结核分枝杆菌H37Ra感染小鼠模型建立及其对B细胞发育分化特征的实验研究
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摘要
目的建立减毒结核分枝杆菌H37Ra感染BALB/c小鼠模型并探讨H37Ra感染对小鼠B淋巴细胞分化发育的影响。方法将24只BALB/c小鼠随机分为感染组和对照组,感染组尾静脉注射H37Ra菌液106 CFU/0.2ml,对照组注射等量生理盐水,感染4周及8周分别取小鼠的肝脏、肺脏及肾脏组织,切片HE染色,组织匀浆液接种罗氏培养基培养;取小鼠骨髓细胞和脾脏细胞经荧光抗体染色,采用FACSCalibur分析感染早期及晚期小鼠的B淋巴细胞表型。结果感染组小鼠肝脏、肺脏及肾脏肿大,培养后均有H37Ra菌株生长。感染组与对照组比较,骨髓总B淋巴细胞、脾脏总B淋巴细胞、未成熟B细胞比例低于对照组,差异有统计学意义(均P<0.05);感染组骨髓成熟B淋巴细胞、脾脏成熟B淋巴细胞比例高于对照组,差异有统计学意义(均P<0.05);感染组8周骨髓成熟B淋巴细胞、脾脏成熟B淋巴细胞较感染组4周时增多(均P<0.05)。结论本研究成功建立了H37Ra感染BALB/c鼠模型;小鼠被H37Ra感染后影响了B淋巴细胞的分化发育,促使B淋巴细胞向成熟阶段分化,为机体抵御H37Ra感染奠定了基础。
Objective To generate Mycobacterium tuberculosis H37Ra infected BALB/c mouse model and to understand the effect of infection on B cell development and differentiation. Methods 24BALB/c mice were randomly divided into two groups and were injected with H37Ra(106 CFU/0.2ml)and normal saline(control mice)via tail vein.They were sacrificed at 4and 8weeks post infection and murine lung,kidney,liver were collected,weighed and stained with HE.Tissue homogenates were then cultured using Roche culture medium.Bone marrow cells and spleen cells were stained with fluorescent antibody,and B cell phenotype was analyzed by FACSCalibur both in the early(4weeks)and late(8weeks)stage of infection. Results The liver,kidney and lung were found enlarged in mice infected with H37Ra and were culture positive.Compared to control mice,the ratios of total B cells of bone marrow,spleen and immature B cells of infected mice were lower,the differences were statistically significant(P<0.05).The overall ratios of mature B cells of bone marrow and spleen were significantly higher than control mice(P<0.05).Meanwhile,the total number of mature B cells in bone marrow and spleen increased at 8weeks of infection compared with 4weeks(P<0.05). ConclusionsThe H37Ra infected mice model is successfully established.The infection of H37Ra triggers the differentiation of B cells,which plays an important role in host immune defense.
Objective To generate Mycobacterium tuberculosis H37Ra infected BALB/c mouse model and to understand the effect of infection on B cell development and differentiation. Methods 24BALB/c mice were randomly divided into two groups and were injected with H37Ra(106 CFU/0.2ml)and normal saline(control mice)via tail vein.They were sacrificed at 4and 8weeks post infection and murine lung,kidney,liver were collected,weighed and stained with HE.Tissue homogenates were then cultured using Roche culture medium.Bone marrow cells and spleen cells were stained with fluorescent antibody,and B cell phenotype was analyzed by FACSCalibur both in the early(4weeks)and late(8weeks)stage of infection. Results The liver,kidney and lung were found enlarged in mice infected with H37Ra and were culture positive.Compared to control mice,the ratios of total B cells of bone marrow,spleen and immature B cells of infected mice were lower,the differences were statistically significant(P<0.05).The overall ratios of mature B cells of bone marrow and spleen were significantly higher than control mice(P<0.05).Meanwhile,the total number of mature B cells in bone marrow and spleen increased at 8weeks of infection compared with 4weeks(P<0.05). ConclusionsThe H37Ra infected mice model is successfully established.The infection of H37Ra triggers the differentiation of B cells,which plays an important role in host immune defense.
引文
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[15]张爱霞,江京娣,吕静竹,等.减毒结核分枝杆菌H37Ra感染小鼠模型的实验研究[J].蚌埠医学院学报,2016,41(2):141-144.
[16] O′Garra A,Redford PS,McNab FW,et al.The immune response in tuberculosis[J].Annu Rev Immunol,2013,31:475-527.
[17]李江平,吴长有.结核性胸膜炎的固有免疫应答和适应性免疫应答的研究进展[J].新发传染病电子杂志,2017,2(3):133-142.
[18]刘文文,吕艳,王聪,等.结核分枝杆菌感染对BALB/c小鼠B细胞发育分化的影响[J].中国人兽共患病学报,2018,34(8):703-707.
[19]白丽.B淋巴细胞发育与BCR基因重排[J].大理学院学报,2010,9(6):28-32.
[2]Mlen H,De Souza GA,Pathak S,et al.Comparison of membrane proteins of Mycobacterium tuberculosis H37Rv and H37Ra strains[J].BMC Microbiol,2011,11:18.
[3] Zhang L,Cheng X,Bian S,et al.Utility of Th1-cell immune responses for distinguishing active tuberculosis from non-active tuberculosis:A case-control study[J].PLoS One,2017,12(5):e0177850.
[4] Valentini D,Rao M,Ferrara G,et al.Immune recognition surface construction of Mycobacterium tuberculosis epitope-specific antibody responses in tuberculosis patients identified by peptide microarrays[J].Int J Infect Dis,2017,56:155-166.
[5] Casta1o D,García LF,Rojas M.Differentiation of human mononuclear phagocytes increases their innate response to Mycobacterium tuberculosis infection[J].Tuberculosis(Edinb),2014,94(3):207-218.
[6] Jena L,Kashikar S,Kumar S,et al.Effect of single amino acid mutations on function of Mycobacterium tuberculosis H37RV and H37RA by computational approaches[J].Indian J Tuberc,2014,61(3):200-206.
[7]梁艳,吴雪琼,李宁,等.结核分枝杆菌感染小鼠模型的建立与评价[J].中国病原生物学杂志,2014,9(9):775-778.
[8]吕艳,白丽,刘文文,等.Mtb感染BALB/c鼠淋巴细胞亚群及血清抗体水平变化[J].热带医学杂志,2017,17(5):583-587.
[9]吕艳,白丽,刘文文,等.Mtb感染对BALB/c小鼠B细胞亚群分布的影响[J].中国病原生物学杂志,2017,12(1):25-28.
[10] Kozakiewicz L,Phuah J,Flynn J,et al.The role of B cells and humoral immunity in Mycobacterium tuberculosis infection[J].Adv Exp Med Biol,2013,783:225-250.
[11]苏瑾文.肺结核的保护性免疫和免疫病理机制进展[J].中国热带医学,2015,15(5):635-639.
[12] Maiga M,Ahidjo BA,Maiga MC,et al.Erratum to"efficacy of adjunctive tofacitinib therapy in mouse models of tuberculosis"[EBioMedicine 2(8),August 2015,868-873][J].EBioMedicine,2015,2(11):1834.
[13] Cantrell SA,Pascopella L,Flood J,et al.Community-wide transmission of a strain of Mycobacterium tuberculosis that causes reduced lung pathology in mice[J].J Med Microbiol,2008,57(Pt 1):21-27.
[14]宁唤唤,路延之,康健,等.分枝杆菌持续感染小鼠模型的建立及其特征分析[J].中国病原生物学杂志,2017,12(3):219-223.
[15]张爱霞,江京娣,吕静竹,等.减毒结核分枝杆菌H37Ra感染小鼠模型的实验研究[J].蚌埠医学院学报,2016,41(2):141-144.
[16] O′Garra A,Redford PS,McNab FW,et al.The immune response in tuberculosis[J].Annu Rev Immunol,2013,31:475-527.
[17]李江平,吴长有.结核性胸膜炎的固有免疫应答和适应性免疫应答的研究进展[J].新发传染病电子杂志,2017,2(3):133-142.
[18]刘文文,吕艳,王聪,等.结核分枝杆菌感染对BALB/c小鼠B细胞发育分化的影响[J].中国人兽共患病学报,2018,34(8):703-707.
[19]白丽.B淋巴细胞发育与BCR基因重排[J].大理学院学报,2010,9(6):28-32.