932 微生物学通报 2009, Vol.36, No.6,,,,,,,,,, [1 3], ES ES,, 1000 mg/ml [4] ES, 1 材料和方法 1.1 材料与设备菌种 :,, 试剂 : RMI1640 (Gibco ), /

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1 微生物学通报 JUN 20, 2009, 36(6): 931~935 Microbiology 2009 by Institute of Microbiology, CAS 主编点评文章一种双歧杆菌胞外多糖免疫调节功能研究 * 李伟欣陈倩李平兰程静 ( ) 摘要 : 根据保健食品功能评价规范功能学评价程序研究一种双歧杆菌胞外多糖 (Bifidobacterium spp. exopolysaccharide, ES) 的免疫调节功能通过脾淋巴细胞增殖反应绵羊红细胞诱导小鼠迟发型变态反应 (DTH) 血清溶血素测定以及巨噬细胞吞噬实验探讨该 ES 的免疫调节活性 ES 分别以高中低剂量组连续口服给药 10 天, 结果发现低剂量的 ES[100 mg/(kg d)] 可以促进脾淋巴细胞增殖 ; 高中低剂量对绵羊红细胞诱导的小鼠迟发型变态反应均无促进作用 ; 但是, ES 高中低剂量组均能明显提高小鼠血清半数溶血值 HC 50 以及增强小鼠腹腔巨噬细胞吞噬鸡红细胞的吞噬能力根据规范可知, 该双歧杆菌 ES 具有一定的免疫调节活性关键词 : 双歧杆菌, 胞外多糖, 免疫 Study on Immunoregulation Effect of Exopolysaccharide roduced by Bifidobacterium spp. LI Wei-Xin CHEN Qian LI ing-lan * CHENG Jing (Food Science and Nutrition Engineering College, China Agricultural University, Beijing , China) Abstract: The immunoregulation effect to polysaccharides from Bifidobacterium spp. was investigated on the base of functional assessment standards of health food. Effects of the ES on immunity were investigated by promoted the proliferation of spleen lymph cells, delayed type hypersensitivity reaction, the HC 50 value and macrophage function assay in mice. Data showed that the ES could obviously increase the ratio of swallowed chicken red blood cell by macrophage and the HC 50 value in mice. However, no significant effect was found on the delayed type hypersensitive induced by sheep red blood cell, for only the low dose of 100 mg/(kg d) ES promoted the proliferation of spleen lymph cells. Bifidobacterium spp. ES can certain immunomodulating function. Keywords: Bifidobacterium, Exopolysaccharide, Immunomodulating,,,, D ( Bifidobacterium spp. exopolysaccharide, ES), 基金项目 : 863 (No. 2008AA10Z324); (No ) * 通讯作者 : Tel: ; : lipinglan@cau.edu.cn 收稿日期 : ; 接受日期 :

exopolysaccharide, ES) 的免疫调节功能通过脾淋巴细胞增殖反应绵羊红细胞诱导小鼠迟发型变态反应 (DTH) 血清溶血素测定以及巨噬细胞吞噬实验探讨该 ES 的免疫调节活性 ES 分别以高中低剂量组连续口服给药 10 天, 结果发

2 932 微生物学通报 2009, Vol.36, No.6,,,,,,,,,, [1 3], ES ES,, 1000 mg/ml [4] ES, 1 材料和方法 1.1 材料与设备菌种 :,, 试剂 : RMI1640 (Gibco ), /( ), (,, : ), (,, : ), ConA( Sigma ), MTT( Sigma ), SDS( Sigma ) 试验动物 : BALB/c ICR [6~8,,, : SCXK( ) ], [, : SCXK( ) ] 仪器设备 : TGL-16C (16000 r/min/17000 g), TGL-20M, SCL-1300, AL-9908S,, CO 2, ACSC alibur, 752,, XDS-1B, LEICA DM2000 (), Anymicro DSSTM YT-7M Digital Shoot System 1.2 方法双歧杆菌粗 ES 的制备 : 2, 3% ( ), 37 C 24 h; 1/3, 6000 r/min 10 min; 3 95%, 4 C, 6000 r/min 10 min,, Sevag(ES: Sevag =1:2(V/V), : =4:1(V/V)), 8000 D~14000 D 48 h, ES [5] 动物分组及给药 : ( ) 5, 8 1 ; 2, (Ginseng polysaccharide, GS) 50 mg/kg, 7 d, (Antitheses) 20 mg/kg, 10 d; 3, 4, 5 ES (400 mg/kg 200 mg/kg 100 mg/kg),, 10 d 小鼠脾淋巴细胞增殖反应 : 1 h,, ml, 96, 100 µl, RMI ConA(5 µg/ml) ConA RMI µl, 37 C 72 h 4 h, MTT(5 mg/ml), 20 µl, 100 µl,, MTT, 570 nm 绵羊红细胞诱导小鼠迟发型变态反应 (Delayed Type Hypersensitivity Reaction, DTH): 2% SRBC 0.2 ml, 4,, 20% SRBC 20 μl, 24 h, 3, DTH 血清溶血素测定 : 2% SRBC 0.2 ml, 5,, ml, 0.5 ml 10% SRBC 1 ml 1:8, 37 C 30 min 2000 r/min 10 min, 1 ml, 3 ml ;, 0.25 ml 10% SRBC, 4 ml,, 10 min, 540 nm HC 50 =(/ SRBC )

2 方法 1.2.1 双歧杆菌粗 ES 的制备 : 2, 3% (8.4 10 9 ), 37 C 24 h; 1/3, 6000 r/min 10 min; 3 95%, 4 C, 6000 r/min 10 min,, Sevag(ES: Sevag =1:2(V/V), : =4:1(V/V)), 8000 D~14000 D 48 h, ES [5] 1.2.2 动物分组及给药 : ( ) 5, 8 1 ; 2, (Ginseng polysaccharide, GS) 50 mg/kg, 7 d, (Antitheses) 20 mg/kg, 10 d; 3, 4, 5 ES (400 mg/kg 200 mg/kg 100 mg/kg),, 10 d 1.

3 : 小鼠单核巨噬细胞吞噬鸡红细胞实验 : 4 d 2% 0.2 ml,, 10% Hank s 4 ml/, 20,, 2 ml 0.5 ml 0.5 ml 1%,, 37 C 15 min, 1 min, 15 min,, (%)=(/ 100 ) 100% 统计学分析 : x s, t 2 结果 :, (, ) (, ) -(, ) NK 4 2,, 4 2, 2, 2.1 小鼠脾淋巴细胞增殖反应,, 4, NK BT 2 [6] 1, ES ConA ConA, ES T,,, [7],, ES 表 1 ES 对淋巴细胞增殖反应的影响 (n=8) Table 1 The effect of ES on lymph cell proliferation (n=8) ConA None ConA ConA ConA CK 0.162± ±0.073 GS(50 mg/kg) 0.179±0.021* ±0.139* ES(400 mg/kg) 0.143± ± ES(200 mg/kg) 0.149± ± ES(100 mg/kg) 0.214±0.058* ±0.078* Note: *: Significantly different ( 0.05 ) compared with untreated controls; **: Significantly different ( 0.01 ) compared with untreated 2.2 绵羊红细胞诱导小鼠迟发型变态反应,,, [8 10] 2, ES, , ES 2.3 血清溶血素测定, - B, T 表 2 ES 对绵羊红细胞诱导小鼠迟发型变态反应的影响 (n=8) Table 2 The effect of ES on delayed type hypersensivity in mice induced by SRBC (n=8) Delayed type hypersensitivity reaction CK 0.020±0.012 GS(50 mg/kg) 0.056±0.030** ES(400 mg/kg) 0.026± ES(200 mg/kg) 0.029± ES(100 mg/kg) 0.037± Note: *: Significantly different( 0.05 ) compared with untreated controls; **: Significantly different ( 0.01 ) compared with untreated

0.162±0.009 0.972±0.073 GS(50 mg/kg) 0.179±0.021* 0.044 1.141±0.139* 0.019 ES(400 mg/kg) 0.143±0.041 0.314 1.061±0.111 0.101 ES(200 mg/kg) 0.149±0.034 0.275 0.930±0.080 0.332 ES(100 mg/kg) 0.214±0.

4 934 微生物学通报 2009, Vol.36, No.6,,, [11,12] 3, ES 3 HC 50,,, ES 表 3 对小鼠半数溶血值的影响 (n=8) Table 3 The effect on HC 50 in mice (n=8) HC 50 CK ±19.2 Antitheses(20 mg/kg) ±12.47* ES(400 mg/kg) ±15.68* ES(200 mg/kg) ±19.33* ES(100 mg/kg) ±14.3* Note: *: Significantly different( 0.05 ) compared with untreated controls; **: Significantly different( 0.01 ) compared with untreated 2.4 小鼠单核巨噬细胞吞噬鸡红细胞实验 4, ES 3, ES - 表 4 ES 对巨噬细胞吞噬鸡红细胞的影响 (n=8) Table 4 The effect of ES on macrophage swallowing CRBC (n=8) (%) ercentage of macrophage phagocytosis (%) CK 19.4±5.46 GS(50 mg/kg) 40.0±10.58** ES(400 mg/kg) 27.5±3.27* ES(200 mg/kg) 38.0±9.65** ES(100 mg/kg) 31.3±7.27* Note: *: Significantly different( 0.05 ) compared with untreated controls; **: Significantly different( 0.01 ) compared with untreated,,, ES,, ES ;, p ES, 2,, 3 讨论 [13] : 1) :, 2) : ( ), 3) T B :, 4) :, TT,,,, [14],,, ES,, T B,,,, [15 18] ES[100 mg/(kg d)], ES,,, ES ; ES HC 50, ES

05 ) compared with untreated controls; **: Significantly different( 0.01 ) compared with untreated 2.

5 : 935,,, ES, ES T B,,,, B, T [19] Neumann, [20],, B T,, ES,, ES-,, ES,,,, 参考文献 [1] Adriana Arena, Teresa Lu, Bernadette, et al. Bernadette pavone antiviral and immunoregulatory effect of a novel exopolysaccharide from a marine thermotolerant Bacillus licheniformis. International Immunopharmacology, 2006, (6): [2] XueSong Wang, Qun Dong, Jianing Zuo, et al. Structure and potential immunological activity of a pectin from Centella asiatica (L.) Urban. Carbohydrate Research, 2003, 338: [3],,.., 2006, 18(3): [4], (ES)., 2005, 33(3): [5],,, (ES)., 2005, 31(6): [6],,,. Z222 (ESⅠ)., 2003, 23(6): [7],.., 2003, 23(2): [8],,,.., 2004, 25(10): [9],.., 2006, 34(10): [10],,,.., 2004, 20(12): [11],,.., 2006, 4: [12],,,.., 2004, 27(3): [13],.., 2006, 15(5): [14],.., 2007, 17(1): [15],.., 2003, 23(1): [16],,,.., 2002, 18(1): [17],,.., 1995, 15(2): [18] Jin M, Kim S, Kim BK. Induction of B cell proliferation and NF-kappa B activation by a water solubie from Lentinus lepideus. International Journal of Immunopharmacology, 1996, 18(8/9): [19],,.., 2002(12): 4 6. [20] Neumann NF, Fagan D, Belosevic M. Macrophage activating factor(s) secreted by mitogien stimulatied goldfish kidney leukocytes syneirgize with bacterial lipopolysaccharide to induce nitric oxide production in teleost macrophages. Development and Comparative Immunology, 1995, 19(6):

[2] XueSong Wang, Qun Dong, Jianing Zuo, et al. Structure and potential immunological activity of a pectin from Centella asiatica (L.) Urban. Carbohydrate Research, 2003, 338: 2393 2402. [3],,.

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932 微 生 物 学 通 报 2009, Vol.36, No.6,,,,,,,,,, [1 3], ES ES,, 1000 mg/ml [4] ES, 1 材 料 和 方 法 1.1 材 料 与 设 备 菌 种 :,, 试 剂 : RMI1640 (Gibco ), /

932 微生物学通报 2009, Vol.36, No.6,,,,,,,,,, [1 3], ES ES,, 1000 mg/ml [4] ES, 1 材料和方法 1.1 材料与设备菌种 :,, 试剂 : RMI1640 (Gibco ), /