鼠脾T細(xì)胞,免疫組小鼠脾細(xì)胞均發(fā)生增殖。各組脾細(xì)胞增殖活性如下(加ConA孔的A值與不加ConA孔的A值的比值):pcDNA3-W2b4a:1.23，pcDNA3-W2b4a+黃芪多糖組:1.98, pcDNA3-W2b4a+香菇多糖組：2.23,各免疫組脾淋巴細(xì)胞增殖能力與兩對(duì)照組比較明顯增強(qiáng)(P<0.05)。

2.4 抗攻擊感染的免疫保護(hù)作用 末次免疫后第四周每只小鼠經(jīng)腹腔注射500個(gè)弓形蟲速殖子，實(shí)驗(yàn)小鼠均未能耐受弓形蟲攻擊感染。疫苗組小鼠存活時(shí)間明顯長(zhǎng)于pcDNA3組及生理鹽水組(P<0.05),且pcDNA3-W2b4a+黃芪多糖組、pcDNA3-W2b4a+香菇多糖組小鼠存活時(shí)間長(zhǎng)于pcDNA3-W2b4a組(P<0.05 )(表1)。

組 別Groups小鼠數(shù)量/只No.ofmice不同時(shí)間小鼠存活的數(shù)量/hNo.ofmiceindifferenttime/h144168192216240264288平均存活時(shí)間/hpcDNA3-W2b4a1515963110189±28*pcDNA3-W2b4a+黃芪多糖15151496411220±31*#pcDNA3-W2b4a+AstragalanpcDNA3-W2b4a+香菇多糖15151487411218±26*#pcDNA3-W2b4a+LentinanpcDNA315141420000164±11生理鹽水1510000000151±7physiologicalsaline

注：*與對(duì)照組比較，P<0.05；#與pcDNA3-W2b4a比較，P<0.05。

Note: *As compared with the control group,P<0.05; # As compared with pcDNA3-W2b4a group,P<0.05.

3 討 論

本課題選用黃芪多糖、香菇多糖作為弓形蟲wx2b4a表位疫苗的佐劑，它們具有廣泛的藥理作用，也具有很強(qiáng)的免疫調(diào)節(jié)作用，可從多個(gè)方面發(fā)揮免疫調(diào)節(jié)作用：黃芪多糖可顯著增強(qiáng)非特異性免疫和體液免疫功能,提高免疫抑制小鼠的IL-2、IFN-γ、TNF-a的水平，并可促進(jìn)T淋巴細(xì)胞增殖[4]。代巧妹[5]等對(duì)急性弓形蟲感染的BALB/c小鼠采用香菇多糖預(yù)處理之后能有效的調(diào)節(jié)Tregs的數(shù)量和功能，從而調(diào)控Th1/Th2之間的動(dòng)態(tài)平衡達(dá)到治療弓形蟲的作用。

表位疫苗已在寄生蟲疫苗的研究中獲得可喜的免疫效果，本課題組前期成功構(gòu)建了弓形蟲多表位疫苗pcDNA3-W2b4a，并證實(shí)誘導(dǎo)小鼠產(chǎn)生一定的抗弓形蟲感染的作用[2]。本研究結(jié)果顯示：各免疫組小鼠血清IgG抗體水平顯著高于對(duì)照組，且pcDNA3-W2b4a+黃芪多糖組高于pcDNA3-W2b4a+香菇多糖組，各免疫組抗體水平均在末次免疫后第4周最高；各免疫組T細(xì)胞增殖活性與對(duì)照組比較明顯增強(qiáng)，且pcDNA3-W2b4a+香菇多糖組要大于pcDNA3-W2b4a+黃芪多糖組，這與王國秀[6]等研究認(rèn)為協(xié)同ConA促進(jìn)脾淋巴細(xì)胞增殖作用是香菇多糖大于黃芪多糖，促進(jìn)脾淋巴細(xì)胞分泌IgG的是黃芪多糖大于香菇多糖的結(jié)果相一致。黃德尚[7]等研究黃芪多糖作為免疫佐劑能提高豬瘟疫苗對(duì)仔豬的免疫力,其作用機(jī)制與增加免疫球蛋白和促進(jìn)豬瘟抗體形成有關(guān)。各免疫組小鼠脾細(xì)胞IL-2、IFN-γ水平高于對(duì)照組，pcDNA3-W2b4a+黃芪多糖組、pcDNA3-W2b4a+香菇多糖組小鼠脾細(xì)胞IL-2、IFN-γ水平高于pcDNA3-W2b4a組，提示黃芪多糖、香菇多糖結(jié)合wx2b4a表位疫苗傾向于Th1型優(yōu)勢(shì)的免疫應(yīng)答。葛璞[8]等研究香菇多糖能夠有效激發(fā)弓形蟲RH株感染的BALB/c小鼠Th1型免疫應(yīng)答的建立，對(duì)抗弓形蟲感染。王庭欣[9]等研究黃芪多糖對(duì)正常小鼠的細(xì)胞免疫、體液免疫及巨噬細(xì)胞功能均有明顯的增強(qiáng)作用。小鼠攻擊試驗(yàn)表明，pcDNA3-W2b4a+黃芪多糖組、pcDNA3-W2b4a+香菇多糖組小鼠存活時(shí)間明顯長(zhǎng)于pcDNA3-W2b4a組和對(duì)照組。由此可見，黃芪多糖、香菇多糖可增強(qiáng)弓形蟲wx2b4a表位疫苗免疫小鼠的保護(hù)作用，可望用于弓形蟲亞單位疫苗的研制。

[1]Fan JB, Wu X, Tan K, et al. The construction and characterization of multiple-epitopes vaccine of new genes ofToxoplasmagondii[J]. J Pathog Biol, 2007, 2(2): 118-121. (in Chinese) 范久波,吳翔,譚逵，等.弓形蟲新基因表位疫苗質(zhì)粒的構(gòu)建及鑒定[J].中國病原生物學(xué)雜志,2007,2(2):118-121.

[2]Fan JB, Wu X, Tan K, et al. The protective immunity induced by the epitope vaccines from two new novel genes ofToxoplasmagondiiin mice[J]. J Pathog Biol, 2008, 3(6): 421-424. (in Chinese) 范久波,吳翔,譚逵，等.弓形蟲新基因表位疫苗效果的觀察[J].中國病原生物學(xué)雜志, 2008, 3(6): 421-424.

[3]He YS. Study on the immune adjusting reagent of chinese medicine and its anti-tumor effect[J]. Occupat Health, 2005, 21(10):1454-1459. (in Chinese) 何躍生.中草藥免疫調(diào)節(jié)劑及抗腫瘤作用研究進(jìn)展[J].職業(yè)與健康,2005,21(10):1454-1459.

[4]Zhao D, Chang J. Advances in the studies on the mechanism of immuno-potentiation effect of chinese material medica[J]. J Baotou Med Coll, 2013, 29(4): 122-124. (in Chinese) 趙東,常江.中藥有效成分免疫增強(qiáng)作用機(jī)制的研究概況[J]. 包頭醫(yī)學(xué)院學(xué)報(bào)，2013,29(4):122-124.

[5]Dai QM, Liu L, Cai LS, et al. Regulatory effect of Lentinan on CD4+CD25+Foxp3+regulatory T cells in BALB/ c mice infected withToxoplasmagondii[J]. Chin J Microecol, 2013,25(5): 524-527.(in Chinese) 代巧妹，劉蕾，蔡連順，等.香菇多糖對(duì)急性弓形蟲感染過程中CD4+CD25+Foxp3+調(diào)節(jié)性T 細(xì)胞的調(diào)節(jié)作用研究[J].中國微生態(tài)學(xué)雜志,2013,25(5):524-527.

[6]Wang GX, Lin WM, Zhao RF, et al. Effects of six polysaccharides extracted from plants on the immunological cells of mice[J]. J Hyg Res, 2008, 37(5): 577-580. (in Chinese) 王國秀，藺威鳴，趙瑞芳，等.6 種多糖對(duì)小鼠免疫細(xì)胞活性作用的比較研究[J]. 衛(wèi)生研究，2008，37(5)：577-580.

[7]Huang DS, Guo AZ. Effects of immunity inAstragalanadjuvanon swine plague vaccine on piglets[J]. China Anim Husb Vet Med, 2011, 38(6): 189-192. (in Chinese) 黃德尚，郭愛珍.黃芪多糖佐劑對(duì)豬瘟疫苗免疫效果的影響[J].中國畜牧獸醫(yī)，2011,38(6):189-192.

[8]Guo P, Li CL, Huang KS, et al. Promotion effects of Lentinan on immunologic function of BALB/c Murine peritoneal macrophage againstToxoplasmaGondiiinfection[J]. China Pharm, 2011, 22(3): 215-217. (in Chinese) 葛璞，李春莉，黃開順,等.香菇多糖促BALB/c小鼠腹腔巨噬細(xì)胞抗弓形蟲感染的研究[J].中國藥房，2011,22(3)：215-217.

[9]Wang TX, Wang TX, Wu GC, et al. Effect of Astragalus polysaccharideon immunological function in mice[J]. Lishizhen Med Materia Medica Res, 2009,20(7): 1763-1764.(in Chinese) 王庭欣,王庭祥,吳廣臣，等.黃芪多糖增強(qiáng)小鼠免疫功能的實(shí)驗(yàn)研究[J].時(shí)珍國醫(yī)國藥,2009,20(7)：1763-1764.

DOI:10.3969/cjz.j.issn.1002-2694.2015.08.009

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

Enhancement of epitope vaccines fromwx2b4a

ofToxoplasmagondiipotency using Astragalan and Lentinan in mice

XIE Rong-hua1,FAN Jiu-bo2,SHU Heng-ping3

(1.EnvironmentBiologyofProfessionTechnologyCollegeinHunan,Hengyang421001,China;2.XiangfanCentralHospital,Xiangfan441021,China;3.DepartmentofParositology,XiangyaSchoolofMedicine,CentralSouthUniversity,Changsha410078,China)

We observed the effect of Astragalan and Lentinan to enhance the immune response and protective immunity induced by the epitope vaccines fromwx2b4a ofToxoplasmagondiiin mice. Mice were immunized intramuscularly with Astragalan and pcDNA3-W2b4a, Lentinan and pcDNA3-W2b4a, respectively. The level of IgG antibodies of mice were detected by ELISA.Invitro, the levels of IL-2 and IFN-γ that were secreted from pcDNA3-W2b4a-stimulated splenocytes were also measured by ELISA. Proliferation of T cells was detected by CCK-8 and mouse survival challenged byT.gondiiwas observed. Results showed that after the immunization, the level of IgG antibodies in immunized groups were significantly higher than those in the control group (P<0.05). The level of IgG antibodies in sera of mice inoculated with Astragalan and pcDNA3-W2b4a were higher than that in Lentinan and pcDNA3-W2b4a (P<0.05). After the immunization, the level of IL-2 and IFN-γ in splenoctes of mice inoculated with Astragalan and pcDNA3-W2b4a, Lentinan and pcDNA3-W2b4a were significantly higher than that those in GrouppcDNA3-W2b4a (allP<0.05). After the immunization, proliferation of T cells in immunized groups were significantly higher than those in the control group (P<0.05). The proliferation of T cells inoculated with Lentinan and pcDNA3-W2b4a were higher than Astragalan and pcDNA3-W2b4a (P<0.05). Following challenging with RH tachyzoites, the mean survival time in Astragalan and pcDNA3-W2b4a group, Lentinan and pcDNA3-W2b4a group were significantly longer than those in Group pcDNA3-W2b4a (allP<0.05). It indicated that Astragalan and Lentinan could enhancewx2b4a immune response that exerts a superior immune protection, which might be a new practical strategy for developingToxoplasmagondiivaccines. Keywords:Toxoplasmagondii; Astragalan; Lentinan; epitope vaccine; protective efficacy

10.3969/cjz.j.issn.1002-2694.2015.08.008

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 421001； 2.湖北襄陽中心醫(yī)院，襄樊 441021； 3.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 Province (No. 2014FJ3126), the Social Science Foundation of Hengyang (No. 2014D100), and Science and Technology Development Project of Hengyang City (No. 2014KJ27)

R382

A

1002-2694(2015)08-0724-04

2015-01-28；

2015-04-16

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)、衡陽市社會(huì)科學(xué)基金(No.2014D100)、衡陽市科學(xué)技術(shù)發(fā)展計(jì)劃項(xiàng)目(No.2014KJ27)聯(lián)合資助