乳酸菌胞外多糖对结肠癌HT-29细胞增殖的影响
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摘要
从15株大连工业大学大连市益生菌功能研究重点实验室保藏植物乳杆菌中筛选出对人结肠癌细胞HT-29抑制率最高的乳杆菌胞外多糖,对其进行分离纯化并研究多糖对HT-29细胞增殖的影响。通过苯酚硫酸法测定15种乳酸菌胞外多糖产量、3-(4,5-二甲基吡啶-2-基)-5-(3-羧基甲氧基苯基)-2-(4-磺苯基)-2H-四唑[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,MTS]测定不同浓度的粗多糖对HT-29细胞的抑制率,最终筛选出高产胞外多糖、对HT-29细胞增殖抑制率高的菌株Lactobacillus plantarum12所产胞外多糖,其胞外多糖的糖含量为54%、蛋白含量为3.6%。对其胞外多糖进行DEAE-Sepharose Fast Flow离子柱和Sepharose CL-6B凝胶柱的分离纯化,得到中性多糖组分EPS12-1和酸性多糖组分EPS12-2,2个多糖组分的糖含量分别为31.82%和35.21%,通过MTS法测定L. plantarum12菌所产粗多糖、多糖组分EPS12-1和EPS12-2分别在50、100、250、500μg/mL浓度时对HT-29细胞的抑制率,发现多糖浓度为250μg/mL时,对HT-29细胞增殖达到了最佳抑制效果,抑制率分别为27.58%、7.11%、12.00%。当多糖浓度为250μg/mL时,HT-29细胞凋亡率、活性氧(reactive oxygen species,ROS)水平、caspase 8活性,均处在较高水平。L. plantarum12胞外多糖通过增加ROS酶活和激活caspase 8来抑制HT-29增殖。
The exopolysaccharide(EPS)from Lactobacillus strains with the higher antiproliferative effect on colon cancer cell line HT-29 was screened from 15 strains of Lactobacillus plantarum provided by Dalian probiotic functional property key laboratory in Dalian Polytechnical University.Lactobacillus strains with higher exopolysaccharide yield was selected by using phenol sulfuric acid method,and the antiproliferative effects of EPS on HT-29 cells were determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)method.The results showed that L.plantarum 12 had the highest EPS yield and the EPS showed the higher antiproliferative effect among the 15 strains.The carbohydrate content of the crude L.plantarum 12 EPS was 54%and the protein content was 3.6%.The crude EPS of the L.plantarum12 was separated and purified by DEAE-Sepharose Fast flow ion column and Sepharose CL-6B gel column,and two components were obtained,neutral polysaccharide fraction EPS12-1 and acidic polysaccharide group EPS12-2.The carbohydrate contents of the two components were 31.82%and 35.21%.The inhibiting rates on HT-29 cells of the crude EPS,EPS12-1 and EPS12-2 of L.plantarum 12 at the concentration of 250μg/m L were 27.58%,7.11%and 12.00%respectively,higher than those at the concentrations of 50μg/m L,100μg m L and 500μg/m L,which was consistent with the higher apoptosis,reactive oxygen species(ROS)levels and caspase 8 activity of the HT-29 cells treated by EPS at the concentration of 250μg/m L.L.plantarum12 EPS showed antiproliferative by increased ROS activity and activated caspase 8.
The exopolysaccharide(EPS)from Lactobacillus strains with the higher antiproliferative effect on colon cancer cell line HT-29 was screened from 15 strains of Lactobacillus plantarum provided by Dalian probiotic functional property key laboratory in Dalian Polytechnical University.Lactobacillus strains with higher exopolysaccharide yield was selected by using phenol sulfuric acid method,and the antiproliferative effects of EPS on HT-29 cells were determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS)method.The results showed that L.plantarum 12 had the highest EPS yield and the EPS showed the higher antiproliferative effect among the 15 strains.The carbohydrate content of the crude L.plantarum 12 EPS was 54%and the protein content was 3.6%.The crude EPS of the L.plantarum12 was separated and purified by DEAE-Sepharose Fast flow ion column and Sepharose CL-6B gel column,and two components were obtained,neutral polysaccharide fraction EPS12-1 and acidic polysaccharide group EPS12-2.The carbohydrate contents of the two components were 31.82%and 35.21%.The inhibiting rates on HT-29 cells of the crude EPS,EPS12-1 and EPS12-2 of L.plantarum 12 at the concentration of 250μg/m L were 27.58%,7.11%and 12.00%respectively,higher than those at the concentrations of 50μg/m L,100μg m L and 500μg/m L,which was consistent with the higher apoptosis,reactive oxygen species(ROS)levels and caspase 8 activity of the HT-29 cells treated by EPS at the concentration of 250μg/m L.L.plantarum12 EPS showed antiproliferative by increased ROS activity and activated caspase 8.
引文
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[2]Wu N,Yang X,Zhang R,et al.Dysbiosis signature of fecal microbiota in colorectal cancer patients[J].Microbial Ecology,2013,66(2):462-470
[3]Konstantinov S R,Kuipers E J,Peppelenbosch M P.Functional genomic analyses of the gut microbiota for CRC screening[J].Nat Rev Gastroenterol Hepatol,2013,10(12):741-745
[4]Guo C F,Li J Y.Hypocholesterolaemic action of Lactobacillus casei,F0822 in rats fed acholesterol-enriched diet[J].International Dairy Journal,2013,32(2):144-149
[5]Hoarau C,Lagaraine C,Martin L,et al.Supernatant of Bifidobacterium breve induces dendritic cell maturation,activation,and survival through a Toll-like receptor 2 pathway[J].Journal of Allergy&Clinical Immunology,2006,117(3):696-702
[6]Xue C,Zhang L,Li H,et al.Functionality of the S-layer proteins from Lactobacillus,in the competitive against enteropathogens infection[J].European Food Research&Technology,2013,236(2):249-255
[7]Minelli E B,Benini A,Marzotto M,et al.Assessment of novel probiotic Lactobacillus casei strains for the production of functional dairy foods[J].International Dairy Journal,2004,14(8):723-736
[8]De M d L A,Matar C,Perdigón G.The application of probiotics in cancer[J].British Journal of Nutrition,2007,98 Suppl 1(S1):S105-S110
[9]Linsalata M,Russo F.Nutritional factors and polyamine metabolism in colorectal cancer[J].Nutrition,2008,24(4):382-389
[10]Francesco Russo,Antonella Orlando,Michele Linsalata,et al.Effects of Lactobacillus Rhamnosus GG on the Cell Growth and Polyamine Metabolism in HGC-27 Human Gastric Cancer Cells[J].Nutrition&Cancer-an International Journal,2007,59(1):106-114
[11]Oda M,Hasegawa H,Komatsu S,et al.Anti-tumor Polysaccharide from Lactobacillus sp.[J].Journal of the Agricultural Chemical Society of Japan,1983,47(7):1623-1625
[12]Liu C T,Chu F J,Chou C C,et al.Antiproliferative and anticytotoxic effects of cell fractions and exopolysaccharides from Lactobacillus casei 01.[J].Mutation Research,2011,721(2):157-162
[13]Choi S S,Kim Y,Han K S,et al.Effects of Lactobacillus strains on cancer cell proliferation and oxidative stress in vitro[J].Letters in Applied Microbiology,2006,42(5):452-458
[14]Ohta T,Nakatsugi S,Watanabe K,et al.Inhibitory effects of Bifidobacterium-fermented soy milk on 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced rat mammary carcinogenesis,with a partial contribution of its component isoflavones[J].Carcinogenesis,2000,21(5):937-941
[15]Bermudes D,Zheng L M,King I C.Live bacteria as anticancer agents and tumor-selective protein delivery vectors[J].Current opinion in drug discovery&development,2002,5(2):194-199
[16]王鑫,马桂荣.几株乳酸菌的分离鉴定[J].生物技术,1994(1):37-39
[17]张惟杰.糖复合物生化研究技术[M].杭州:浙江大学出版社,1999
[18]Kim Y,Oh S,Yun H S,et al.Cell-bound exopolysaccharide from probiotic bacteria induces autophagic cell death of tumour cells[J].Letters in Applied Microbiology,2010,51(2):123-130
[19]Zhang L,Liu C,Li D,et al.Antioxidant activity of an exopolysaccharide isolated from Lactobacillus plantarum C88[J].International Journal of Biological Macromolecules,2013,54(1):270-275
[20]王文平,郭祀远,李琳,等.苯酚-硫酸法测定野木瓜中多糖含量的研究[J].食品科学,2007,28(4):276-279
[21]Wang K,Li W,Rui X,et al.Characterization of a novel exopolysaccharide with antitumor activity from Lactobacillus plantarum 70810[J].International Journal of Biological Macromolecules,2014,63:133-139
[22]Jie S,Rui H L.Cranberry phytochemical extracts induce cell cycle arrest and apoptosis in human MCF-7 breast cancer cells[J].Cancer Letters,2006,241(1):124-134
[23]Ashwaq A S,Alqubaisi M S,Rasedee A,et al.Inducing G2/M Cell Cycle Arrest and Apoptosis through Generation Reactive Oxygen Species(ROS)-Mediated Mitochondria Pathway in HT-29 Cells by Dentatin(DEN)and Dentatin Incorporated in Hydroxypropyl-β-Cyclodextrin(DEN-HPβCD)[J].International Journal of Molecular Sciences,2016,17(10):1653
[24]Asker M M S,Ahmed Y M,Ramadan M F.Chemical characteristics and antioxidant activity of exopolysaccharide fractions from Microbacterium terregens[J].Carbohydrate Polymers,2009,77(3):563-567
[25]刘宇,孟祥晨.乳酸菌胞外多糖及其抗肿瘤活性[J].中国乳品工业,2008,36(1):39-43
[26]Bleau C,Monges A,Rashidan K,et al.Intermediate chains of exopolysaccharides from Lactobacillus rhamnosus RW-9595M increase IL-10 production by macrophages[J].Journal of Applied Microbiology,2010,108(2):666-675
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[28]Hu P,Song W,Shan Y,et al.Lactobacillus paracasei subsp.paracasei M5L induces cell cycle arrest and calreticulin translocation via the generation of reactive oxygen species in HT-29 cell apoptosis[J].Food&function,2015,6(7):2257-2265
[29]Ashkenazi A,Dixit V M.Death receptors:signaling and modulation[J].Science,1998,281(5381),1305-1308
[30]Zhuang H,Zhou Z Y,Song C J.Angelica sinensis polysaccharides promotes apoptosis in human breast cancer cells via creb-regulated caspase-3 activation[J].Biochemical&Biophysical Research Communications,2015,467(3):562-569
[31]Zhou X,Hong T,Yu Q,et al.Exopolysaccharides from Lactobacillus plantarum NCU116 induce c-Jun dependent Fas/Fasl-mediated apoptosis via TLR2 in mouse intestinal epithelial cancer cells[J].Scientific Reports,2017,7(1):14247
[32]Zhang J H,Tong H X,Teng S C,et al.Experimental study on role of Caspase 8 in apoptosis of neuroblastoma cells[J].China Journal of Modern Medicine,2006(13):13-17