胶体金对小鼠免疫活性影响的研究
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摘要
胶体金(Colloidal gold)是由氯金酸(HAuCl4)在还原剂的作用下聚合成的带负电的疏水胶溶液,具有高电子密度、介电特性和催化作用,能与多种生物大分子结合,且不影响其生物活性。1971年胶体金(CG)被Faulk正式引入免疫化学中后,在免疫金标记方面被广泛应用。近年来,科技工作者对胶体金的研究扩展到了免疫原性方面,但是目前国内外对胶体金的研究报导仅限于CG和其他物质(半抗原和抗原)结合后对试验动物免疫功能的影响方面,而对单独使用CG对试验动物免疫功能影响的研究,尚未见报导。
为了明确不同胶体金直径和剂量对动物机体免疫功能的影响,为胶体金在免疫原性方面的应用寻求理论依据。本研究在参考Dykman等人研究资料的基础上,通过对试验小鼠机体注射不同直径、不同剂量的CG,采取免疫学研究的测试方法,对试验小鼠机体的T淋巴细胞增殖活性、血清IL-2含量、腹腔巨噬细胞活性及脾脏NK细胞杀伤活性等能够衡量机体特异和非特异性免疫的重要指标进行了检测,研究结果如下:
实验一胶体金对小鼠脾脏和外周血T淋巴细胞增殖反应的影响。通过直径、剂量、时间三个因素对小鼠特异性细胞免疫水平的综合影响的研究,结果表明:与对照相比,不同直径的胶体金对小鼠脾脏和外周血T淋巴细胞增殖反应均有一定影响,但不同直径和剂量组合对其影响不同,其中5nm胶体金影响不显著, 10nm和15nm胶体金影响显著,而且剂量越大,效果越明显;以15nm,0.3 mL的胶体金对T淋巴细胞的影响最为显著。从时间上看,在14d时各处理组胶体金的效果最为明显。
实验二胶体金对小鼠外周血血清和脾脏淋巴细胞诱生白介素2(IL-2)含量的影响。采用免疫放射法,对试验小鼠外周血血清和脾脏淋巴细胞诱生IL-2含量的进行了测试,结果显示:脾脏淋巴细胞诱生的IL-2的规律性比血清中的规律明显,但是总体趋势相同。10nm和15nm组的CG在各个剂量组都可以使血清及其淋巴细胞诱导的IL-2分泌增多,且与剂量呈明显的正相关,但是5nm和20nm组的效果均不明显。
实验三免疫胶体金对小鼠腹腔巨噬细胞功能和脾脏NK细胞活性的影响。试验结果表明,与对照组相比,各直径组中的低、中、高剂量组均可明显增强巨嗜和NK细胞活性,并以15nm 0.2mL组最为显著,但是同直径组CG低、中、高三个剂量组之间,高剂量组优于中、低剂量组。
综合这三个试验的结果分析,可以说明,不同直径和剂量的CG对机体免疫活性有不同程度的影响,其中直径15nm、10nmCG的0.3mL和0.2mL剂量组不仅对T淋巴细胞增殖活性和血清中IL-2含量有显著促进作用,而且可显著提高腹腔巨噬细胞活性及脾脏NK细胞杀伤活性,而0.1mL剂量组虽有提高但不显著;直径5nm、20nm各个剂量组对机体免疫活性的影响效果波动较大,其中中、高剂量组对四项指标均有一定的促进作用,但效果不显著,低剂量组则没有促进作用。表明15nm和10nm中、高剂量的胶体金可显著提高小鼠机体特异性和非特异性细胞免疫功能。
Colloidal gold (CG) is an kind of hydrophobe solution gathered by the hydrochloro-auric acid under the electronegative circumstance, It has high electron density, electricity medium ability and the catalytic effect. It can combine many kinds of biological macro-molecule and does not affect its biological activity. After the colloidal gold (CG) was introduced into immunochemistry by the Faulk in 1971, it is widely applied in the area of immunity mark. In recent years, the researchers developed the immunity colloid gold and began to research the immunity original application. Rencently the reports of domestic and foreign research are only restricted in influence of combination of CG and other matter (half antigen and antigen) on the experimental animal immunity function, but it has no reported about influence of single CG on the experimental animal immunity function.
In view of this, from the reference to the former work of Dykman’s studies, in this paper we have injected different diameter, different dosage CG to the mice organism , using the test method of the immunology research. We tested the mice organism’s T-lymphocyte multiplication activeness, IL-2 content in the blood serum, abdominal cavity macrophage activeness and spleen NK cell activeness and so on, which are important indexes that can judge of the special and the non- specificity immunity of the organism. The examination has been carried on for the purpose of definitude the influence of CG on the animal organism immunity function , through the comparison between the different experimental group results and the comparison group results, and then we can carry on the preliminary discussion for the application of colloid gold in the immunity original area. The main results of this paper include:
Results 1: The influence of colloid gold on the mice spleen and serum T lymphocyte proliferating response. We synthetically studied the influence of colloid gold on the mice body fluid immunity level and the cellular immunity level through three factors that is the diameter, the dosage, the time. Result shows that the influence of colloid gold on the mice spleen and serum T lymphocyte proliferating response is different because of different diameter, the influence of 5nm colloid gold is not remarkable, 10nm and the 15nm is remarkable (p<0.05), and when the dosage is bigger, the effect is more remarkable. The influence of colloid gold which is 15nm, 0.3 mL on the T-lymphocyte is most remarkable. Measured by days, the influence of colloid gold every diameter which are14 days are most remarkable.
Result 2: The influence of colloid gold on content of IL-2 in the mice serum and spleen lymphocyte.This experimentation adopts RIA assays to mensurate the content of IL-2 in the mice serum and spleen lymphocyte.The spleen lymphocyte IL-2 has a better orderliness than the mice serum IL-2 ,but they have the same direction . Compared with the comparison group, content of IL-2 in the blood serum and lymphocyte of all kinds of 10 and 15nm groups are in the majority, and assume obvious direct ratio relations with the dosage.
Result 3: The influence of immune colloids gold on the activity of abdominal cavity macrophage and NK cell.The experiment shows that, compared with the comparison group, all the low, middle, the high doses team in various diameters group can notably strengthen the activity of abdominal cavity macrophage and NK cell, and the 15nm 0.2mL group is most remarkable. And in the same diameter group, the high doses team surpasses the low and middle monitoring team.
All the results shows that different effects on mice which injects CG for mice.0.3mL、0.2mL of 15 nm、10 nm CG can enhance the activity of lymphocyte proliferating and increase content of IL-2 in serum and induction in spleen lymphocyte, meanwhile they can also enhance the activity of abdominal cavity macrophage and NK cell notably; 0.1 mL doses can improve also but unconspicuous. The effects on the doses of 5nm、20nmCG is fluctuate,the group of middle and high doses can improve the four criterions but unconspicuous; the group of low doses have no effect. We can learn that the middle and high doses of 15 nm and 10 nm colloidal gold can enhancing specific and nonspecific immune response in laboratory mice.
为了明确不同胶体金直径和剂量对动物机体免疫功能的影响,为胶体金在免疫原性方面的应用寻求理论依据。本研究在参考Dykman等人研究资料的基础上,通过对试验小鼠机体注射不同直径、不同剂量的CG,采取免疫学研究的测试方法,对试验小鼠机体的T淋巴细胞增殖活性、血清IL-2含量、腹腔巨噬细胞活性及脾脏NK细胞杀伤活性等能够衡量机体特异和非特异性免疫的重要指标进行了检测,研究结果如下:
实验一胶体金对小鼠脾脏和外周血T淋巴细胞增殖反应的影响。通过直径、剂量、时间三个因素对小鼠特异性细胞免疫水平的综合影响的研究,结果表明:与对照相比,不同直径的胶体金对小鼠脾脏和外周血T淋巴细胞增殖反应均有一定影响,但不同直径和剂量组合对其影响不同,其中5nm胶体金影响不显著, 10nm和15nm胶体金影响显著,而且剂量越大,效果越明显;以15nm,0.3 mL的胶体金对T淋巴细胞的影响最为显著。从时间上看,在14d时各处理组胶体金的效果最为明显。
实验二胶体金对小鼠外周血血清和脾脏淋巴细胞诱生白介素2(IL-2)含量的影响。采用免疫放射法,对试验小鼠外周血血清和脾脏淋巴细胞诱生IL-2含量的进行了测试,结果显示:脾脏淋巴细胞诱生的IL-2的规律性比血清中的规律明显,但是总体趋势相同。10nm和15nm组的CG在各个剂量组都可以使血清及其淋巴细胞诱导的IL-2分泌增多,且与剂量呈明显的正相关,但是5nm和20nm组的效果均不明显。
实验三免疫胶体金对小鼠腹腔巨噬细胞功能和脾脏NK细胞活性的影响。试验结果表明,与对照组相比,各直径组中的低、中、高剂量组均可明显增强巨嗜和NK细胞活性,并以15nm 0.2mL组最为显著,但是同直径组CG低、中、高三个剂量组之间,高剂量组优于中、低剂量组。
综合这三个试验的结果分析,可以说明,不同直径和剂量的CG对机体免疫活性有不同程度的影响,其中直径15nm、10nmCG的0.3mL和0.2mL剂量组不仅对T淋巴细胞增殖活性和血清中IL-2含量有显著促进作用,而且可显著提高腹腔巨噬细胞活性及脾脏NK细胞杀伤活性,而0.1mL剂量组虽有提高但不显著;直径5nm、20nm各个剂量组对机体免疫活性的影响效果波动较大,其中中、高剂量组对四项指标均有一定的促进作用,但效果不显著,低剂量组则没有促进作用。表明15nm和10nm中、高剂量的胶体金可显著提高小鼠机体特异性和非特异性细胞免疫功能。
Colloidal gold (CG) is an kind of hydrophobe solution gathered by the hydrochloro-auric acid under the electronegative circumstance, It has high electron density, electricity medium ability and the catalytic effect. It can combine many kinds of biological macro-molecule and does not affect its biological activity. After the colloidal gold (CG) was introduced into immunochemistry by the Faulk in 1971, it is widely applied in the area of immunity mark. In recent years, the researchers developed the immunity colloid gold and began to research the immunity original application. Rencently the reports of domestic and foreign research are only restricted in influence of combination of CG and other matter (half antigen and antigen) on the experimental animal immunity function, but it has no reported about influence of single CG on the experimental animal immunity function.
In view of this, from the reference to the former work of Dykman’s studies, in this paper we have injected different diameter, different dosage CG to the mice organism , using the test method of the immunology research. We tested the mice organism’s T-lymphocyte multiplication activeness, IL-2 content in the blood serum, abdominal cavity macrophage activeness and spleen NK cell activeness and so on, which are important indexes that can judge of the special and the non- specificity immunity of the organism. The examination has been carried on for the purpose of definitude the influence of CG on the animal organism immunity function , through the comparison between the different experimental group results and the comparison group results, and then we can carry on the preliminary discussion for the application of colloid gold in the immunity original area. The main results of this paper include:
Results 1: The influence of colloid gold on the mice spleen and serum T lymphocyte proliferating response. We synthetically studied the influence of colloid gold on the mice body fluid immunity level and the cellular immunity level through three factors that is the diameter, the dosage, the time. Result shows that the influence of colloid gold on the mice spleen and serum T lymphocyte proliferating response is different because of different diameter, the influence of 5nm colloid gold is not remarkable, 10nm and the 15nm is remarkable (p<0.05), and when the dosage is bigger, the effect is more remarkable. The influence of colloid gold which is 15nm, 0.3 mL on the T-lymphocyte is most remarkable. Measured by days, the influence of colloid gold every diameter which are14 days are most remarkable.
Result 2: The influence of colloid gold on content of IL-2 in the mice serum and spleen lymphocyte.This experimentation adopts RIA assays to mensurate the content of IL-2 in the mice serum and spleen lymphocyte.The spleen lymphocyte IL-2 has a better orderliness than the mice serum IL-2 ,but they have the same direction . Compared with the comparison group, content of IL-2 in the blood serum and lymphocyte of all kinds of 10 and 15nm groups are in the majority, and assume obvious direct ratio relations with the dosage.
Result 3: The influence of immune colloids gold on the activity of abdominal cavity macrophage and NK cell.The experiment shows that, compared with the comparison group, all the low, middle, the high doses team in various diameters group can notably strengthen the activity of abdominal cavity macrophage and NK cell, and the 15nm 0.2mL group is most remarkable. And in the same diameter group, the high doses team surpasses the low and middle monitoring team.
All the results shows that different effects on mice which injects CG for mice.0.3mL、0.2mL of 15 nm、10 nm CG can enhance the activity of lymphocyte proliferating and increase content of IL-2 in serum and induction in spleen lymphocyte, meanwhile they can also enhance the activity of abdominal cavity macrophage and NK cell notably; 0.1 mL doses can improve also but unconspicuous. The effects on the doses of 5nm、20nmCG is fluctuate,the group of middle and high doses can improve the four criterions but unconspicuous; the group of low doses have no effect. We can learn that the middle and high doses of 15 nm and 10 nm colloidal gold can enhancing specific and nonspecific immune response in laboratory mice.
引文
[1] 冯娟,陈义平,王志亮.胶体金的应用研究新进展[J].中国动物检疫 2005,22(7)48～51.
[2] Faulk W, Taylorn G M. An immunocolloidal gold method for the electron microscope[J]. Immuno-chemistry, 1971,8:1081～1087.
[3] L.A.Dykman, M.V.Sumaroka, S.A. Staroverov, I.S. Zaitseva, V.A. Bogatyrev:Immunogenic Properties of Colloidal Gold [J]. Russian Academy of Sciences, January 2004, 31(1): 75～79.
[4] L.A. Dykman, Matora, L.Yu. , and Bogatyrev, V.A., Use of Colloidal Gold to Obtain Antibiotin Antibodies[J] Microbiol. Meth., 1996, 24:247~248.
[5] L.A. Dykman, Bogatyrev, V.A. Colloidal Gold in Solid-Phase Assays [J] Biochemistry (Moscow), 1997, 62 :411～418.
[6] Moses S, Brewer S H, Lowe L B, et al. Characterization of single-and double-stranded DNA on gold surfaces[J]. Langmuir, 2004, 20(25):34~40.
[7] 朱前勇,李志,陶生策等.胶体金标记/银染信号放大法在寡核苷酸芯片检测人HCM 突变的应用[J]. 第三军医大学学报,2004, 19 : 9~12.
[8] Boehmer. Immunogold silver staining:new method of immuno-staining with enhanced sensitivity[J]. Histochem cytochem, 1983, 31: 938～944.
[9] 杨中汉,陈朋,邵邻相等.玉米精细胞膜特异糖蛋自胶体金免疫定位[J].htip ://w w w .pku.edu.cn/aca-demis/xb/2000/ OOc208.html,1999-03-01/2000-03-20.
[10] 兰小鹏,黄俏佳,叶小份等.用快速斑点免疫金渗滤法检测抗双链 DMA 抗体[J]. 中华医学杂志,1996,2(19):92～94.
[11] 黄瑜,甘孟侯,刘尚高.银加强胶体金技术检测猪细小病毒的研究[J]. 畜牧兽医学报,1995,26(3):23～24.
[12] Shyu R H, Shyu H F, LiuH W et al. Colloidal gold 一 based immunochrom atographic assay for detection of ricinc[J].Toxicon, 2002, 40(3):255～258.
[13] Bucchler K F, Mols, Noar B et al. Simulianeous detection of seven dings of abuse by the triage panel for drug of abuse[J]. ClinChem, 1992, 38:1678～1684.
[14] Lou S C,Patel C, Chingsal. One step competitive immunochrom autographic assay for semiquantitative determination of lipoprotein in plasma [J]. Clin Chem,1993,39:619～624.
[15] 喻海燕,陶义训,曾瑞云等. 甲胎蛋白半定量金免疫层析测定 [J]. 中国检验医学与临床,2000(1):15～17.
[16] Cho J H,Paek S H. Semiquantitative barcodcvcrsion of immunochrom atographic assay system for human serum album in asmodel analyte[J]. Biotechnol B ioeng, 200175(6): 725～732.
[17] Kim J H, Joung-Hwan C,Sig CG,et al. Conductimetric membrane Scrip immunoscnsor with polyaniline-bound gold colloides assingnal generator [J]. Biotechnol B ioeng, 2000, 14:907～915.
[18] Braining K, Kozitsina A,BeikinJ. Electrochcmical immunosensor for Forest-Spring encephalitis based on protein A labeled with colloidal gold[J]. Anal BioanalChem,2003, 376(4):481～485.
[19]Mena M L, Yanez-Sedeno P, Pingarron J M .A comparison of different strategics for the construction of amperometric enzymebioscesors using gold nanopariscle-modified electrodes[J].Anal Biochcm,2005,336(1):20～7.
[20] Tang D, Yuan R, ChaiYet al. A novel immunosensor based on immobilization of hepatitis B surface antiody on platinum electrode modified colloidal gold and polyvinyl butyral as mairices via electrochemical impedance spec-troscopy[J] Biolectrochcm istry, 2004, 65 (1):15～22.
[21] Kitano H, Makino Y, Kawasaki H et al. Sel-assembled monolayer of a pepstatin fragment as a sensing element for aspartyl proteases [J]. Anal Chem, 2005,77(6):1588～1595
[22] Shiosaka S., Kiyama H, Wanaka A and Tohyama M. A New Method for Producing a Specific and High Titre Antibody against Glutamate Using Colloidal Gold as a Carrier, Brain Res[J].1998, 382, 399~403.
[23] Chen J H,Zou F, W ang N D,et al. Production and application of LPA polyclonal antibody[J]. BioorgMed ChemLets, 2000, 10 (15):1691~1693.
[24] Zhao Z,Wakita T, Yasui K. InocμLation of plasmids encoding Japanese encephalitis virus PM-E proteins with colloidal goldelicits a protective immune response in BALB /c mice [J]. Viro1, 2003, 77 (16):9107.
[25] Zhang L,Widera G. Rabussay D. Enhancement of the effectiveness of electroporation-augmented cutaneous DNA vaccination by a particμLate adjuvant[J]. Bioelectrochemistry,2004,63(18):369～373
[26] Hainfeld J F, Slaikin D N, Smilowitz H M. The use of gold nanoparticles to enhance radioiherapy in mice[J]. Phys med Biol, 2004, 49(18):309～315.
[27] Paciotti G F, Mycr L, Weinreich et al. Colloidal gold:a nove1 nanoparticle vector for tumor directed drug delivery[J] Drug Deliv, 2004, 11(3):169～83.
[28] 杜念兴,兽医免疫学[M]. 上海科学技术出版社, 1984, 272～274.
[29]Kirchner H, and Oppenheim J J. Stimulation of chicken lymphocytes in a serum-free medium[J]. CellIm-munol, 1972, 33(3):695～699.
[30] Hovi T, Suni J. Stimulation of chicken lymphocytes by T- and B-cell mitogens[J]. Cell Immunol, 1978, 39(1):70～78.
[31] Lee L F. In vitro assay of mitogen stimulation of avian peripheral lymphocytes[J].Avian Dis. 1974.18:602～609.
[32] Barta O, Barta V. Optimum conditions for the chicken lymphocyte transformation test[J]. Avian Diseases, 1992, 36(2):945～955.
[33] Maheswaran S K. Thies E S. Development of a microculture system for stimulation of chicken peripheral blood lymphocytes with concanavalin A. Am. [J]. Vet. Res, 1975, 36(3): 1053～1055.
[34] Lee L F, Sharma J M, Mazerian K. Suppression of mitogen-induced proliferation of normal spleen cells by macrophages from chickens inoculated with Marek's disease virus[J]. Immunol. 1978, 120:1554-1559.
[35] Powell P C. The influence of erythrocytes on the stimulation of chicken lymphocytes by phytohaemag-glutinin[J]. Avian Pathol. 1980, 9(2): 465～470.
[36] Lassila, O, Eskola,J. A micromethod for stimulation of chicken lymphocytes in vitro using whole blood. Clin. Exp[J]. Immunol. 1976, 26(4): 641～646.
[37] Lee L F, Liu X, and Witter R L. Monoclonal antibodies with specificity for three different serotypes of Marek’s disease viruses in chickens [J]. Immunol. 1983,130:1003～1006.
[38] 李庆章,刘忠贵. MTT 比色分析法检测鸡 T 淋巴细胞体外增殖反应的研究[J]. 中国兽医杂志, 1994, 9:18～20
[39] Lassila O, Eskola J. A micromethod for stimulation of chicken lymphocytes in vitro using whole blood. Clin. Exp[J]. Immunol. 1976, 26(4): 641～646.
[40] Nathanson R M. Transformation of chicken lymphocytes stimulated by concanavalin A: Response Pa-rameters. Can [J]. Comp. Med, 1982,46:57～59.
[41] 吴建设, 张日俊, 周毓平. 全血法鸡淋巴细胞转化试验最佳试验条件的研究. [J].畜牧兽医学报, 1997, 28(3), 212～216
[42] Thiel H J, Burkhardt E. Development of optimal conditions for the stimulation of chicken peripheralblood lymphocytes by phytohaemagglutimin (PHA) in the microculture system. Vet[J]. Immunol. Immunopathol, 1984. 6(3): 327～340.
[43] 刘华忠, 黄银燕, 陈绍红. 谷氨酰胺对雏鸡外周血 T 淋巴细胞转化作用的影响[J]. 饲料研究. 2004.6: 35～36
[44] Talebi, A. Torgerson,P,R. Optimal conditions for measurement of blastogenic responses of chickens to concanavalin A in whole blood assays. Vet[J]. Immunol. Immunopathol, 1995. 46(2): 293～301.
[45] Bounous V V. Immunological effects of reticuloen-dotheliosis virus as potential contaminant of Marek's disease vaccines[J]. Avian Pathol. 1977. 6:383-393.
[46] Maslak, Thies E S. Development of a microculture system for stimulation of chicken peripheral blood lymphocytes with concanavalin [J]. Vet. Res, 1975, 36(3): 1053～1055.
[47] 李祥瑞, 金红, 王秀丽等. 以 MTT 比色法检测鸡脾淋巴细胞转化效果.[J]. 畜牧与兽医,1996.28(1):3～5.
[48] Sung Y J.et al. [J] Leukoc Biol.1991. 50:49～60
[49] Lambrecht B.et al, Proceeding of Xith International Congress of the World Veterinary [J]. Poultry Association. 1997, 163～179
[50] Pertile T L, Karaca K, Sharma J M , Walser M M, An antiviral effect of nitric oxide: Inhibition of reovirus replication[J]. Avian Dis. 1996, 40: 342～348.
[51] Karaca K., Kim I. J, Reddy S K. Nitric oxide inducing factor as measure of antigen and mitogen specific T cell responses in chickens. [J]. Immunol. Methods. 1996, 192, 97～103.
[52] Ferrari M, Fornasicro MC, Isetta AM. MTT colorimetric assay for testing macroPHAge cytotoxic activity in vitro.[J]. Immunol Methods, 1990; 131: 165
[53] 田志刚, 张建华. MTT 法在检测细胞因子与细胞毒效应中的应用[J].中国肿瘤生物治疗杂志. 1994.1(1): 74-79
[54] 赖清金,林涛,王志平.免疫胶体金及其在畜牧生产中的应用[J].福建畜牧兽医,2005,27(1): 7～9
[55] 陈凤梅,李娟,曲原君.免疫胶体金技术的应用及研究进展[J].中国兽药杂志,2004,38(8): 33～35
[56] 陈哲生.唾哇蓝比色法用来研究人淋巴细胞和巨噬细胞活性. [J]上海免疫学杂志,1992,12(5):269～270
[57] 沈关心,周汝麟.现代免疫学试验技术(第二版) [M] 武汉:湖北科学技术出版社,2002:407～410
[58] 陶义训,杨振修.免疫诊断技术[J].中华消化杂志, 1995, 15:226
[59] 张成裕.免疫增强剂在动物医学上的研究进展及应用[J].福建畜牧兽医,2004,26(12):6～64.
[60] 温顺妮.治疗性疫苗的研究进展[J].广州医药,1999,30(1):8～10.
[61] 钱锋.佐剂的研究进展[J].国外医学军事医学分册,1995,12(1):8～11.
[62] 张汉荆.免疫佐剂 IRIV 的研究进展[J].国外医学微生物学分册,1995,28(9):4～6.
[64] 程富胜,胡庭俊,梁纪兰等.黄芪多糖对小鼠腹腔巨噬细胞一氧化氮生成的影响[J].中兽医医药杂志,2001,1:3～4.
[65] 王世若,王兴龙,韩文瑜.现代动物免疫学[M].第二版.吉林:吉林科学技术出版社,2001.
[66] 孙云,邓阳梅,蔡元培.复方三七口服液对小鼠免疫功能和抗氧化功能的影响[J]江苏临床医学杂志,2000,6(6):518
[67] 胡香云,许川山. Coγ 射线辐射小鼠脾淋巴细胞增殖活性的改变[J].激光杂志,2001,22(3):67
[68] 吴红彦,刘永琦,苏韫.复合生命肽对衰老模型小鼠免疫功能及过氧化作用的研究[J].中医药学刊,2002,20(6):823
[69] 凌小曼,丁虹,罗顺德.当归多糖对血瘀症动物免疫功能和抗氧化功能的影响[J].中国医院药学杂志,2002,22(10):584
[70] 何宏文,周丽华.人参对老年大鼠淋巴细胞功能的影响[J].中山大学学报(医学科学院报),2003,24(5):425
[71] 伍利民,袁晓辉,王雅贤.参芪王浆制剂对大龄大鼠血清 IL-2 和 T 淋巴细胞增殖功能的影响[J].哈尔滨医科大学学报,2005,10(5):415～416
[72] 胡东维.胶体金蛋自复合体制备中此问题的探讨[J].山东农业人学学报,1997,28:125～127.
[73] 丁明孝,梁凤霞,焦干杰,等.胶体金标记 DNA .RNA .蛋白质和糖的定性定位的研究[J].电子显微学报,1994,13(5):377
[74]上龙生,郭仁舆.胶体金标记术的原理与应用[J].解剖学杂志,1990, 13(2):164～167.
[75]张蜀秋,贾文琐,等.用胶体金免疫电镜技术观察水分胁迫对蚕豆根中 ABA分布与含量的影响[J].857～868.
[76]常国权,杨盛华.检测抗原抗体反应的新制剂一胶体金探针[[ ,T].中国兽医学报,1994,14(2) : 139～145.
[77]蒋治良.冯忠伟.李廷盛.等.金纳米粒子的共振散射光谱[M].中国科学(B 辑),2001,31(2):183～188
[78] 李祥瑞,闫慎飞..动物细胞因子的应用研究进展[J].畜牧与兽医, 1999,31(5):36～37
[79] 李庆章, 刘忠贵. MTT 比色分析法检测鸡 T 淋巴细胞体外增殖反应的研究[J]. 中国兽医杂志, 1994,9:18～20.
[80] 高春义,张建,赵跃然,等.蜂胶提取物抗肿瘤作用及对免疫功能的影响[J].中国中医药信息杂志,2000, 7 (6) : 27～28.
[81] 徐彩菊,傅剑云,夏勇,等.皇浆蜂胶粉对小鼠免疫功能的影响[J].实用预防医学,1996,6(6):410～411.
[82] 钟丕,李庆章,等.黄茂多糖和香菇多糖对雏鸡淋巴细胞化学发光的影响[J].中兽医医药杂志,1998,4(3): 5～7.
[83] 程富胜,胡庭俊,梁纪兰,等.黄茂多糖对小鼠腹腔巨噬细胞一氧化氮生成的影响仁[J].中兽医医药杂志,2001,3: 3～5.
[84]Mosmann T. Rapid colorimetric assay for cellar growth and survival:application to proliferation and cytotoxicity assays [J].Immunol. Methods, 1983, 65(1):55～63
[85] Abraham G E, Himmel P B. Management of Rheumatoid Arthritis: Rationale for the Use of Colloidal Metallic Gold [J]. Nutr. Med, 1997, 7:295～305.
[86] Dykman L A, Matora, L.Yu, Bogatyrev VA. Use of Colloidal Gold to Obtain Antibiotin Antibodies. [J] Microbiol. Meth,1996, 24:247～248.
[87] Gechegan WD. Passiver gold agglutination—an alternative to passive hemagglutination[J]. Immnol Methods, 1980, 34: 11.
[88] Beeley J E. Immunocytochemistry: A practical approach, 1st ed.[M]. Oxford: Oxford University Press Inc,1993. 115.
[89] Leuvering JHW. Sol particle immunoassay [J]. Immunoassay, 1980, 1: 77.
[90] Rocks BF, Patel N, Bailey MP. Use of a silver enhanced goldlabeled immunoassay for detection of antibodies to human Immun-odeficiency virvus[J]. Ann Clin Biochem, 1991, 27: 114.
[91] Osikowicz G. One-step chromatographic immunoassay for qualitative determination of choriogonadotropin in urine[J]. Clin Chem,1990,36:1586.
[92] Fowler SJ. The detection of proteins on blot using gold or immuno-gold[J]. Methods Mol Biol, 1994, 32: 239
[93] Ryan EJ,DalyM,Mills K H,et al.Immunomodulators and Del6ivery systems for vaccination by mucosal routes[J].Trends Biotechnol, 2001, 19(8):293～304.
[94] Hu K F,Lovgren Bengts son K,Morein B,et al.Immuno- stimulating complexes for nasal vaccination[J].AdvDrug Deliever, 2001, 51(13): 149~159
[95] Frens G.Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions [J]. Nat.Phys.Sci, 1973, 241(105):20.
[96]Abraham, G.E. and Himmel, P.B, Management of Rheumatoid Arthritis: Rationale for the Use of Colloidal Metallic Gold[J]. Nutr. Med, 1997, 7:295～305.
[97]Andreev S M, Babakhin A A, Petrukhina A O, Roma-nova V S. Immunogenic and Allergenic Properties of Fulleren Conjugates with Amino Acids and Proteins, Dokl. Ross. Akad. Nauk, 2000, 370:261~264.
[99]Dou X, Jung Y M, Yamamoto H. Near-Infrared Excited Surface-Enhanced Raman Scattering of Biological Molecules on Gold Colloid I: Effects of pH of the Solutions of Amino Acids and of Their Polymerization[J]. Appl. Spectrosc, 1999, 53:133~138.
[100]Frens G,Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions[J]. Nature Phys. Sci, 1973, 241:20~22.
[101]Fukasawa M, Liposome Oligomannan-Coated with Neoglycolipid, a New Candidate for a Safe Adjuvant for Induction of CD8+ Cytotoxic T-Lymphocytes, [J].FEBS Lett, 1998. 441:353~356.
[2] Faulk W, Taylorn G M. An immunocolloidal gold method for the electron microscope[J]. Immuno-chemistry, 1971,8:1081～1087.
[3] L.A.Dykman, M.V.Sumaroka, S.A. Staroverov, I.S. Zaitseva, V.A. Bogatyrev:Immunogenic Properties of Colloidal Gold [J]. Russian Academy of Sciences, January 2004, 31(1): 75～79.
[4] L.A. Dykman, Matora, L.Yu. , and Bogatyrev, V.A., Use of Colloidal Gold to Obtain Antibiotin Antibodies[J] Microbiol. Meth., 1996, 24:247~248.
[5] L.A. Dykman, Bogatyrev, V.A. Colloidal Gold in Solid-Phase Assays [J] Biochemistry (Moscow), 1997, 62 :411～418.
[6] Moses S, Brewer S H, Lowe L B, et al. Characterization of single-and double-stranded DNA on gold surfaces[J]. Langmuir, 2004, 20(25):34~40.
[7] 朱前勇,李志,陶生策等.胶体金标记/银染信号放大法在寡核苷酸芯片检测人HCM 突变的应用[J]. 第三军医大学学报,2004, 19 : 9~12.
[8] Boehmer. Immunogold silver staining:new method of immuno-staining with enhanced sensitivity[J]. Histochem cytochem, 1983, 31: 938～944.
[9] 杨中汉,陈朋,邵邻相等.玉米精细胞膜特异糖蛋自胶体金免疫定位[J].htip ://w w w .pku.edu.cn/aca-demis/xb/2000/ OOc208.html,1999-03-01/2000-03-20.
[10] 兰小鹏,黄俏佳,叶小份等.用快速斑点免疫金渗滤法检测抗双链 DMA 抗体[J]. 中华医学杂志,1996,2(19):92～94.
[11] 黄瑜,甘孟侯,刘尚高.银加强胶体金技术检测猪细小病毒的研究[J]. 畜牧兽医学报,1995,26(3):23～24.
[12] Shyu R H, Shyu H F, LiuH W et al. Colloidal gold 一 based immunochrom atographic assay for detection of ricinc[J].Toxicon, 2002, 40(3):255～258.
[13] Bucchler K F, Mols, Noar B et al. Simulianeous detection of seven dings of abuse by the triage panel for drug of abuse[J]. ClinChem, 1992, 38:1678～1684.
[14] Lou S C,Patel C, Chingsal. One step competitive immunochrom autographic assay for semiquantitative determination of lipoprotein in plasma [J]. Clin Chem,1993,39:619～624.
[15] 喻海燕,陶义训,曾瑞云等. 甲胎蛋白半定量金免疫层析测定 [J]. 中国检验医学与临床,2000(1):15～17.
[16] Cho J H,Paek S H. Semiquantitative barcodcvcrsion of immunochrom atographic assay system for human serum album in asmodel analyte[J]. Biotechnol B ioeng, 200175(6): 725～732.
[17] Kim J H, Joung-Hwan C,Sig CG,et al. Conductimetric membrane Scrip immunoscnsor with polyaniline-bound gold colloides assingnal generator [J]. Biotechnol B ioeng, 2000, 14:907～915.
[18] Braining K, Kozitsina A,BeikinJ. Electrochcmical immunosensor for Forest-Spring encephalitis based on protein A labeled with colloidal gold[J]. Anal BioanalChem,2003, 376(4):481～485.
[19]Mena M L, Yanez-Sedeno P, Pingarron J M .A comparison of different strategics for the construction of amperometric enzymebioscesors using gold nanopariscle-modified electrodes[J].Anal Biochcm,2005,336(1):20～7.
[20] Tang D, Yuan R, ChaiYet al. A novel immunosensor based on immobilization of hepatitis B surface antiody on platinum electrode modified colloidal gold and polyvinyl butyral as mairices via electrochemical impedance spec-troscopy[J] Biolectrochcm istry, 2004, 65 (1):15～22.
[21] Kitano H, Makino Y, Kawasaki H et al. Sel-assembled monolayer of a pepstatin fragment as a sensing element for aspartyl proteases [J]. Anal Chem, 2005,77(6):1588～1595
[22] Shiosaka S., Kiyama H, Wanaka A and Tohyama M. A New Method for Producing a Specific and High Titre Antibody against Glutamate Using Colloidal Gold as a Carrier, Brain Res[J].1998, 382, 399~403.
[23] Chen J H,Zou F, W ang N D,et al. Production and application of LPA polyclonal antibody[J]. BioorgMed ChemLets, 2000, 10 (15):1691~1693.
[24] Zhao Z,Wakita T, Yasui K. InocμLation of plasmids encoding Japanese encephalitis virus PM-E proteins with colloidal goldelicits a protective immune response in BALB /c mice [J]. Viro1, 2003, 77 (16):9107.
[25] Zhang L,Widera G. Rabussay D. Enhancement of the effectiveness of electroporation-augmented cutaneous DNA vaccination by a particμLate adjuvant[J]. Bioelectrochemistry,2004,63(18):369～373
[26] Hainfeld J F, Slaikin D N, Smilowitz H M. The use of gold nanoparticles to enhance radioiherapy in mice[J]. Phys med Biol, 2004, 49(18):309～315.
[27] Paciotti G F, Mycr L, Weinreich et al. Colloidal gold:a nove1 nanoparticle vector for tumor directed drug delivery[J] Drug Deliv, 2004, 11(3):169～83.
[28] 杜念兴,兽医免疫学[M]. 上海科学技术出版社, 1984, 272～274.
[29]Kirchner H, and Oppenheim J J. Stimulation of chicken lymphocytes in a serum-free medium[J]. CellIm-munol, 1972, 33(3):695～699.
[30] Hovi T, Suni J. Stimulation of chicken lymphocytes by T- and B-cell mitogens[J]. Cell Immunol, 1978, 39(1):70～78.
[31] Lee L F. In vitro assay of mitogen stimulation of avian peripheral lymphocytes[J].Avian Dis. 1974.18:602～609.
[32] Barta O, Barta V. Optimum conditions for the chicken lymphocyte transformation test[J]. Avian Diseases, 1992, 36(2):945～955.
[33] Maheswaran S K. Thies E S. Development of a microculture system for stimulation of chicken peripheral blood lymphocytes with concanavalin A. Am. [J]. Vet. Res, 1975, 36(3): 1053～1055.
[34] Lee L F, Sharma J M, Mazerian K. Suppression of mitogen-induced proliferation of normal spleen cells by macrophages from chickens inoculated with Marek's disease virus[J]. Immunol. 1978, 120:1554-1559.
[35] Powell P C. The influence of erythrocytes on the stimulation of chicken lymphocytes by phytohaemag-glutinin[J]. Avian Pathol. 1980, 9(2): 465～470.
[36] Lassila, O, Eskola,J. A micromethod for stimulation of chicken lymphocytes in vitro using whole blood. Clin. Exp[J]. Immunol. 1976, 26(4): 641～646.
[37] Lee L F, Liu X, and Witter R L. Monoclonal antibodies with specificity for three different serotypes of Marek’s disease viruses in chickens [J]. Immunol. 1983,130:1003～1006.
[38] 李庆章,刘忠贵. MTT 比色分析法检测鸡 T 淋巴细胞体外增殖反应的研究[J]. 中国兽医杂志, 1994, 9:18～20
[39] Lassila O, Eskola J. A micromethod for stimulation of chicken lymphocytes in vitro using whole blood. Clin. Exp[J]. Immunol. 1976, 26(4): 641～646.
[40] Nathanson R M. Transformation of chicken lymphocytes stimulated by concanavalin A: Response Pa-rameters. Can [J]. Comp. Med, 1982,46:57～59.
[41] 吴建设, 张日俊, 周毓平. 全血法鸡淋巴细胞转化试验最佳试验条件的研究. [J].畜牧兽医学报, 1997, 28(3), 212～216
[42] Thiel H J, Burkhardt E. Development of optimal conditions for the stimulation of chicken peripheralblood lymphocytes by phytohaemagglutimin (PHA) in the microculture system. Vet[J]. Immunol. Immunopathol, 1984. 6(3): 327～340.
[43] 刘华忠, 黄银燕, 陈绍红. 谷氨酰胺对雏鸡外周血 T 淋巴细胞转化作用的影响[J]. 饲料研究. 2004.6: 35～36
[44] Talebi, A. Torgerson,P,R. Optimal conditions for measurement of blastogenic responses of chickens to concanavalin A in whole blood assays. Vet[J]. Immunol. Immunopathol, 1995. 46(2): 293～301.
[45] Bounous V V. Immunological effects of reticuloen-dotheliosis virus as potential contaminant of Marek's disease vaccines[J]. Avian Pathol. 1977. 6:383-393.
[46] Maslak, Thies E S. Development of a microculture system for stimulation of chicken peripheral blood lymphocytes with concanavalin [J]. Vet. Res, 1975, 36(3): 1053～1055.
[47] 李祥瑞, 金红, 王秀丽等. 以 MTT 比色法检测鸡脾淋巴细胞转化效果.[J]. 畜牧与兽医,1996.28(1):3～5.
[48] Sung Y J.et al. [J] Leukoc Biol.1991. 50:49～60
[49] Lambrecht B.et al, Proceeding of Xith International Congress of the World Veterinary [J]. Poultry Association. 1997, 163～179
[50] Pertile T L, Karaca K, Sharma J M , Walser M M, An antiviral effect of nitric oxide: Inhibition of reovirus replication[J]. Avian Dis. 1996, 40: 342～348.
[51] Karaca K., Kim I. J, Reddy S K. Nitric oxide inducing factor as measure of antigen and mitogen specific T cell responses in chickens. [J]. Immunol. Methods. 1996, 192, 97～103.
[52] Ferrari M, Fornasicro MC, Isetta AM. MTT colorimetric assay for testing macroPHAge cytotoxic activity in vitro.[J]. Immunol Methods, 1990; 131: 165
[53] 田志刚, 张建华. MTT 法在检测细胞因子与细胞毒效应中的应用[J].中国肿瘤生物治疗杂志. 1994.1(1): 74-79
[54] 赖清金,林涛,王志平.免疫胶体金及其在畜牧生产中的应用[J].福建畜牧兽医,2005,27(1): 7～9
[55] 陈凤梅,李娟,曲原君.免疫胶体金技术的应用及研究进展[J].中国兽药杂志,2004,38(8): 33～35
[56] 陈哲生.唾哇蓝比色法用来研究人淋巴细胞和巨噬细胞活性. [J]上海免疫学杂志,1992,12(5):269～270
[57] 沈关心,周汝麟.现代免疫学试验技术(第二版) [M] 武汉:湖北科学技术出版社,2002:407～410
[58] 陶义训,杨振修.免疫诊断技术[J].中华消化杂志, 1995, 15:226
[59] 张成裕.免疫增强剂在动物医学上的研究进展及应用[J].福建畜牧兽医,2004,26(12):6～64.
[60] 温顺妮.治疗性疫苗的研究进展[J].广州医药,1999,30(1):8～10.
[61] 钱锋.佐剂的研究进展[J].国外医学军事医学分册,1995,12(1):8～11.
[62] 张汉荆.免疫佐剂 IRIV 的研究进展[J].国外医学微生物学分册,1995,28(9):4～6.
[64] 程富胜,胡庭俊,梁纪兰等.黄芪多糖对小鼠腹腔巨噬细胞一氧化氮生成的影响[J].中兽医医药杂志,2001,1:3～4.
[65] 王世若,王兴龙,韩文瑜.现代动物免疫学[M].第二版.吉林:吉林科学技术出版社,2001.
[66] 孙云,邓阳梅,蔡元培.复方三七口服液对小鼠免疫功能和抗氧化功能的影响[J]江苏临床医学杂志,2000,6(6):518
[67] 胡香云,许川山. Coγ 射线辐射小鼠脾淋巴细胞增殖活性的改变[J].激光杂志,2001,22(3):67
[68] 吴红彦,刘永琦,苏韫.复合生命肽对衰老模型小鼠免疫功能及过氧化作用的研究[J].中医药学刊,2002,20(6):823
[69] 凌小曼,丁虹,罗顺德.当归多糖对血瘀症动物免疫功能和抗氧化功能的影响[J].中国医院药学杂志,2002,22(10):584
[70] 何宏文,周丽华.人参对老年大鼠淋巴细胞功能的影响[J].中山大学学报(医学科学院报),2003,24(5):425
[71] 伍利民,袁晓辉,王雅贤.参芪王浆制剂对大龄大鼠血清 IL-2 和 T 淋巴细胞增殖功能的影响[J].哈尔滨医科大学学报,2005,10(5):415～416
[72] 胡东维.胶体金蛋自复合体制备中此问题的探讨[J].山东农业人学学报,1997,28:125～127.
[73] 丁明孝,梁凤霞,焦干杰,等.胶体金标记 DNA .RNA .蛋白质和糖的定性定位的研究[J].电子显微学报,1994,13(5):377
[74]上龙生,郭仁舆.胶体金标记术的原理与应用[J].解剖学杂志,1990, 13(2):164～167.
[75]张蜀秋,贾文琐,等.用胶体金免疫电镜技术观察水分胁迫对蚕豆根中 ABA分布与含量的影响[J].857～868.
[76]常国权,杨盛华.检测抗原抗体反应的新制剂一胶体金探针[[ ,T].中国兽医学报,1994,14(2) : 139～145.
[77]蒋治良.冯忠伟.李廷盛.等.金纳米粒子的共振散射光谱[M].中国科学(B 辑),2001,31(2):183～188
[78] 李祥瑞,闫慎飞..动物细胞因子的应用研究进展[J].畜牧与兽医, 1999,31(5):36～37
[79] 李庆章, 刘忠贵. MTT 比色分析法检测鸡 T 淋巴细胞体外增殖反应的研究[J]. 中国兽医杂志, 1994,9:18～20.
[80] 高春义,张建,赵跃然,等.蜂胶提取物抗肿瘤作用及对免疫功能的影响[J].中国中医药信息杂志,2000, 7 (6) : 27～28.
[81] 徐彩菊,傅剑云,夏勇,等.皇浆蜂胶粉对小鼠免疫功能的影响[J].实用预防医学,1996,6(6):410～411.
[82] 钟丕,李庆章,等.黄茂多糖和香菇多糖对雏鸡淋巴细胞化学发光的影响[J].中兽医医药杂志,1998,4(3): 5～7.
[83] 程富胜,胡庭俊,梁纪兰,等.黄茂多糖对小鼠腹腔巨噬细胞一氧化氮生成的影响仁[J].中兽医医药杂志,2001,3: 3～5.
[84]Mosmann T. Rapid colorimetric assay for cellar growth and survival:application to proliferation and cytotoxicity assays [J].Immunol. Methods, 1983, 65(1):55～63
[85] Abraham G E, Himmel P B. Management of Rheumatoid Arthritis: Rationale for the Use of Colloidal Metallic Gold [J]. Nutr. Med, 1997, 7:295～305.
[86] Dykman L A, Matora, L.Yu, Bogatyrev VA. Use of Colloidal Gold to Obtain Antibiotin Antibodies. [J] Microbiol. Meth,1996, 24:247～248.
[87] Gechegan WD. Passiver gold agglutination—an alternative to passive hemagglutination[J]. Immnol Methods, 1980, 34: 11.
[88] Beeley J E. Immunocytochemistry: A practical approach, 1st ed.[M]. Oxford: Oxford University Press Inc,1993. 115.
[89] Leuvering JHW. Sol particle immunoassay [J]. Immunoassay, 1980, 1: 77.
[90] Rocks BF, Patel N, Bailey MP. Use of a silver enhanced goldlabeled immunoassay for detection of antibodies to human Immun-odeficiency virvus[J]. Ann Clin Biochem, 1991, 27: 114.
[91] Osikowicz G. One-step chromatographic immunoassay for qualitative determination of choriogonadotropin in urine[J]. Clin Chem,1990,36:1586.
[92] Fowler SJ. The detection of proteins on blot using gold or immuno-gold[J]. Methods Mol Biol, 1994, 32: 239
[93] Ryan EJ,DalyM,Mills K H,et al.Immunomodulators and Del6ivery systems for vaccination by mucosal routes[J].Trends Biotechnol, 2001, 19(8):293～304.
[94] Hu K F,Lovgren Bengts son K,Morein B,et al.Immuno- stimulating complexes for nasal vaccination[J].AdvDrug Deliever, 2001, 51(13): 149~159
[95] Frens G.Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions [J]. Nat.Phys.Sci, 1973, 241(105):20.
[96]Abraham, G.E. and Himmel, P.B, Management of Rheumatoid Arthritis: Rationale for the Use of Colloidal Metallic Gold[J]. Nutr. Med, 1997, 7:295～305.
[97]Andreev S M, Babakhin A A, Petrukhina A O, Roma-nova V S. Immunogenic and Allergenic Properties of Fulleren Conjugates with Amino Acids and Proteins, Dokl. Ross. Akad. Nauk, 2000, 370:261~264.
[99]Dou X, Jung Y M, Yamamoto H. Near-Infrared Excited Surface-Enhanced Raman Scattering of Biological Molecules on Gold Colloid I: Effects of pH of the Solutions of Amino Acids and of Their Polymerization[J]. Appl. Spectrosc, 1999, 53:133~138.
[100]Frens G,Controlled Nucleation for the Regulation of the Particle Size in Monodisperse Gold Suspensions[J]. Nature Phys. Sci, 1973, 241:20~22.
[101]Fukasawa M, Liposome Oligomannan-Coated with Neoglycolipid, a New Candidate for a Safe Adjuvant for Induction of CD8+ Cytotoxic T-Lymphocytes, [J].FEBS Lett, 1998. 441:353~356.