Sj26基因转染的树突状细胞对日本血吸虫感染的免疫保护机制研究

中国寄生虫学与寄生虫病杂志 ›› 2007, Vol. 25 ›› Issue (1): 4-21.

Sj26基因转染的树突状细胞对日本血吸虫感染的免疫保护机制研究

沈定文1;罗金萍1;李雍龙2;刘文琪2;龙小纯2

1 咸宁学院医学院，咸宁 437100； 2 华中科技大学同济医学院病原生物学系, 武汉 430030

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2007-02-28 发布日期:2007-02-28
通讯作者: 李雍龙

Studies on Mechanism of Protective Immunity Against Infection of Schistosoma japonicum Induced by Sj26 Gene Transfected Dendritic Cell

SHEN Ding-wen1;LUO Jin-ping1;LI Yong-long2; LIU Wen-qi2;LONG Xiao-chun2

1 Medical College of Xianning College, Xianning 437100, China; 2 Department of Pathogenic Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

Received:1900-01-01 Revised:1900-01-01 Online:2007-02-28 Published:2007-02-28
Contact: LI Yong-long

摘要/Abstract

摘要： 目的探讨日本血吸虫Sj26基因转染的树突状细胞（DC）对日本血吸虫感染的保护性免疫作用机制。方法 BALB/c小鼠48只随机均分4组, 于小鼠耳廓分别注射细胞悬液(浓度1×10⁶/ml)0.2 ml, A组注射Sj26基因转染的DC、B组注射质粒pcDNA3转染的DC、 C组注射未处理的DC, D组注射RPMI-1640。共免疫3次，间隔2周。末次免疫后2周，每鼠经皮肤感染40±2条尾蚴。分别于免疫前、末次免疫后第 2 周以及攻击感染后第 6 周采血, ELISA法检测血清IgG抗体、γ干扰素（IFN-γ）和白细胞介素-4（IL-4）水平，免疫印迹法（Western blot）检测血清特异性抗Sj26 IgG抗体，双夹心ELISA法检测脾淋巴细胞经伴刀豆球蛋白A(ConA)和可溶性虫卵抗原(SEA)刺激后培养上清中IFN-γ和IL-4含量。噻唑蓝法（MTT）检测脾淋巴细胞增殖情况。结果 A组血清IgG抗体水平(吸光度A₄₉₁值)，免疫后（A₄₉₁=0.117）显著高于免疫前（A₄₉₁=0.049）（t=2.73，P<0.05），也显著高于B组（A₄₉₁=0.061）和C组（A₄₉₁=0.058）（t值为2.48和2.56，P<0.05）。A组血清能特异识别血吸虫成虫抗原Mr 26 000蛋白。血清IL-4水平，各组免疫前、后均无明显变化。 IFN-γ水平，A组血清免疫后为（101.4±4.9)pg/ml, 明显高于免疫前的(15.0±1.9) pg/ml（t=5.80，P<0.01），亦高于免疫后的B组（40.1±3.1) pg/ml和C组（35.6±1.2) pg/ml (t值为3.98和4.13，P<0.01)。脾淋巴细胞经ConA刺激诱生的IFN-γ， A组（171.2 pg/ml）显著高于 D组（91.0 pg/ml）（t=4.25，P<0.01）。经SEA刺激诱生的IFN-γ， A组（70.8 pg/ml）显著高于D组（49.7 pg/ml）（t=2.83，P<0.01）。经ConA刺激诱生的IL-4水平，A组（79.7 pg/ml）明显低于D组（125.2 pg/ml）（t=4.40, P<0.01）。经SEA刺激诱生的IL-4，A组（50.7 pg/ml）明显低于D组（70.5 pg/ml）（t=2.62，P<0.05）。A组脾淋巴细胞经ConA和SEA刺激后的刺激指数分别为4.1和2.82，均高于其他各组（与D组比较，t=3.20, P<0.01和t=2.15，P<0.05）。结论 Sj26基因转染的树突状细胞免疫小鼠主要诱导Th1型免疫应答。

关键词: 日本血吸虫, 树突状细胞, Sj26基因, 基因转染, 保护性免疫

Abstract: Objective To explore the mechanism of protective immunity against Schistosoma japonicum infection induced by Sj26 gene transfected dendritic cell (DC). Methods 48 BALB/c mice were divided randomly into 4 groups with 12 each. The mice were injected through auricle for three times with Sj26 gene transfected DC (Group A), pcDNA3 transfected DC (Group B), untreated DC (Group C) and RPMI-1640 (Group D) respectively, and challenged with 40±2 cercariae of S. japonicum per mouse 2 weeks after the last immunization. Sera from mice were examined for IgG antibody, IFN-γ and IL-4 by ELISA. Western blot was used for detecting specific anti-Sj26 IgG antibody. The production of IFN-γ and IL-4 in the supernatant of spleen cells stimulated with soluble egg antigen (SEA) and ConA was quantified by sandwich ABC-ELISA. The proliferation of spleen cells were measured with MTT method. Results IgG antibody increased significantly in the mice of group A at 2 weeks after the last immunization（absorbency A₄₉₁=0.117）, higher than that of group B（A₄₉₁=0.061） and group C（A₄₉₁=0.058）（P<0.05）. The Mr 26 000 antigen of S. japonicum was strongly recognized by sera from group A by Western blot. The level of IL-4 in mice of each group showed no significant difference before and after immunization. The level of IFN-γ in group A（101.4±4.9 pg/ml） was significantly higher than that before immunization (15.0±1.9 pg/ml) and that of group B（40.1±3.1 pg/ml） and group C（35.6±1.2 pg/ml）（P<0.01）. The level of IFN-γ in spleen cells from group A in response to ConA and SEA (171.2 and 70.8 pg/ml, respectively) was higher than that of group D (91 and 49.7 pg/ml, respectively) （P<0.01）. The level of IL-4 in spleen cells from group A in response to ConA and SEA (79.7 and 50.7 pg/ml, respectively) was lower than that of group D (125.2 and 70.5 pg/ml, respectively) （P<0.01）. The stimulating index of spleen cells from group A was 4.1 and 2.82 in response to ConA and SEA respectively, higher than that of other groups (compared with group D, P<0.05). Conclusion Sj26 gene transfected dendritic cell induces predominant Th1 type immune response which might play a role in protection against S. japonicum infection.

Key words: Schistosoma japonicum, Dendritic cell, Sj26 gene, Gene transfection, Protective immunity

沈定文;罗金萍;李雍龙;刘文琪;龙小纯. Sj26基因转染的树突状细胞对日本血吸虫感染的免疫保护机制研究[J]. 中国寄生虫学与寄生虫病杂志, 2007, 25(1): 4-21.

SHENDing-wen;LUOJin-ping;LIYong-long;LIUWen-qi;LONGXiao-chun. Studies on Mechanism of Protective Immunity Against Infection of Schistosoma japonicum Induced by Sj26 Gene Transfected Dendritic Cell[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2007, 25(1): 4-21.

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