SCP抽提物合剂诱导感染小鼠产生的抗肝内日本血吸虫卵肉芽肿效应的分子机理研究
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摘要
第一部分 鲨鱼软骨粉（SCP）抽提物的制备及其免疫原性特征的
研究
目的 本研究应用SCP的生理盐水抽提物（SCP1）和盐酸胍抽提物
（SCP2）免疫小鼠后，观察其诱导的T淋巴细胞亚群表型和特异性抗体
应答的免疫原性特征。材料和方法 50只健康昆明种雄性小鼠，随机
均分5组。组Ⅰ和组Ⅱ分别用SCP1小剂量（50μg/0.2ml）和大剂量
（100μg/0.2ml）免疫小鼠；组Ⅲ和组Ⅳ分别用与上述相同小剂量和大
剂量的SCP2免疫小鼠；组Ⅴ注射生理盐水作为对照。在第2次加强
免疫后2wk（首次免疫后6wk）剖杀小鼠，采用流式细胞仪测定小鼠
脾T淋巴细胞亚群CD_4~+和CD_8~+百分数及其比值；并用ELISA检测小
鼠血清中特异性IgG及其亚类IgG_1和IgG_3抗体水平。结果 实验结
果表明，组Ⅰ至组Ⅳ的CD_4~+和CD_8~+细胞百分数均数分别高于和低于
正常对照小鼠及其CD_4~+/CD_8~+比值（1.45～1.92），均显著地高于正常对照
组Ⅴ的1.15；应用大剂量（组Ⅱ和组Ⅳ）的两种抽提物诱导出的细
胞免疫应答均强于小剂量者（组Ⅰ和组Ⅲ），但SCP1诱导的细胞免疫
应答要强于SCP2者。SCP1和SCP2大剂量组小鼠均能诱导出高水平
的特异性抗体IgG（1.22±0.09～1.72±0.12）及其亚类IgG_1和
IgG_3（0.26±0.09至0.39±0.05），但SCP1诱导出的特异性体液免疫应答
要强于SCP2者。结论 研究结果表明，应用SCP生理盐水抽提物
（SCP1）和盐酸胍抽提物（SCP2）免疫小鼠后6wk时均显示明显的
SCP抽挞物合剂诱导妞染小鼠产生的抗肝内【］本血吸虫卵肉芽肿效应的分了机理研究 中文摘要
免疫原性应答，用大剂量者要强于小剂量者，但SCPI免疫小鼠后诱
导出的具有细胞免疫和体液免疫应答的免疫原性特征要强于SCPZ
者。认为SCP应用生理盐水冷抽提的SCPI，似含有能诱导出显著免
疫原性应答特征的免疫因子，值得持续深入研究。
Part I Preparation of Shark Cartilage Powder (SCP) Extraction
and their immunogenicity characterization
Objective To investigate the immunogenicity characterization of T
lymphocyte phenotypes and specific antibody responses induced by SCP
extraction in normal saline (SCP1) and in guatudine hydrochloride
solution (SCP2). Methods Fifty Kunming male mice were divided
evenly into 5 groups (from group I to V). Mice were immunized with
SCP1 at the smaller dose of 5O~g/O.2ml protein content per mouse (group
I ) or at the larger one of 1OO~tg/O.2ml (group II), respectively. Group III
and group IV were immunized respectively with the smaller or larger
dosage of SCP2. the dosage was the same as the above~mentioned. Group
V was injected with normal saline as a control group. Two weeks later
after the second boosted immunization (six weeks after first
immunization), mice were killed for determinations of the following
indices. The percentage of splenic T lymphocyte phenotypes of mice was
detected by flowcytometer. The serum specific antibody IgG and its
subclass IgG1 and IgG3 levels were determined by ELISA. Results The
results showed that the percentages of splenic CD or CD8~ in group I ~. II
III and IV were significantly higher or lower than those in group V. The
ratios of CD4~ / CDg~(1.45--1.92) in group V.. IL.. III and IV were obviously
higher than that (1.15) in group V (p responses induced by the larger dosage of SCP1 (group II) and SCP2
VI
Efiects ~md ~kI~ctiIm Mechanisms of Anti ~shutosoe,a,apcirnosim E~ ilTanuloma h,duncd by SCP Extnidisui Con鼅utm~ keap~is
Abstracts
(group IV) were more intensive than those by the smaller dosage of SCPI
(group I) and SCP2(group III), respectively (p induced by SCPI were stronger than those by SCP2. The specific antibody
IgG(l.22?.09-A.72?.12) and its subclass IgG1 and IgG3(0.26?.09 to
0.39?.05) levels could be induced by the larger dosage of SCPI and
SCP2, and the specific humoral immune responses induced by SCPI were
more intensive than those by SCP2. Conclusion The results of this study
indicate that tile significant immunogenicity responses can be induced by
SCP extraction in normal saline (SCPI) and in guanidine hydrochloride
solution (SCP2). The responses induced by the larger dosage of SCPI and
SCP2 are stronger than those by tile smaller ones. Tile imrnunogenicity
characterization of cellular and humoral immune responses induced by
SCPI are more intensive than those by SCP2. It is suggested that SCP1
seems to contain irnmunomodulators which can induce obvious
umnunogenlcltv characterizaton. It is worth further researchina.
response
研究
目的 本研究应用SCP的生理盐水抽提物（SCP1）和盐酸胍抽提物
（SCP2）免疫小鼠后，观察其诱导的T淋巴细胞亚群表型和特异性抗体
应答的免疫原性特征。材料和方法 50只健康昆明种雄性小鼠，随机
均分5组。组Ⅰ和组Ⅱ分别用SCP1小剂量（50μg/0.2ml）和大剂量
（100μg/0.2ml）免疫小鼠；组Ⅲ和组Ⅳ分别用与上述相同小剂量和大
剂量的SCP2免疫小鼠；组Ⅴ注射生理盐水作为对照。在第2次加强
免疫后2wk（首次免疫后6wk）剖杀小鼠，采用流式细胞仪测定小鼠
脾T淋巴细胞亚群CD_4~+和CD_8~+百分数及其比值；并用ELISA检测小
鼠血清中特异性IgG及其亚类IgG_1和IgG_3抗体水平。结果 实验结
果表明，组Ⅰ至组Ⅳ的CD_4~+和CD_8~+细胞百分数均数分别高于和低于
正常对照小鼠及其CD_4~+/CD_8~+比值（1.45～1.92），均显著地高于正常对照
组Ⅴ的1.15；应用大剂量（组Ⅱ和组Ⅳ）的两种抽提物诱导出的细
胞免疫应答均强于小剂量者（组Ⅰ和组Ⅲ），但SCP1诱导的细胞免疫
应答要强于SCP2者。SCP1和SCP2大剂量组小鼠均能诱导出高水平
的特异性抗体IgG（1.22±0.09～1.72±0.12）及其亚类IgG_1和
IgG_3（0.26±0.09至0.39±0.05），但SCP1诱导出的特异性体液免疫应答
要强于SCP2者。结论 研究结果表明，应用SCP生理盐水抽提物
（SCP1）和盐酸胍抽提物（SCP2）免疫小鼠后6wk时均显示明显的
SCP抽挞物合剂诱导妞染小鼠产生的抗肝内【］本血吸虫卵肉芽肿效应的分了机理研究 中文摘要
免疫原性应答，用大剂量者要强于小剂量者，但SCPI免疫小鼠后诱
导出的具有细胞免疫和体液免疫应答的免疫原性特征要强于SCPZ
者。认为SCP应用生理盐水冷抽提的SCPI，似含有能诱导出显著免
疫原性应答特征的免疫因子，值得持续深入研究。
Part I Preparation of Shark Cartilage Powder (SCP) Extraction
and their immunogenicity characterization
Objective To investigate the immunogenicity characterization of T
lymphocyte phenotypes and specific antibody responses induced by SCP
extraction in normal saline (SCP1) and in guatudine hydrochloride
solution (SCP2). Methods Fifty Kunming male mice were divided
evenly into 5 groups (from group I to V). Mice were immunized with
SCP1 at the smaller dose of 5O~g/O.2ml protein content per mouse (group
I ) or at the larger one of 1OO~tg/O.2ml (group II), respectively. Group III
and group IV were immunized respectively with the smaller or larger
dosage of SCP2. the dosage was the same as the above~mentioned. Group
V was injected with normal saline as a control group. Two weeks later
after the second boosted immunization (six weeks after first
immunization), mice were killed for determinations of the following
indices. The percentage of splenic T lymphocyte phenotypes of mice was
detected by flowcytometer. The serum specific antibody IgG and its
subclass IgG1 and IgG3 levels were determined by ELISA. Results The
results showed that the percentages of splenic CD or CD8~ in group I ~. II
III and IV were significantly higher or lower than those in group V. The
ratios of CD4~ / CDg~(1.45--1.92) in group V.. IL.. III and IV were obviously
higher than that (1.15) in group V (p
VI
Efiects ~md ~kI~ctiIm Mechanisms of Anti ~shutosoe,a,apcirnosim E~ ilTanuloma h,duncd by SCP Extnidisui Con鼅utm~ keap~is
Abstracts
(group IV) were more intensive than those by the smaller dosage of SCPI
(group I) and SCP2(group III), respectively (p
IgG(l.22?.09-A.72?.12) and its subclass IgG1 and IgG3(0.26?.09 to
0.39?.05) levels could be induced by the larger dosage of SCPI and
SCP2, and the specific humoral immune responses induced by SCPI were
more intensive than those by SCP2. Conclusion The results of this study
indicate that tile significant immunogenicity responses can be induced by
SCP extraction in normal saline (SCPI) and in guanidine hydrochloride
solution (SCP2). The responses induced by the larger dosage of SCPI and
SCP2 are stronger than those by tile smaller ones. Tile imrnunogenicity
characterization of cellular and humoral immune responses induced by
SCPI are more intensive than those by SCP2. It is suggested that SCP1
seems to contain irnmunomodulators which can induce obvious
umnunogenlcltv characterizaton. It is worth further researchina.
response
引文
1．李允鹤，陈家旭，庞智，等。中药“425”合剂诱导的抗血吸虫卵肉芽肿效应及其机制研究。苏州医学院学报，1998；18(11)：1111-1114
2．陈家旭，李允鹤，龚唯，等。鲨鱼软骨粉合剂诱导的抗血吸虫性肝纤维化效应的研究。中西医结合肝病杂志，1999；9(6)：17-20
3．李允鹤、骆伟、陈家旭，等。鲨鱼软骨粉合剂诱导的抗肝血吸虫性纤维化效应机制的研究。中国寄生虫病防治杂志，1999；12(2)：137-139
4．李允鹤、龚唯、骆伟，等。SCP合剂治疗血吸虫病患者前后超声影像及特异性抗体的变化。苏州医学院学报，2000；20(12)：1118-1120
5．李允鹤、夏超明、陈家旭，等。日本血吸虫感染动物血清中循环抗体(CAb)及循环抗原(CAg)水平的动态研究。中国人兽共患病杂志，1994；5(1)：12-14
6. Li YH, Xie KM, Mao ZH, et al, Separation and kinetics of non-free circulating antigen on erythrocytes of rabbits infected with Schistosoma japonicum. Chinese Journal of Parasitic Disease Control, 1996: 9(3): 185-187
7．毛佐华，施云松，李允鹤，等。日本血吸虫感染兔红细胞上非游离性循环抗原的分子基础及其性质研究。中国人兽共患病杂志，1996；12(4)：11-13
8．夏超明，李允鹤，龚唯，等。非游离性循环抗原组分对血吸虫感染宿主虫卵肉芽肿保护性免疫调节机制的研究。中国血吸虫病防治杂志，1998；10(增)：66-71
9．施云松，李允鹤，夏超明，等。活化剂HYP协同“425”制剂的抗日本血吸虫卵肉芽肿病变免疫效应机理的研究。中国寄生虫病防治杂志，1998；11(2)：121-125
10．夏超明，李允鹤。免疫增强剂“425”对日本血吸虫感染小鼠虫卵肉芽肿病变作用的研究。中国寄生虫学与寄生虫病杂志，1992；10(1)：18-21
11．刘述先。血吸虫病免疫发病机制中的几种免疫调节作用。见：李允鹤主编。寄生虫病免疫学及免疫诊断，第1版 南京：江苏省科学技术出版社，1991，185
12．李允鹤。血吸虫卵肉芽肿病变的形成机制及免疫调节作用。苏州医学院学报，1999；19(11)：1141-1144
13. Townsend MJ, Fallon PG, Matthews DJ, et al. T_1/ST_2—deficient mice demonstrate the importance of T_1/ST_2 in developing primary T helper cell type 2 responses. J Exp Med, 2000; 191(6): 1069-1076
14. Infante-Duarte C and Kamradt T. Th_1/Th_2 balance in infection. Springer Semin Immunopathol, 1999; 21 (3): 317-318
15. Yang JQ, Tasaka k, Chuang CK, et al. Dynamic analysis of T-lymphocyte function in relation to hepatopathologic changes and effect of interleukin-12 treatment in mice infected with Schistosorna japonicum. J Parasitol, 1999; 85(2): 257-262
16. Hassan MM, Ramadan MA, Mostafa NE, et al. Different Cytokines profiles in spleen cells and liver granuloma of Schistosorna mansoni experimentally infected mice during disease development. J Egypt Soc Parasitol, 2000; 30(1): 245-256
17. Cheever AW, Jankovic D, Yap GS, et al. Role of cytokines in the formation and downregulation of hepatic circumoval granulomas and hepatic fibrosis in Schistosoma mansoni-infected mice. Mem Inst Oswaldo Cruz, 1998; 93(Suppll): 25-32
18. Lohning M, Grogan JL. Coyle AJ. et al. T_1/ST_2 expression is enhanced on CD_4~+ T cells from Schistosome egg-induced granulomas: analysis of Th cell cytokine coexpression ex vivo. J Immunol, 1999; 162(7): 3882-3889
19. Fallon PG, Richardson EJ, Mckenzie GJ, et al. Schistosome infection of transgenic mice defines distinct and contrasting pathogenic roles for IL-4 and IL-13: IL-13 is a profibrotic agent. J Immunol, 2000; 164(5): 2585-2591
20. Kaplan MH, Whitfield, IR, Boros DL, et al. Th_2 cells are required for the Schistosoma mansoni egg-induced granulomatous response. J Immunol, 1998: 160(4): 1850-1860
21．刘莉，沈一平，管晓虹，等。日本血吸虫感染小鼠脾细胞IL-2及IFN-γ动态观察。中国寄生虫学与寄生虫病杂志，1995；13(1)：35-38
22．胡永秀和薛燕萍。血吸虫卵肉芽肿的形成机制及免疫调节。寄生虫与医学昆虫学报，1997；4(2)：119-124
23. Hu Yongxiu, Xue Yangping, Tian Xiaojun, et al. Modulation of in vivo granuloma formation related to regulation of in vitro IFN-γ and IL-4 expressions in experimental Schistosomiasis japonica. Chinese Medical Journal. 1999: 112(8): 681-685.
24. Mwatha JK. Kimani G, Kaman T, et al. High levels of TNF. soluble TNF receptors, soluble ICAM-1, and IFN-γ. but low levels of IL-5, are associated with hepatosplenic disease in human Schistosomiasis mansoni. J Immunol, 1998: 160(4): 1992-1999
25. Montenegro SML, Miranda P, Mahanty S, et al. Cytokine production in acute versus chronic human Schistosomiasis mansoni: the cross-regulatory role of interferon-γ and interleukin-10 in the responses of peripheral blood mononuclear cells and splenocytes to parasite antigens. J Infect Dis, 1999; 179(6): 1502-1514
26. Falcao PL, Malaquias LCC, Martins-Filho DA, et al. Human Schistosomiasis mansoni: IL-10 modulates the in vitro granuloma formation. Parasite Immnuol, 1998; 20: 447-454
27. Wynn TA, Cheever AW, Williams ME, et al. IL-10 regulates liver vascular endothelial growth factor in cancer patients: which is the optimal specimen. Int J Oncol, 2000; 17(1): 149-152.
pathology in acute murine Schistosomiasis mansoni but is not required for immune down-modulation of chronic disease. J Immunol, 1998; 160(9) :4473-4480
28． Chiaramonte MG, Donaldson DD, Cheever AW, et al. An IL-13 inhibitor blocks the development of hepatic fibrosis during a T-helper type-2 dominated inflammatory response. J Clin Invest, 1999; 104(6) :777-785
2．陈家旭，李允鹤，龚唯，等。鲨鱼软骨粉合剂诱导的抗血吸虫性肝纤维化效应的研究。中西医结合肝病杂志，1999；9(6)：17-20
3．李允鹤、骆伟、陈家旭，等。鲨鱼软骨粉合剂诱导的抗肝血吸虫性纤维化效应机制的研究。中国寄生虫病防治杂志，1999；12(2)：137-139
4．李允鹤、龚唯、骆伟，等。SCP合剂治疗血吸虫病患者前后超声影像及特异性抗体的变化。苏州医学院学报，2000；20(12)：1118-1120
5．李允鹤、夏超明、陈家旭，等。日本血吸虫感染动物血清中循环抗体(CAb)及循环抗原(CAg)水平的动态研究。中国人兽共患病杂志，1994；5(1)：12-14
6. Li YH, Xie KM, Mao ZH, et al, Separation and kinetics of non-free circulating antigen on erythrocytes of rabbits infected with Schistosoma japonicum. Chinese Journal of Parasitic Disease Control, 1996: 9(3): 185-187
7．毛佐华，施云松，李允鹤，等。日本血吸虫感染兔红细胞上非游离性循环抗原的分子基础及其性质研究。中国人兽共患病杂志，1996；12(4)：11-13
8．夏超明，李允鹤，龚唯，等。非游离性循环抗原组分对血吸虫感染宿主虫卵肉芽肿保护性免疫调节机制的研究。中国血吸虫病防治杂志，1998；10(增)：66-71
9．施云松，李允鹤，夏超明，等。活化剂HYP协同“425”制剂的抗日本血吸虫卵肉芽肿病变免疫效应机理的研究。中国寄生虫病防治杂志，1998；11(2)：121-125
10．夏超明，李允鹤。免疫增强剂“425”对日本血吸虫感染小鼠虫卵肉芽肿病变作用的研究。中国寄生虫学与寄生虫病杂志，1992；10(1)：18-21
11．刘述先。血吸虫病免疫发病机制中的几种免疫调节作用。见：李允鹤主编。寄生虫病免疫学及免疫诊断，第1版 南京：江苏省科学技术出版社，1991，185
12．李允鹤。血吸虫卵肉芽肿病变的形成机制及免疫调节作用。苏州医学院学报，1999；19(11)：1141-1144
13. Townsend MJ, Fallon PG, Matthews DJ, et al. T_1/ST_2—deficient mice demonstrate the importance of T_1/ST_2 in developing primary T helper cell type 2 responses. J Exp Med, 2000; 191(6): 1069-1076
14. Infante-Duarte C and Kamradt T. Th_1/Th_2 balance in infection. Springer Semin Immunopathol, 1999; 21 (3): 317-318
15. Yang JQ, Tasaka k, Chuang CK, et al. Dynamic analysis of T-lymphocyte function in relation to hepatopathologic changes and effect of interleukin-12 treatment in mice infected with Schistosorna japonicum. J Parasitol, 1999; 85(2): 257-262
16. Hassan MM, Ramadan MA, Mostafa NE, et al. Different Cytokines profiles in spleen cells and liver granuloma of Schistosorna mansoni experimentally infected mice during disease development. J Egypt Soc Parasitol, 2000; 30(1): 245-256
17. Cheever AW, Jankovic D, Yap GS, et al. Role of cytokines in the formation and downregulation of hepatic circumoval granulomas and hepatic fibrosis in Schistosoma mansoni-infected mice. Mem Inst Oswaldo Cruz, 1998; 93(Suppll): 25-32
18. Lohning M, Grogan JL. Coyle AJ. et al. T_1/ST_2 expression is enhanced on CD_4~+ T cells from Schistosome egg-induced granulomas: analysis of Th cell cytokine coexpression ex vivo. J Immunol, 1999; 162(7): 3882-3889
19. Fallon PG, Richardson EJ, Mckenzie GJ, et al. Schistosome infection of transgenic mice defines distinct and contrasting pathogenic roles for IL-4 and IL-13: IL-13 is a profibrotic agent. J Immunol, 2000; 164(5): 2585-2591
20. Kaplan MH, Whitfield, IR, Boros DL, et al. Th_2 cells are required for the Schistosoma mansoni egg-induced granulomatous response. J Immunol, 1998: 160(4): 1850-1860
21．刘莉，沈一平，管晓虹，等。日本血吸虫感染小鼠脾细胞IL-2及IFN-γ动态观察。中国寄生虫学与寄生虫病杂志，1995；13(1)：35-38
22．胡永秀和薛燕萍。血吸虫卵肉芽肿的形成机制及免疫调节。寄生虫与医学昆虫学报，1997；4(2)：119-124
23. Hu Yongxiu, Xue Yangping, Tian Xiaojun, et al. Modulation of in vivo granuloma formation related to regulation of in vitro IFN-γ and IL-4 expressions in experimental Schistosomiasis japonica. Chinese Medical Journal. 1999: 112(8): 681-685.
24. Mwatha JK. Kimani G, Kaman T, et al. High levels of TNF. soluble TNF receptors, soluble ICAM-1, and IFN-γ. but low levels of IL-5, are associated with hepatosplenic disease in human Schistosomiasis mansoni. J Immunol, 1998: 160(4): 1992-1999
25. Montenegro SML, Miranda P, Mahanty S, et al. Cytokine production in acute versus chronic human Schistosomiasis mansoni: the cross-regulatory role of interferon-γ and interleukin-10 in the responses of peripheral blood mononuclear cells and splenocytes to parasite antigens. J Infect Dis, 1999; 179(6): 1502-1514
26. Falcao PL, Malaquias LCC, Martins-Filho DA, et al. Human Schistosomiasis mansoni: IL-10 modulates the in vitro granuloma formation. Parasite Immnuol, 1998; 20: 447-454
27. Wynn TA, Cheever AW, Williams ME, et al. IL-10 regulates liver vascular endothelial growth factor in cancer patients: which is the optimal specimen. Int J Oncol, 2000; 17(1): 149-152.
pathology in acute murine Schistosomiasis mansoni but is not required for immune down-modulation of chronic disease. J Immunol, 1998; 160(9) :4473-4480
28． Chiaramonte MG, Donaldson DD, Cheever AW, et al. An IL-13 inhibitor blocks the development of hepatic fibrosis during a T-helper type-2 dominated inflammatory response. J Clin Invest, 1999; 104(6) :777-785