CD34~+CD38~+造血干细胞移植MISTRG小鼠的人源化免疫特征研究
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摘要
目的研究人脐带血CD34~+CD38~+造血干细胞移植入MISTRG免疫缺陷小鼠体内的人源化免疫特征。方法利用Ficoll密度梯度离心法从新生儿脐带血中分离单个核细胞,并结合免疫磁珠法分选hCD34~+造血干细胞,然后经体外扩增培养后利用流式细胞技术检测CD34~+CD38~+细胞的比例。取2×105细胞数注射至辐照后的新生MISTRG小鼠肝脏内,分别于第3、6、9、12周采血检测人源免疫细胞的分化与表达情况。结果免疫磁珠法分选人的CD34~+CD38~+造血干细胞纯度高达92.0%。移植小鼠外周血中的hCD45~+细胞初始浓度水平大于10.0%,淋巴细胞群包含人源T(hCD3~+CD45~+)、B(hCD3-CD19~+)和NK(hCD3-CD56~+)淋巴细胞,其中B(hCD3-CD19~+)淋巴细胞所占比值最大(初始水平为49.0%±7.4%),髓系细胞群则主要由巨噬细胞组成(初始水平为82.3%±4.5%)。小鼠外周血中hCD45~+细胞比值和T细胞占免疫细胞的比值随着时间的推移逐渐下降,B淋巴细胞和巨噬细胞所占的比例逐渐升高,而NK细胞基本完全消失。结论人源CD34~+CD38~+造血干细胞在新生MISTRG小鼠中可有效分化为人源淋系和髓系免疫细胞。在移植后的12周内hCD45~+细胞的比例逐渐下降,MISTRG免疫缺陷小鼠最佳的人源化免疫系统维持时间为第3～6周。
This study was performed to investigate the differentiation and immunophenotypic characterization of purified lineage CD34~+CD38~+cord blood hematopoietic stem cells(HSCs) in MISTRG mice. Mononuclear cells were isolated from the whole blood via ficoll density gradient method and then CD34~+HSCs were separated through magnetic activated cell sorting kit. The cell surface phenotype of culture-expanded CD34~+HSCs was analyzed by multiparameter flow cytometry and the purity of CD34~+CD38~+HSCs was above 92.0%. Those cultured CD34~+cells(2×105) were then injected into the liver of newborn MISTRG mice, and then the blood samples were collected at 3,6, 9 and 12 weeks after the injection. After cell transplantation, the initial level of hCD45~+cells was above 10.0%.The human T(hCD3~+CD45~+), B(hCD3-CD19~+) and NK(hCD3-CD56~+) cells were both detected, and the B(hCD3-CD19~+) cells were present with highest abundance(the initial level was 49.0%±7.4%). Meanwhile, the human myeloid lineage cell population was mainly composed of macrophages(hCD14~+CD68~+, the initial level was82.3% ± 4.5%). The percent of B(hCD3-CD19~+) cells and macrophages increased gradually over time, while hCD45~+, T(hCD3~+CD45~+) and NK(hCD3-CD56~+) cellsdecreased gradually, especially NK(hCD3-CD56~+) cells disappeared completely. In conclusion, the purified lineage CD34~+CD38~+cord blood HSCs can effectively differentiate into both lymphocyte and myeloid lineage cells in MISTRG mice. The percent of hCD45~+cells decrease gradually within 12 weeks and the optimal humanization time is between 3 and 6 weeks.
This study was performed to investigate the differentiation and immunophenotypic characterization of purified lineage CD34~+CD38~+cord blood hematopoietic stem cells(HSCs) in MISTRG mice. Mononuclear cells were isolated from the whole blood via ficoll density gradient method and then CD34~+HSCs were separated through magnetic activated cell sorting kit. The cell surface phenotype of culture-expanded CD34~+HSCs was analyzed by multiparameter flow cytometry and the purity of CD34~+CD38~+HSCs was above 92.0%. Those cultured CD34~+cells(2×105) were then injected into the liver of newborn MISTRG mice, and then the blood samples were collected at 3,6, 9 and 12 weeks after the injection. After cell transplantation, the initial level of hCD45~+cells was above 10.0%.The human T(hCD3~+CD45~+), B(hCD3-CD19~+) and NK(hCD3-CD56~+) cells were both detected, and the B(hCD3-CD19~+) cells were present with highest abundance(the initial level was 49.0%±7.4%). Meanwhile, the human myeloid lineage cell population was mainly composed of macrophages(hCD14~+CD68~+, the initial level was82.3% ± 4.5%). The percent of B(hCD3-CD19~+) cells and macrophages increased gradually over time, while hCD45~+, T(hCD3~+CD45~+) and NK(hCD3-CD56~+) cellsdecreased gradually, especially NK(hCD3-CD56~+) cells disappeared completely. In conclusion, the purified lineage CD34~+CD38~+cord blood HSCs can effectively differentiate into both lymphocyte and myeloid lineage cells in MISTRG mice. The percent of hCD45~+cells decrease gradually within 12 weeks and the optimal humanization time is between 3 and 6 weeks.
引文
[1]Buque A,Galluzzi L.Modeling tumor immunology and immunotherapy in mice[J].Trends Cancer,2018,4(9):599-601.
[2]Carrillo MA,Zhen A,Kitchen SG.The use of the humanized mouse model in gene therapy and immunotherapy for HIV and cancer[J].Front Immunol,2018,9:746.
[3]Yong K,Her Z,Chen Q.Humanized mice as unique tools for human-specific studies[J].Arch Immunol Ther Ex,2018,66(4):245-266.
[4]Fujiwara S.Humanized mice:A brief overview on their diverse applications in biomedical research[J].J Cell Physiol,2018,233(4):2889-2901.
[5]De La Rochere P,Guil Luna S,Decaudin D,et al.Humanized mice for the study of immuno-oncology[J].Trends Immunol,2018,39(9):748-763.
[6]Rongvaux A,Willinger T,Martinek J,et al.Development and function of human innate immune cells in a humanized mouse model[J].Nat Biotechnol,2014,32(4):364-372.
[7]Huey DD,Niewiesk S.Production of humanized mice through stem cell transfer[J].Curr Protoc Mouse Biol,2018,8(1):17-27.
[8]Skelton JK,Ortega Prieto AM,Dorner M.A Hitchhiker’s guide to humanized mice:new pathways to studying viral infections[J].Immunology,2018,154(1):50-61.
[9]Hetzel M,Mucci A,Blank P,et al.Hematopoietic stem cell gene therapy for IFNγR1 deficiency protects mice from mycobacterial infections[J].Blood,2017,131(5):533-545.
[10]张梦军,韩清娟,陈晓玲,等.NOD小鼠人源化后CD4+Tregs和CD8+Tregs频率和功能变化及意义[J].免疫学杂志,2018,34(1):19-25.
[11]Astle J,Xiang Y,Rongvaux A,et al.Developing a model of human pluripotent to hematopoietic stem cell development in Mistrg mice[J].Blood,2015,126(23):4755.
[12]Saito Y,Ellegast JM,Rafiei A,et al.Peripheral blood CD34(+)cells efficiently engraft human cytokine knock-in mice[J].Blood,2016,128(14):1829-1833.
[13]Alben?z I,Türker?ener L,Ba?A,et al.Isolation of hematopoietic stem cells and the effect of CD38 expression during the early erythroid progenitor cell development process[J].Oncol Lett,2012,3(1):55-60.
[14]Higuchi Y,Zeng HM.CD38 expression by hematopoietic stem cells of newborn and juvenile mice[J].Leukemia,2003,17(1):171-174.
[15]买文丽,韩清娟,曹文轩,等.人源化NOD小鼠中CD8+CD25+Tregs的频率和功能研究[J].免疫学杂志,2016,32(1):7-11.
[2]Carrillo MA,Zhen A,Kitchen SG.The use of the humanized mouse model in gene therapy and immunotherapy for HIV and cancer[J].Front Immunol,2018,9:746.
[3]Yong K,Her Z,Chen Q.Humanized mice as unique tools for human-specific studies[J].Arch Immunol Ther Ex,2018,66(4):245-266.
[4]Fujiwara S.Humanized mice:A brief overview on their diverse applications in biomedical research[J].J Cell Physiol,2018,233(4):2889-2901.
[5]De La Rochere P,Guil Luna S,Decaudin D,et al.Humanized mice for the study of immuno-oncology[J].Trends Immunol,2018,39(9):748-763.
[6]Rongvaux A,Willinger T,Martinek J,et al.Development and function of human innate immune cells in a humanized mouse model[J].Nat Biotechnol,2014,32(4):364-372.
[7]Huey DD,Niewiesk S.Production of humanized mice through stem cell transfer[J].Curr Protoc Mouse Biol,2018,8(1):17-27.
[8]Skelton JK,Ortega Prieto AM,Dorner M.A Hitchhiker’s guide to humanized mice:new pathways to studying viral infections[J].Immunology,2018,154(1):50-61.
[9]Hetzel M,Mucci A,Blank P,et al.Hematopoietic stem cell gene therapy for IFNγR1 deficiency protects mice from mycobacterial infections[J].Blood,2017,131(5):533-545.
[10]张梦军,韩清娟,陈晓玲,等.NOD小鼠人源化后CD4+Tregs和CD8+Tregs频率和功能变化及意义[J].免疫学杂志,2018,34(1):19-25.
[11]Astle J,Xiang Y,Rongvaux A,et al.Developing a model of human pluripotent to hematopoietic stem cell development in Mistrg mice[J].Blood,2015,126(23):4755.
[12]Saito Y,Ellegast JM,Rafiei A,et al.Peripheral blood CD34(+)cells efficiently engraft human cytokine knock-in mice[J].Blood,2016,128(14):1829-1833.
[13]Alben?z I,Türker?ener L,Ba?A,et al.Isolation of hematopoietic stem cells and the effect of CD38 expression during the early erythroid progenitor cell development process[J].Oncol Lett,2012,3(1):55-60.
[14]Higuchi Y,Zeng HM.CD38 expression by hematopoietic stem cells of newborn and juvenile mice[J].Leukemia,2003,17(1):171-174.
[15]买文丽,韩清娟,曹文轩,等.人源化NOD小鼠中CD8+CD25+Tregs的频率和功能研究[J].免疫学杂志,2016,32(1):7-11.
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