乳酸杆菌对宫颈癌Hela细胞抑制作用机制的研究
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摘要
目的:观察乳酸杆菌对宫颈癌细胞株Hela细胞的抑制作用,进一步探讨乳酸杆菌对宫颈癌细胞杀伤作用的机制。从而为开发出安全、有效、低成本的预防和治疗宫颈癌的药物打下基础。
方法:1.胎盘兰染色在油镜下观察并计数不同状态的乳酸杆菌与宫颈癌细胞株Hela细胞共培养后在不同时间阶段的粘附指数及粘附后细胞活力的变化。2.用流式细胞仪(抗体直接免疫荧光技术)检测乳酸杆菌对宫颈癌细胞表面表型(CD80、CD86、MHC-1)的影响。3.用MTT法观察乳酸杆菌调节免疫细胞(NK细胞、CTL细胞)对宫颈癌细胞杀伤作用的影响。
结果:
1.乳酸杆菌与宫颈癌Hela细胞株作用后粘附指数及细胞活力的
1.1油镜下观察乳酸杆菌对Hela细胞的黏附抑制作用:肿瘤细胞培养12小时后,培养液均清亮,对照组细胞生长密集,生长状态良好,光泽度好;细胞伸展,呈不规则梭形。各乳酸杆菌组细胞密度较对照组低,光泽度下降。灭活组乳酸杆菌对Hela细胞的黏附密集度明显高于活菌组,细胞活力较好。48小时后见对照组细胞,已有重叠生长,乳酸杆菌组细胞,出现细胞减少,稀疏,细胞形态破坏,细胞变形,细胞皱缩,体积变小,细胞间隙增大,变圆。胞膜破裂,肿胀等。灭活组较活菌组更加显著,并且随着时间延长变化更加明显。
1.2两组乳酸杆菌黏附指数比较,活菌组乳酸杆菌黏附指数随着培养时间延长逐渐增加,在培养12小时达高峰,随后随着时间延长,黏附指数逐渐降低,灭活组与活菌组相似,在每一个培养时间比较,灭活组乳酸杆菌粘附指数均高于活菌组。1.3乳酸杆菌黏附后Hela细胞活力比较:在共培养1 h、3 h、6 h阶段两组Hela细胞活力无差异,但在培养12h时,活菌组细胞活力突然下降,而灭活组细胞活力仍保持在高水平,再随着时间的延长,灭活组共培养24h宫颈癌细胞活力开始下降,并低于活菌组(P<0.01)。2.乳酸杆菌对宫颈癌细胞表面抗原(MHC-1类分子、CD80和CD86分子)表达的影响测定(流式细胞技术):2.1不同状态的乳酸杆菌对宫颈癌细胞表面抗原表达的影响:经乳酸杆菌作用后两种状态的乳酸杆菌组的宫颈癌细胞表面抗原表达率均明显高于对照组,灭活菌组与活菌组对促进宫颈癌细胞表面抗原表达作用具有显著差异性,与灭活的乳酸杆菌作用后宫颈癌Hela细胞表面抗原表达率明显高于活菌组。2.2对照组与乳酸杆菌宫颈癌细胞表面抗原表达的影响:对照组宫颈癌细胞表面抗原(MHC-1、CD80、CD86)表达率极低。经乳酸杆菌作用后两种状态的乳酸杆菌组的宫颈癌细胞表面抗原表达率均明显高于对照组(P<0.05)。
3.MTT法测定乳酸杆菌调节免疫细胞对宫颈癌细胞杀伤作用的结果: 3.1不同浓度乳酸杆菌作用后NK细胞、CTL细胞对Hela细胞的杀伤活性实验结果表明:乳酸杆菌作用后明显增强NK细胞、CTL细胞对宫颈癌Hela细胞的杀伤活性,并且NK细胞、CTL细胞与不同浓度乳酸杆菌作用后杀伤活性存在显著差异性(P〈0.05),以效靶比是20:1时杀伤活性最强,具有浓度依赖性。3.2不同时间阶段乳酸杆菌作用后NK细胞、CTL细胞对Hela细胞的杀伤活性结果表明:NK细胞、CTL细胞与乳酸杆菌作用不同时间后其杀伤活性存在显著差异性,并且随着时间延长(12、24、48、72)其杀伤活性随之增强,有时间依赖性。
结论:1.乳酸杆菌能很好的黏附并抑制宫颈癌Hela细胞的生长,灭活的乳酸杆菌对宫颈癌细胞有着更好的黏附抑制作用,并且具有时间依赖性。
2.乳酸杆菌能上调宫颈癌细胞表面抗原CD80、CD86、MHC-1的表达,灭活的乳酸杆菌作用后宫颈癌Hela细胞表面抗原表达率明显高于活菌组。
3.用MTT法实验结果表明乳酸杆菌能促进免疫细胞(NK细胞、CTL细胞)对宫颈癌细胞的杀伤作用,并且呈剂量-时间依赖性。
Objective: Lactic acid bacteria observed in cervical cancer cell lines Hela cell line, Lactobacillus further explore the role of cervical cancer cell killing mechanisms So as to develop safe, effective, low-cost prevention and treatment of cervical cancer drug basis.
Methods:1 Placental blue staining were observed in the oil and Lactobacillus counts in different states of Hela cells and cervical cancer cell lines were cultured at different time stages of the adhesion index and changes in cell viability after adhesion.
2 Using flow cytometry (antibody direct immunofluorescence) cervical cancer cells by Lactobacillus on the surface of the table type (CD80, CD86, MHC-1) effects.
3 MTT was observed with Lactobacillus immune cells (NK cells, CTL cells) in cervical cancer cell killing effects.
Results:
1 Lactic acid bacteria and Hela cervical cancer cell lines and cell viability after the adhesion index results:
1.1 Oil microscope Lactobacillus inhibited the adhesion of Hela cells: Tumor cells cultured for 12 hours, medium are clear, and control cell growth-intensive, growth in good condition, good gloss; Cell extension, irregular spindle. The density of Lactobacillus cells compared with control group, decreased gloss. Inactivated Lactobacillus group on the adhesion of Hela cell density was significantly higher than viable group, cell viability better. After 48 hours, see the control cells, have overlapping growth of Lactobacillus cells, cells appeared less sparse, destruction of cell morphology, cell shape, cell shrinkage, smaller size, cell gap increases, and round. Membrane rupture, swelling. Inactivated group more significant than the viable group, and the more obvious changes with time. Adhesion index of .
1.2 compared two groups of lactobacilli, Lactobacillus viable group adherence index increased gradually with culture time, A peak at 12 hours of incubation, and then with time, adhesion decreased gradually Inactivated and viable group was similar, in every culture time compared with inactivated Lactobacillus adhesion index group were higher than viable group.
1.3 Lactobacillus adhesion Hela cell viability after the comparison: in the co-culture 1 h, 3 h, 6 h phase difference between the two groups no Hela cell viability, 12h in culture, the vitality of a sudden drop in viable cells, while the activity of cells inactivated remained at a high level. Then with time, inactivation of group co-cultured cancer cell viability began to decrease 24h and less than the viable group (P <0.01).
2 actic acid bacteria on the cancer cell surface antigen (MHC-1 molecules, CD80 and CD86 molecules) expression assay (flow cytometry): Cells in the negative control group well grew adhesively,most in shuttle shape,moderately sized,clear nuceolus,nulear division phase found.Cells shrinkage after treatment by acitretin,the breakdown was irregular in shape,some cells appear at both ends of the pseudo-foot slender.INF-stimulated cells showed the number of bipolar cells increase.The cells treated by acitretin and INF were present in the supernatant.The density of adherence cells was diminished when compared with the control.
2.1 Lactobacillus different states of cervical cancer cell surface antigen expression: The role of lactic acid bacteria Lactobacillus two states after the group's cervical cancer cell surface antigen expression were significantly higher. Inactivated bacteria group and viable group to promote the role of cervical cancer cell surface antigen expression in a significant difference, and after inactivation of the role of Lactobacillus Hela cervical cancer cell surface antigen expression was significantly higher than the viable group.
2.2 in control group and the Lactobacillus cervical cancer cell surface antigen expression: Control cervical cancer cell surface antigen (MHC-1, CD80, CD86) expression was extremely low. Control cervical cancer cell surface antigen (MHC-1, CD80, CD86) expression was extremely low. The role of lactic acid bacteria Lactobacillus two states after the group's cervical cancer cell surface antigen expression were significantly higher (P <0.05).
3 Determination of lactic acid bacteria 3.MTT regulate immune cells in cervical cancer cell killing effect of:
3.1 The role of different concentration of lactic acid bacteria after the NK cells, CTL cell killing activity of Hela cells results show that: Lactobacillus markedly enhanced the role of NK cells after, CTL cell killing activity of cervical cancer Hela cells, and NK cells, CTL cells with different concentrations of the role of lactic acid bacteria killing activity after the significant difference (P <0.05). To effectively target ratio is 20:1 strongest cytotoxic activity, with concentration-dependent.
3.2 The role of different time stages after Lactobacillus NK cells, CTL cell killing activity of Hela cells results show that: NK cells, CTL cells with Lactobacillus different time of their killing activity of significant differences, and with time (12,24,48,72) the killing activity increases, and there are time-dependent.
Conclusion:
1 Lactobacillus well adhesion and inhibit cervical Hela cell growth, live Lactobacillus cervical cells and cervical cell has better adhesion inhibition and has time dependent.
2 Lactobacillus could upregulate cervical cell surface antigen CD80, CD86, MHC-1 expression inactivated Lactobacillus role cervical carcinoma after Hela cell surface antigen expression rate significantly higher than viable group.
3 Using MTT method results show Lactobacillus can promote immune cells (NK cell, CTL cells) on cervical Cells and dose - time dependent.
方法:1.胎盘兰染色在油镜下观察并计数不同状态的乳酸杆菌与宫颈癌细胞株Hela细胞共培养后在不同时间阶段的粘附指数及粘附后细胞活力的变化。2.用流式细胞仪(抗体直接免疫荧光技术)检测乳酸杆菌对宫颈癌细胞表面表型(CD80、CD86、MHC-1)的影响。3.用MTT法观察乳酸杆菌调节免疫细胞(NK细胞、CTL细胞)对宫颈癌细胞杀伤作用的影响。
结果:
1.乳酸杆菌与宫颈癌Hela细胞株作用后粘附指数及细胞活力的
1.1油镜下观察乳酸杆菌对Hela细胞的黏附抑制作用:肿瘤细胞培养12小时后,培养液均清亮,对照组细胞生长密集,生长状态良好,光泽度好;细胞伸展,呈不规则梭形。各乳酸杆菌组细胞密度较对照组低,光泽度下降。灭活组乳酸杆菌对Hela细胞的黏附密集度明显高于活菌组,细胞活力较好。48小时后见对照组细胞,已有重叠生长,乳酸杆菌组细胞,出现细胞减少,稀疏,细胞形态破坏,细胞变形,细胞皱缩,体积变小,细胞间隙增大,变圆。胞膜破裂,肿胀等。灭活组较活菌组更加显著,并且随着时间延长变化更加明显。
1.2两组乳酸杆菌黏附指数比较,活菌组乳酸杆菌黏附指数随着培养时间延长逐渐增加,在培养12小时达高峰,随后随着时间延长,黏附指数逐渐降低,灭活组与活菌组相似,在每一个培养时间比较,灭活组乳酸杆菌粘附指数均高于活菌组。1.3乳酸杆菌黏附后Hela细胞活力比较:在共培养1 h、3 h、6 h阶段两组Hela细胞活力无差异,但在培养12h时,活菌组细胞活力突然下降,而灭活组细胞活力仍保持在高水平,再随着时间的延长,灭活组共培养24h宫颈癌细胞活力开始下降,并低于活菌组(P<0.01)。2.乳酸杆菌对宫颈癌细胞表面抗原(MHC-1类分子、CD80和CD86分子)表达的影响测定(流式细胞技术):2.1不同状态的乳酸杆菌对宫颈癌细胞表面抗原表达的影响:经乳酸杆菌作用后两种状态的乳酸杆菌组的宫颈癌细胞表面抗原表达率均明显高于对照组,灭活菌组与活菌组对促进宫颈癌细胞表面抗原表达作用具有显著差异性,与灭活的乳酸杆菌作用后宫颈癌Hela细胞表面抗原表达率明显高于活菌组。2.2对照组与乳酸杆菌宫颈癌细胞表面抗原表达的影响:对照组宫颈癌细胞表面抗原(MHC-1、CD80、CD86)表达率极低。经乳酸杆菌作用后两种状态的乳酸杆菌组的宫颈癌细胞表面抗原表达率均明显高于对照组(P<0.05)。
3.MTT法测定乳酸杆菌调节免疫细胞对宫颈癌细胞杀伤作用的结果: 3.1不同浓度乳酸杆菌作用后NK细胞、CTL细胞对Hela细胞的杀伤活性实验结果表明:乳酸杆菌作用后明显增强NK细胞、CTL细胞对宫颈癌Hela细胞的杀伤活性,并且NK细胞、CTL细胞与不同浓度乳酸杆菌作用后杀伤活性存在显著差异性(P〈0.05),以效靶比是20:1时杀伤活性最强,具有浓度依赖性。3.2不同时间阶段乳酸杆菌作用后NK细胞、CTL细胞对Hela细胞的杀伤活性结果表明:NK细胞、CTL细胞与乳酸杆菌作用不同时间后其杀伤活性存在显著差异性,并且随着时间延长(12、24、48、72)其杀伤活性随之增强,有时间依赖性。
结论:1.乳酸杆菌能很好的黏附并抑制宫颈癌Hela细胞的生长,灭活的乳酸杆菌对宫颈癌细胞有着更好的黏附抑制作用,并且具有时间依赖性。
2.乳酸杆菌能上调宫颈癌细胞表面抗原CD80、CD86、MHC-1的表达,灭活的乳酸杆菌作用后宫颈癌Hela细胞表面抗原表达率明显高于活菌组。
3.用MTT法实验结果表明乳酸杆菌能促进免疫细胞(NK细胞、CTL细胞)对宫颈癌细胞的杀伤作用,并且呈剂量-时间依赖性。
Objective: Lactic acid bacteria observed in cervical cancer cell lines Hela cell line, Lactobacillus further explore the role of cervical cancer cell killing mechanisms So as to develop safe, effective, low-cost prevention and treatment of cervical cancer drug basis.
Methods:1 Placental blue staining were observed in the oil and Lactobacillus counts in different states of Hela cells and cervical cancer cell lines were cultured at different time stages of the adhesion index and changes in cell viability after adhesion.
2 Using flow cytometry (antibody direct immunofluorescence) cervical cancer cells by Lactobacillus on the surface of the table type (CD80, CD86, MHC-1) effects.
3 MTT was observed with Lactobacillus immune cells (NK cells, CTL cells) in cervical cancer cell killing effects.
Results:
1 Lactic acid bacteria and Hela cervical cancer cell lines and cell viability after the adhesion index results:
1.1 Oil microscope Lactobacillus inhibited the adhesion of Hela cells: Tumor cells cultured for 12 hours, medium are clear, and control cell growth-intensive, growth in good condition, good gloss; Cell extension, irregular spindle. The density of Lactobacillus cells compared with control group, decreased gloss. Inactivated Lactobacillus group on the adhesion of Hela cell density was significantly higher than viable group, cell viability better. After 48 hours, see the control cells, have overlapping growth of Lactobacillus cells, cells appeared less sparse, destruction of cell morphology, cell shape, cell shrinkage, smaller size, cell gap increases, and round. Membrane rupture, swelling. Inactivated group more significant than the viable group, and the more obvious changes with time. Adhesion index of .
1.2 compared two groups of lactobacilli, Lactobacillus viable group adherence index increased gradually with culture time, A peak at 12 hours of incubation, and then with time, adhesion decreased gradually Inactivated and viable group was similar, in every culture time compared with inactivated Lactobacillus adhesion index group were higher than viable group.
1.3 Lactobacillus adhesion Hela cell viability after the comparison: in the co-culture 1 h, 3 h, 6 h phase difference between the two groups no Hela cell viability, 12h in culture, the vitality of a sudden drop in viable cells, while the activity of cells inactivated remained at a high level. Then with time, inactivation of group co-cultured cancer cell viability began to decrease 24h and less than the viable group (P <0.01).
2 actic acid bacteria on the cancer cell surface antigen (MHC-1 molecules, CD80 and CD86 molecules) expression assay (flow cytometry): Cells in the negative control group well grew adhesively,most in shuttle shape,moderately sized,clear nuceolus,nulear division phase found.Cells shrinkage after treatment by acitretin,the breakdown was irregular in shape,some cells appear at both ends of the pseudo-foot slender.INF-stimulated cells showed the number of bipolar cells increase.The cells treated by acitretin and INF were present in the supernatant.The density of adherence cells was diminished when compared with the control.
2.1 Lactobacillus different states of cervical cancer cell surface antigen expression: The role of lactic acid bacteria Lactobacillus two states after the group's cervical cancer cell surface antigen expression were significantly higher. Inactivated bacteria group and viable group to promote the role of cervical cancer cell surface antigen expression in a significant difference, and after inactivation of the role of Lactobacillus Hela cervical cancer cell surface antigen expression was significantly higher than the viable group.
2.2 in control group and the Lactobacillus cervical cancer cell surface antigen expression: Control cervical cancer cell surface antigen (MHC-1, CD80, CD86) expression was extremely low. Control cervical cancer cell surface antigen (MHC-1, CD80, CD86) expression was extremely low. The role of lactic acid bacteria Lactobacillus two states after the group's cervical cancer cell surface antigen expression were significantly higher (P <0.05).
3 Determination of lactic acid bacteria 3.MTT regulate immune cells in cervical cancer cell killing effect of:
3.1 The role of different concentration of lactic acid bacteria after the NK cells, CTL cell killing activity of Hela cells results show that: Lactobacillus markedly enhanced the role of NK cells after, CTL cell killing activity of cervical cancer Hela cells, and NK cells, CTL cells with different concentrations of the role of lactic acid bacteria killing activity after the significant difference (P <0.05). To effectively target ratio is 20:1 strongest cytotoxic activity, with concentration-dependent.
3.2 The role of different time stages after Lactobacillus NK cells, CTL cell killing activity of Hela cells results show that: NK cells, CTL cells with Lactobacillus different time of their killing activity of significant differences, and with time (12,24,48,72) the killing activity increases, and there are time-dependent.
Conclusion:
1 Lactobacillus well adhesion and inhibit cervical Hela cell growth, live Lactobacillus cervical cells and cervical cell has better adhesion inhibition and has time dependent.
2 Lactobacillus could upregulate cervical cell surface antigen CD80, CD86, MHC-1 expression inactivated Lactobacillus role cervical carcinoma after Hela cell surface antigen expression rate significantly higher than viable group.
3 Using MTT method results show Lactobacillus can promote immune cells (NK cell, CTL cells) on cervical Cells and dose - time dependent.
引文
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21 Bontkes HJ Walboomers Jmmeijer cj.et alspecific HLAclass I down-negulation is an early event in cervical clysplasia associated with clinical Pmgression Lancet 1998.351(13-14):187-188
22 Fema AP,Luceri C,Dolara P,et al.Antitumorigenic activity of the prebiotic inulin enriched with oligofructose in combination with the probiotics Lactobacillus rhamnosus and Bifidobacterium lactis on a- zoxymethane-induce colon carcinogenesis in rats. Carcinogenesis, 2002, 23 (11): 1953-1956
23 Coconnier M H, Bernet MF, Kerneis S, et al. Inhibition of adhesion ofenteroinvasive pathogens to human intestinal Caco-2 cells by Lactobacillus acidophilus strain LB decreases bacterial invasion. FEMS Microbiology, 1993, 116(4):299-306
24 OPCH,OOSTERHOFA,VEERKAMP JH. Interaction ofbifidobac-terial lipoteichoic acid with human intestinal epithelial cells. Infect Immun, 1985, 47(1): 332-334
25姜国湖,董全江,纪令士.干酪乳杆菌对免疫效应细胞杀伤作用影响的研究.医学检验与临床,2008, 19(5): 7-8
26罗毅平,陈慎仁,扬伟. CD80和CD86在食管癌组织中的表达.海南医学院学报.2006,12(3):204-206
27刘彤,唐立,王林美,袁杰利,李华军,张婵,文姝.乳酸杆菌发酵滤液对人宫颈癌细胞株Hela细胞的抑制作用.中国微生态学杂志. 2009, 21(9): 773-775
28 Cario E, Brown D, Mckee M, et a.l Commensal-associated molecular patterns induce selective toll-like receptor-trafficking from apicalmem -brane to cytoplasmic compartments in polarized intestinal epithelium. TheAmerican JournalofPathology, 2002,160(1): 165~173
29 Biffi A,Coradini D,Larsen R,et al.Antiproliferative effect of fer-mented milk on the growth of a human brest cancer cell line.Nutr- Cancer, 1997,28(1):93-96
30 Ohashi Y,Nakai S,Tsukamoto T,et al.habitual intake of lactic acid bacteria and risk reduction of bladder cancei.Urol Int,2002,68(4):273-275
1 Cocommier M H, Bernet MF, Kerneis S, et al. Inhibition of adhesion ofenteroinvasive pathogens to human intestinal Caco-2 cells by Lactobacillus acidophilus strain LB decreases bacterial invasion. FEMS Microbiology, 1993, 116(4):299-306
2 Fema AP,Luceri C,Dolara P,et al.Antitumorigenic activity of the prebiotic inulin enriched with oligofructose in combination with the probiotics Lactobacillus rhamnosus and Bifidobacterium lactis on a- zoxymethane- induce colon carcinogenesis rats. Carcinogenesis,2002,23(11):1953-1956
3 Biffi A,Coradini D,Larsen R,et al.Antiproliferative effect of fer-mented milk on the growth of a human brest cancer cell line.Nutr- Cancer, 1997,28(1):93-96
4 Eastcott JW, Hdmberg CJ, Dewhirst FE,et al. Oligonucleotide containing CpGmotifs enhances immune response to mucosally or systemically administered tetanus toxoid. [J] Vaccine 2001, 19 (13-14):1636-1642
5 Zabala A, Martin MR,Haza AI, et al. Anti-proliferative effect of towlactic acid bacteria strains of human origin on the growth of a myeloma cell line.Lett Appl Microbiol, 2001,32(4):287-292.
6 Ohashi Y,Nakai S, Tsukamoto T,et al. Habitual intake of lactic acidbacteria and risk reduction of bladder cancer. Urol Int, 2002,68(4):273-280.
7 Seow SW, Rahmat JN, Mohamed AA, et al. Lactobacillus species ismore cytotoxic to human bladder cancer cells than Mycobacterium Bovis(bacillus Calmette-Guerin).J Urol, 2002,168(5):2236-2239.
8 OPCH,OOSTERHOFA,VEERKAMP JH. Interaction ofbifidobac-terial lipoteichoic acid with human intestinal epithelial cells. Infect Immun, 1985, 47(1): 332-334
9 Ahmed KA,Xie Y,Zhang X,et al.Acquired pMHC I complexes greatly enhance CD4(+)Th cell's stimulatory effect on CD8(+)T cell-mediated diabetes in transgenic RIP-mOVA mice.Cell MolImmunol,2008,5(6):407-415.
10 Cario E, Brown D, Mckee M, et a.l Commensal-associatedmolecular patterns induce selective toll-like receptor-traffickingfrom apicalmem- branetocytoplasmic compartments in polarizedintestinal epithelium. TheAmerican JournalofPathology, 2002,160(1): 165-173
11 AROTTA F,NAlTO Y,MINELLI E,et a1.Chemopreventive effect of a probiotic preparation on the development of preneoplastic and neoplastic colonic lesions:an experimental study[J].Hepatogastroenterology,2003,50(54):1914-1918
12 IM BK,MAHENDRAN R,LEE YK,et al.Chemopreventive ef-fect of Lactobacillus rhamnosus on growth of a subcutaneously im-planted bladder cancer cell line in the mouse[J].Jpn J Cancer Res, 2002 , 93(1):36-41
13 De WAARD R,CAASSEN E,BOKKEN GC,et al.Enhanced Im- munological Memory Responses to Listeria monocytogenes in Ro-dents, as Measured by Delayed-Type Hypersensitivity (DTH), Adoptive Transfer of DTH, and Protective Immunity, followingLactobacillus casei Shirota Ingestion Clinical and Diagnostic Labo-ratory Immunology, 2003,10(1):59-65
14 Cario E, Brown D, Mckee M, et a.l Commensal-associated molecular patterns induce selective toll-like receptor-trafficking from apicalmembrane to cytoplasmic compartments in polarized intestinal epithelium. TheAmerican JournalofPathology, 2002,160(1): 165~173
15 GILL HS,RUTHERFURD KJ,et al.Enhancement of naturaland acquired immunity by Lactobacillus rhamnosus (HN001),Lactobacillus acidophilus (HN017) and Bifidobacterium lactis(HN019)[J].Br J Nutr.2000 Feb, 83 (2):167-176
16 POCHARD P,GOSSET P,et al.Lactic acid bacteria inhibit TH2 cytokine production by mononuclear cells from allergic patients [J].J Allergy Clin Immunol,2002 ,110(4):617-623
17 PERDIGON G,ALVAREZ S,RACHID M,et al.Immune system-stimulation by probiotics[J].J Dairy Sci,1995,78:1597-1606
18 Mochizuki K, Hayashi N, Katayama K, et al. B7 /BB21 exp ression and hepatitis activity in liver tissues of patientswith chronic hepatitis C. Hepatology, 1997, 25 (3) : 713- 718
19 Li ZS, Yang XW, Chen Z, et al. In vivo tumor co-transfection withsuperantigen and CD80 induces systemic immunity without toleranceand p rolongs survival in mice with hepatocellular carcinoma. CancerB iol Ther, 2004, 3 (7) : 660- 666
20李秀真,朱万浮,曹卉,等.乳酸杆菌诱导小鼠宫颈鳞癌U14细胞凋亡的动物实验.中华微生物和免疫学杂志,2004,24(4):268-271
21 CHU R S,HEMMIO H,TAKEUCHIO, et a.l A toll-like receptor rec- ognizes bacterialDNANature,2000, 408(6813): 740-745.
22任美玉,吴欣怡. Toll样受体的研究进展[J]现代免疫学, 2006,26(04):340-342.
23马艳琴,李光富,朱鸿飞,等.体外筛选对猪具有免疫刺激活性的CpG ODN[J].现代免疫学, 2003,23(4):252-259.
24 4Vinderola G,Matar C,Perdigon G·Role of intestinal epithelial cells in immune effectsmediated by gram-positive probiotic bacteria:involvement of toll-like receptors[J]·Clin Diagn Lab Immunol,2005;12(9):1075-1084·
25 Grangette C, Muller-Alouf H, Hols P, et al. Enhancedmucosal delivery of antigen with cell wall mutants of lacticacid bacteria[J]. Infect Immun, 2004,72(5):2731-2737
26 KIM H A,KOHM, JUHW, eta.l CpG-ODN-based immunotherapy is effective in controlling the growth ofmetastasized tumor cells[J]. Canc-erLett, 2009, 274(1): 160-164
27 Macpherson AJ, Uhr T. Induction of protective IgA by in- testinal dendritic cells carrying commensal bacteria[J]. Sci-ence, 2004, 303 (5664): 1662-1665
28 Litonjua AA, Sparrow D, Guevarra L,et al. Serum interferon-gamma is associated with longitudinal decline in lung function among asthmatic patients: the Normative Aging Study[ J]. AnnAllergyAsthma Immuno,l 2003, 90(4): 422-428
29 BiazeME, Boniface S, KoscherV,etal. T cell activation, from atopy to asthma: more a paradox than a paradigm[ J]. Allergy,2003, 58(9): 844-853.
30 Takahashi T,KushiroA,Nomoto K,et al.Antitumor effects of the in-instillation of heat killed cell of the Lactobacillus casei strain Shirota on th orthotopic bladder tumor MBT-2.J Urol,2001,(166)6:2506-2511
31 Galdeano C M,Perdigon G·Role of viability of probiotic strains in their persistence in the gut and inmucosal immune stimulation[J]·JApplMi- crobiol,2004;97(4):673-681
32 HANNE R·Lactobacilli Differentially Modulate Expression of Cytokines and Maturation Surface Markers in Murine Dendritic Cells [J]·The Journal of Immunology, 2002, 168: 171-178
2 Eastcott JW, Hdmberg CJ, Dewhirst FE,et al. Oligonucleotide containing CpGmotifs enhances immune response to mucosally or systemically administered tetanus toxoid. Vaccine 2001,19(13-14):1636-1642
3张卓然等,三种细菌DNA及CpG寡核苷酸抗肿瘤免疫的比较实验研究.微生物学杂志.2005,25(1):5-10
4 Kitazawa H, Watanabe H, Shimosato T, et al. Immunostimulatory oligonucleotide, CpG-like motif exists in Lactobacillus delbrueckii ssp. bulgaricus NIAI B6. Int J Food Microbiol, 2003, (85)122:11-21
5 Lammers KM ,Brigidi P ,Vit B ,et a1. Immunondulatory effects of probi-otic bacteria DNA: IL - 1 and IL - 10 response in human peripheral blood mononudear cells. FEMS Immunol Med Microbiol ,2003 ,38 (2) : 165 -172
6 Chen T, Isomaki P, Rimpilainen M, et al. Human cytokine responsesinduced by gram-positive cell walls of normal intestinal microbiota. Clin Exp Immunol,1999,118(2):261-267
7 Mochizuki K, Hayashi N, Katayama K, et al. B7 /BB21 exp ression and hepatitis activity in liver tissues of patientswith chronic hepatitis C. Hepatology, 1997, 25 (3) : 713 - 718
8 Li ZS, Yang XW, Chen Z, et al. In vivo tumor co2transfection withsuperantigen and CD80 induces systemic immunity without toleranceand p rolongs survival in mice with hepatocellular carcinoma. CancerB iol Ther, 2004, 3 (7) : 660 - 666
9 Johnson BD, Gershan JA, Natalia N, et al. Neuroblastoma cells tran2siently transfected to simultaneously exp ress the co2stimulatory mole2cules CD54, CD80, CD86, and CD137L generate antitumor immunity n mice. J Imm unother, 2005, 28 (5) : 449 - 460
10 Lustgarten J, Dominguez AL, Thoman M, et al. Aged mice develop p rotective antitumor immune responses with app rop riate costimulation. J Imm unol, 2004, 173 (7) : 4510 - 4515
11王佑民,杨明功,刘树琴.长期高血糖对继发磺脲类药物失效患者胰岛β细胞功能的影响.中华糖尿病杂志,2004, 12( 1) :2-4
12 Cenuth S. Management of the adult onset diabetic with sulfonylurea drug failure. Endocrinol Metab Clin North Am,1992,21:351- 370
13 Cascinelli N,Rovini D,Illeni M T,et al.In consistent expression ofHLA-B antigens on peripheral blood lymphocytes of stage I melanomapatients:an indicator of poor prognosis.Eur J Cancer,1994,30A(3):294~298
14 Ferrone S,Marincola F M.Lost of HLA class I antigens by melanomacells : molecular functional significance and clinical revelance.Immunol Today , 1995 , 16:487
15陈家伦,陈家伟,陈璐路,等.磺脲类药物应用专家共识.国外医学(内分泌分册) , 2004,24( 4):255-259
16 Helmich BK, Dutton R W. The role of adoptively trans- ferred CD8 T cells and host cells in the control of thegrowth of the EG7 thymoma: factors that determine therelative effectiveness and homing properties of Tc1 andTc2 effectors. J Immuno,l 2001, 166 (11): 6500-6508
17张百川,孟祥晨,益生菌抗肿瘤作用的分子机制的研究进展.现代食品科技,2005,21(4):88-92
18 SHIDA K,TAKAHASHI R,IWADATE E,et al.Lactobacillus casei strain Shirota suppresses serum immunoglobulin E and im-munoglobulin G1 responses and systemic anaphylaxis in a food al-lergy model.Clin Exp Allergy,2002,32(4):563-570
19 BiazeME, Boniface S, KoscherV,etal. T cell activation, from atopy to asthma: more a paradox than a paradigm. Allergy,2003, 58(9): 844-853
20 Weillemien JVD Tjiong MY,Himders CGJM、etal. Associa-tion of allelespefic HLA.expression and histopathollogic progression of cervical carcinoma Gynecol oncol. 1996.62(1): 330-341
21 Bontkes HJ Walboomers Jmmeijer cj.et alspecific HLAclass I down-negulation is an early event in cervical clysplasia associated with clinical Pmgression Lancet 1998.351(13-14):187-188
22 Fema AP,Luceri C,Dolara P,et al.Antitumorigenic activity of the prebiotic inulin enriched with oligofructose in combination with the probiotics Lactobacillus rhamnosus and Bifidobacterium lactis on a- zoxymethane-induce colon carcinogenesis in rats. Carcinogenesis, 2002, 23 (11): 1953-1956
23 Coconnier M H, Bernet MF, Kerneis S, et al. Inhibition of adhesion ofenteroinvasive pathogens to human intestinal Caco-2 cells by Lactobacillus acidophilus strain LB decreases bacterial invasion. FEMS Microbiology, 1993, 116(4):299-306
24 OPCH,OOSTERHOFA,VEERKAMP JH. Interaction ofbifidobac-terial lipoteichoic acid with human intestinal epithelial cells. Infect Immun, 1985, 47(1): 332-334
25姜国湖,董全江,纪令士.干酪乳杆菌对免疫效应细胞杀伤作用影响的研究.医学检验与临床,2008, 19(5): 7-8
26罗毅平,陈慎仁,扬伟. CD80和CD86在食管癌组织中的表达.海南医学院学报.2006,12(3):204-206
27刘彤,唐立,王林美,袁杰利,李华军,张婵,文姝.乳酸杆菌发酵滤液对人宫颈癌细胞株Hela细胞的抑制作用.中国微生态学杂志. 2009, 21(9): 773-775
28 Cario E, Brown D, Mckee M, et a.l Commensal-associated molecular patterns induce selective toll-like receptor-trafficking from apicalmem -brane to cytoplasmic compartments in polarized intestinal epithelium. TheAmerican JournalofPathology, 2002,160(1): 165~173
29 Biffi A,Coradini D,Larsen R,et al.Antiproliferative effect of fer-mented milk on the growth of a human brest cancer cell line.Nutr- Cancer, 1997,28(1):93-96
30 Ohashi Y,Nakai S,Tsukamoto T,et al.habitual intake of lactic acid bacteria and risk reduction of bladder cancei.Urol Int,2002,68(4):273-275
1 Cocommier M H, Bernet MF, Kerneis S, et al. Inhibition of adhesion ofenteroinvasive pathogens to human intestinal Caco-2 cells by Lactobacillus acidophilus strain LB decreases bacterial invasion. FEMS Microbiology, 1993, 116(4):299-306
2 Fema AP,Luceri C,Dolara P,et al.Antitumorigenic activity of the prebiotic inulin enriched with oligofructose in combination with the probiotics Lactobacillus rhamnosus and Bifidobacterium lactis on a- zoxymethane- induce colon carcinogenesis rats. Carcinogenesis,2002,23(11):1953-1956
3 Biffi A,Coradini D,Larsen R,et al.Antiproliferative effect of fer-mented milk on the growth of a human brest cancer cell line.Nutr- Cancer, 1997,28(1):93-96
4 Eastcott JW, Hdmberg CJ, Dewhirst FE,et al. Oligonucleotide containing CpGmotifs enhances immune response to mucosally or systemically administered tetanus toxoid. [J] Vaccine 2001, 19 (13-14):1636-1642
5 Zabala A, Martin MR,Haza AI, et al. Anti-proliferative effect of towlactic acid bacteria strains of human origin on the growth of a myeloma cell line.Lett Appl Microbiol, 2001,32(4):287-292.
6 Ohashi Y,Nakai S, Tsukamoto T,et al. Habitual intake of lactic acidbacteria and risk reduction of bladder cancer. Urol Int, 2002,68(4):273-280.
7 Seow SW, Rahmat JN, Mohamed AA, et al. Lactobacillus species ismore cytotoxic to human bladder cancer cells than Mycobacterium Bovis(bacillus Calmette-Guerin).J Urol, 2002,168(5):2236-2239.
8 OPCH,OOSTERHOFA,VEERKAMP JH. Interaction ofbifidobac-terial lipoteichoic acid with human intestinal epithelial cells. Infect Immun, 1985, 47(1): 332-334
9 Ahmed KA,Xie Y,Zhang X,et al.Acquired pMHC I complexes greatly enhance CD4(+)Th cell's stimulatory effect on CD8(+)T cell-mediated diabetes in transgenic RIP-mOVA mice.Cell MolImmunol,2008,5(6):407-415.
10 Cario E, Brown D, Mckee M, et a.l Commensal-associatedmolecular patterns induce selective toll-like receptor-traffickingfrom apicalmem- branetocytoplasmic compartments in polarizedintestinal epithelium. TheAmerican JournalofPathology, 2002,160(1): 165-173
11 AROTTA F,NAlTO Y,MINELLI E,et a1.Chemopreventive effect of a probiotic preparation on the development of preneoplastic and neoplastic colonic lesions:an experimental study[J].Hepatogastroenterology,2003,50(54):1914-1918
12 IM BK,MAHENDRAN R,LEE YK,et al.Chemopreventive ef-fect of Lactobacillus rhamnosus on growth of a subcutaneously im-planted bladder cancer cell line in the mouse[J].Jpn J Cancer Res, 2002 , 93(1):36-41
13 De WAARD R,CAASSEN E,BOKKEN GC,et al.Enhanced Im- munological Memory Responses to Listeria monocytogenes in Ro-dents, as Measured by Delayed-Type Hypersensitivity (DTH), Adoptive Transfer of DTH, and Protective Immunity, followingLactobacillus casei Shirota Ingestion Clinical and Diagnostic Labo-ratory Immunology, 2003,10(1):59-65
14 Cario E, Brown D, Mckee M, et a.l Commensal-associated molecular patterns induce selective toll-like receptor-trafficking from apicalmembrane to cytoplasmic compartments in polarized intestinal epithelium. TheAmerican JournalofPathology, 2002,160(1): 165~173
15 GILL HS,RUTHERFURD KJ,et al.Enhancement of naturaland acquired immunity by Lactobacillus rhamnosus (HN001),Lactobacillus acidophilus (HN017) and Bifidobacterium lactis(HN019)[J].Br J Nutr.2000 Feb, 83 (2):167-176
16 POCHARD P,GOSSET P,et al.Lactic acid bacteria inhibit TH2 cytokine production by mononuclear cells from allergic patients [J].J Allergy Clin Immunol,2002 ,110(4):617-623
17 PERDIGON G,ALVAREZ S,RACHID M,et al.Immune system-stimulation by probiotics[J].J Dairy Sci,1995,78:1597-1606
18 Mochizuki K, Hayashi N, Katayama K, et al. B7 /BB21 exp ression and hepatitis activity in liver tissues of patientswith chronic hepatitis C. Hepatology, 1997, 25 (3) : 713- 718
19 Li ZS, Yang XW, Chen Z, et al. In vivo tumor co-transfection withsuperantigen and CD80 induces systemic immunity without toleranceand p rolongs survival in mice with hepatocellular carcinoma. CancerB iol Ther, 2004, 3 (7) : 660- 666
20李秀真,朱万浮,曹卉,等.乳酸杆菌诱导小鼠宫颈鳞癌U14细胞凋亡的动物实验.中华微生物和免疫学杂志,2004,24(4):268-271
21 CHU R S,HEMMIO H,TAKEUCHIO, et a.l A toll-like receptor rec- ognizes bacterialDNANature,2000, 408(6813): 740-745.
22任美玉,吴欣怡. Toll样受体的研究进展[J]现代免疫学, 2006,26(04):340-342.
23马艳琴,李光富,朱鸿飞,等.体外筛选对猪具有免疫刺激活性的CpG ODN[J].现代免疫学, 2003,23(4):252-259.
24 4Vinderola G,Matar C,Perdigon G·Role of intestinal epithelial cells in immune effectsmediated by gram-positive probiotic bacteria:involvement of toll-like receptors[J]·Clin Diagn Lab Immunol,2005;12(9):1075-1084·
25 Grangette C, Muller-Alouf H, Hols P, et al. Enhancedmucosal delivery of antigen with cell wall mutants of lacticacid bacteria[J]. Infect Immun, 2004,72(5):2731-2737
26 KIM H A,KOHM, JUHW, eta.l CpG-ODN-based immunotherapy is effective in controlling the growth ofmetastasized tumor cells[J]. Canc-erLett, 2009, 274(1): 160-164
27 Macpherson AJ, Uhr T. Induction of protective IgA by in- testinal dendritic cells carrying commensal bacteria[J]. Sci-ence, 2004, 303 (5664): 1662-1665
28 Litonjua AA, Sparrow D, Guevarra L,et al. Serum interferon-gamma is associated with longitudinal decline in lung function among asthmatic patients: the Normative Aging Study[ J]. AnnAllergyAsthma Immuno,l 2003, 90(4): 422-428
29 BiazeME, Boniface S, KoscherV,etal. T cell activation, from atopy to asthma: more a paradox than a paradigm[ J]. Allergy,2003, 58(9): 844-853.
30 Takahashi T,KushiroA,Nomoto K,et al.Antitumor effects of the in-instillation of heat killed cell of the Lactobacillus casei strain Shirota on th orthotopic bladder tumor MBT-2.J Urol,2001,(166)6:2506-2511
31 Galdeano C M,Perdigon G·Role of viability of probiotic strains in their persistence in the gut and inmucosal immune stimulation[J]·JApplMi- crobiol,2004;97(4):673-681
32 HANNE R·Lactobacilli Differentially Modulate Expression of Cytokines and Maturation Surface Markers in Murine Dendritic Cells [J]·The Journal of Immunology, 2002, 168: 171-178