脐带间充质干细胞体外调节CD34~+CD126~-细胞向巨核系诱导的研究
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摘要
目的:研究CD34~+CD126~-细胞向巨核细胞诱导分化及间充质干细胞对诱导过程的调节。方法:免疫磁珠分选获得CD34~+CD126~-细胞,设置3个实验组:无间充质组、间充质直接接触组和非直接接触组,以脐带血单个核细胞作对照,诱导向巨核细胞分化14 d,分别在7 d末与14 d末进行悬浮细胞数计数,流式测定CD34与CD41表型及镜下细胞形态观察。胶原蛋白凝胶培养基诱导CD34~+CD126~-细胞形成巨核细胞集落形成单位(CFU-Mk),12 d末CD41特异性抗原染色并镜下计数CFU-Mk。结果:(1)诱导7 d或14 d末,间充质组(CD34~+CD126~-细胞与MSC接触或非接触共培养组)获得悬浮细胞数均显著高于无间充质组(均为P<0.001),但MSC接触与非接触组间获得悬浮细胞数差异无统计学意义(P>0.05);(2)诱导7 d末,间充质组CD34阳性细胞数均显著大于无间充质组(均为P<0.001),MSC接触组CD34阳性细胞数显著小于非接触组(P<0.05),诱导14 d末,各组CD34阳性细胞率均下降至2%左右;(3)诱导7 d或14 d末,间充质组CD41阳性细胞数均显著大于无间充质组(均为P<0.001),而MSC接触与非接触组间CD41阳性细胞数均无显著性差异(P>0.05),CD34~+CD126~-细胞各组CD41阳性数均显著大于CBMCs的对应各组(均为P<0.001);(4)CD34~+CD126~-细胞组诱导得到的大CFU-Mk(≥50个细胞)与中CFU-Mk(21～49个细胞)数量显著大于CBMCs组(P_(CFU-Mk;≥50 cells)<0.01,P_(CFU-Mk;21-49 cells)<0.05)。结论:脐带间充质干细胞的旁分泌作用对于巨核细胞分化过程中CD34~+细胞或CD41~+细胞的增殖均具有促进作用,但MSC的微环境能显著降低CD34~+细胞的增殖速率。诱导前筛选较为原始的巨核细胞祖细胞作为起始细胞对于巨核细胞诱导效率的提升具有显著作用。
Objective: To study the differentiation of CD34~+CD126~- cells into megakaryocytes and the regulation of mesenchymal stem cells on the induction process. Methods: To obtain CD34~+CD126~- cells by Immunomagnetic activated cell sorting,and three experimental groups were set up: no mesenchymal group,mesenchymal direct contact group and non direct contact group,umbilical cord blood mononuclear cells as control,induced cells to differentiate into megakaryocytes for 14 days,and the number of suspended cells were counted at the end of 7 d and at the end of 14 d respectively,the phenotype of CD34 and CD41 was measured by flow cytometry and observation of cell morphology under the microscope. Collagen gel culture medium induced CD34~+CD126~- cells to megakaryocyte colony forming unit(CFU-Mk),CD41 specific antigen staining and counting CFU-Mk at the end of 12 d. Results: ①At the end of the induction of 7 d or 14 d,the number of suspended cells of the mesenchymal group(CD34~+CD126~- cells with MSC direct contact group and non direct contact group) was significantly higher than that of the no mesenchymal group(all P < 0. 001),but there was no significant difference in the number of suspended cells between the MSC contact and the non contact group(P > 0. 05). ②At the end of the induction of 7 d,the number of CD34 positive cells in the mesenchymal group was significantly higher than that of the no mesenchymal group(all P < 0. 001). The number of CD34 positive cells in the MSC contact group was significantly smaller than that in the non contact group(P < 0. 05). The rate of CD34 positive cells in each group decreased to about 2% at the end of the induction of 14 d. ③At the end of the induction of 7 d or 14 d,the number of CD41 positive cells in the mesenchymal group was significantly greater than that of the no mesenchymal group(all P < 0. 001). There was no significant difference in the number of CD41 positive cells between the MSC contact and the non contact group(P > 0. 05). The number of CD41 positive cells in each group of CD34~+CD126~- cells was significantly higher than that of CBMCs(all P < 0. 001). ④ The number of large CFU-Mk(≥50 cells) and middle CFU-Mk(21-49 cells) induced from CD34~+CD126~- cells group was significantly greater than that of the CBMCs group(P_(CFU-Mk; ≥50 cells)< 0. 01,PCFU-Mk; 21-49 cells< 0. 05). Conclusion: The paracrine effect of umbilical cord mesenchymal stem cells can promote the proliferation of CD34~+cells or CD41~+cells in the process of megakaryocyte differentiation,but the microenvironment of MSC can significantly reduce the proliferation rate of CD34~+cells. Screening of more primitive megakaryocyte progenitors as initiating cells before induction can significantly enhance the efficiency of megakaryocyte induction.
Objective: To study the differentiation of CD34~+CD126~- cells into megakaryocytes and the regulation of mesenchymal stem cells on the induction process. Methods: To obtain CD34~+CD126~- cells by Immunomagnetic activated cell sorting,and three experimental groups were set up: no mesenchymal group,mesenchymal direct contact group and non direct contact group,umbilical cord blood mononuclear cells as control,induced cells to differentiate into megakaryocytes for 14 days,and the number of suspended cells were counted at the end of 7 d and at the end of 14 d respectively,the phenotype of CD34 and CD41 was measured by flow cytometry and observation of cell morphology under the microscope. Collagen gel culture medium induced CD34~+CD126~- cells to megakaryocyte colony forming unit(CFU-Mk),CD41 specific antigen staining and counting CFU-Mk at the end of 12 d. Results: ①At the end of the induction of 7 d or 14 d,the number of suspended cells of the mesenchymal group(CD34~+CD126~- cells with MSC direct contact group and non direct contact group) was significantly higher than that of the no mesenchymal group(all P < 0. 001),but there was no significant difference in the number of suspended cells between the MSC contact and the non contact group(P > 0. 05). ②At the end of the induction of 7 d,the number of CD34 positive cells in the mesenchymal group was significantly higher than that of the no mesenchymal group(all P < 0. 001). The number of CD34 positive cells in the MSC contact group was significantly smaller than that in the non contact group(P < 0. 05). The rate of CD34 positive cells in each group decreased to about 2% at the end of the induction of 14 d. ③At the end of the induction of 7 d or 14 d,the number of CD41 positive cells in the mesenchymal group was significantly greater than that of the no mesenchymal group(all P < 0. 001). There was no significant difference in the number of CD41 positive cells between the MSC contact and the non contact group(P > 0. 05). The number of CD41 positive cells in each group of CD34~+CD126~- cells was significantly higher than that of CBMCs(all P < 0. 001). ④ The number of large CFU-Mk(≥50 cells) and middle CFU-Mk(21-49 cells) induced from CD34~+CD126~- cells group was significantly greater than that of the CBMCs group(P_(CFU-Mk; ≥50 cells)< 0. 01,PCFU-Mk; 21-49 cells< 0. 05). Conclusion: The paracrine effect of umbilical cord mesenchymal stem cells can promote the proliferation of CD34~+cells or CD41~+cells in the process of megakaryocyte differentiation,but the microenvironment of MSC can significantly reduce the proliferation rate of CD34~+cells. Screening of more primitive megakaryocyte progenitors as initiating cells before induction can significantly enhance the efficiency of megakaryocyte induction.
引文
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[16]贡波,徐志国,王绍红,等.脐带间充质干细胞体外对脐带血CD4+T淋巴细胞免疫调节作用的研究[J].中国免疫学杂志,2017,33(2):220-225.Gong B,Xu ZG,Wang SH,et al.Immunoregulation study of UC-MSCs on UCB CD4+T lymphocytes in vitro[J].Chin J Immunol,2017,33(2):220-225.
[17]杨旭巍,徐志国,刘超,等.干细胞资源库信息管理系统的建立及在转化医学中的应用[J].转化医学杂志,2015,4(4):205-207.Yang XW,Xu ZG,Liu C,et al.The establishment of the stem cell resources bank information system and its application in the field of translational medicine[J].Translat Med J,2015,4(4):205-207.
[18]Celebi Betül,Mantovani Diego,Pineault Nicolas.Irradiated mesenchymal stem cells improve the ex vivo expansion of hematopoietic progenitors by partly mimicking the bone marrow endosteal environment[J].J Immunol Methods,2011,370(1-2):93-103.
[19]Jing Duohui,Fonseca Ana-Violeta,Alakel Nael,et al.Hematopoietic stem cells in co-culture with mesenchymal stromal cells-modeling the niche compartments in vitro[J].Haematologica,2010,95(4):542-550.
[2]Desborough Michael JR,Smethurst Peter A,Estcourt Lise J,et al.Alternatives to allogeneic platelet transfusion[J].Br J Haematol,2016,175(3):381-392.
[3]刘超,徐志国,杨旭巍,等.T细胞亚群的生物学特性及临床应用进展[J].转化医学杂志,2014,3(6):377-380.Liu C,Xu ZG,Yang XW,et al.Biological characteristics and clinical application of T cell subsets[J].Translat Med J,2014,3(6):377-380.
[4]Rocha V,Cornish J,Sievers EL,et al.Comparison of outcomes of unrelated bone marrow and umbilical cord blood transplants in children with acute leukemia[J].Blood,2001,97(10):2962-2971.
[5]Piacibello W,Sanavio F,Garetto L,et al.Extensive amplification and self-renewal of human primitive hematopoietic stem cells from cord blood[J].Blood,1997,89(8):2644-2653.
[6]徐志国,刘超,闫铭杰.人胎盘来源干/祖细胞生物学特性研究进展[J].转化医学杂志,2013,2(6):336-340.Xu ZG,Liu C,Yan MJ.The progress of research in biological characteristics of stem/progenitor cells derived from human placenta[J].Translat Med J,2013,2(6):336-340.
[7]刘超,徐志国,王绍红,等.人羊膜上皮细胞培养工艺的优化及生物学特性[J].中国组织工程研究,2017,21(13):2100-2107.Liu C,Xu ZG,Wang SH,et al.Human amniotic epithelial cells:culture technology optimization and biological characteristics[J].Chin J Tissue Engine Res,2017,21(13):2100-2107.
[8]Delehanty Lorrie L,Mogass Michael,Gonias Sara L,et al.Stromal inhibition of megakaryocytic differentiation is associated with blockade of sustained Rap1 activation[J].Blood,2003,101(5):1744-1751.
[9]Huang Nick,Lou Mabel,Liu Hua,et al.Identification of a potent small molecule capable of regulating polyploidization,megakaryocyte maturation,and platelet production[J].J Hematol Oncol,2016,9(1):136.
[10]陈义柱,徐志国,施旭斌,等.脐带血造血干细胞向巨核细胞诱导分化研究进展[J].临床血液学杂志(输血与检验),2018,31(3):481-485.Chen YZ,Xu ZG,Shi XB,et al.Research progress on differentiation of cord blood hematopoietic stem cells into megakaryocytes[J].J Clin Hematol,2018,31(3):481-485.
[11]Shin Joseph Y,Hu Wenhuo,Naramura Mayumi,et al.High c-Kit expression identifies hematopoietic stem cells with impaired selfrenewal and megakaryocytic bias[J].J Exp Med,2014,211(2):217-231.
[12]Ward Daniel,Carter Deborah,Homer Martin,et al.Mathematical modeling reveals differential effects of erythropoietin on proliferation and lineage commitment of human hematopoietic progenitors in early erythroid culture[J].Haematologica,2016,101(3):286-296.
[13]Bruno E,Murray LJ,Di Giusto R,et al.Detection of a primitive megakaryocyte progenitor cell in human fetal bone marrow[J].Exp Hematol,1996,24(4):552-558.
[14]Woolthuis Carolien M,Park Christopher Y.Hematopoietic stem/progenitor cell commitment to the megakaryocyte lineage[J].Blood,2016,127(10):1242-1248.
[15]Chen L,Xie X,Liu D,et al.Study on the induction and differentiation of megakaryocyte progenitor cell derived from umbilical cord blood[J].Zhonghua Xue Ye Xue Za Zhi,2014,35(3):187-190.
[16]贡波,徐志国,王绍红,等.脐带间充质干细胞体外对脐带血CD4+T淋巴细胞免疫调节作用的研究[J].中国免疫学杂志,2017,33(2):220-225.Gong B,Xu ZG,Wang SH,et al.Immunoregulation study of UC-MSCs on UCB CD4+T lymphocytes in vitro[J].Chin J Immunol,2017,33(2):220-225.
[17]杨旭巍,徐志国,刘超,等.干细胞资源库信息管理系统的建立及在转化医学中的应用[J].转化医学杂志,2015,4(4):205-207.Yang XW,Xu ZG,Liu C,et al.The establishment of the stem cell resources bank information system and its application in the field of translational medicine[J].Translat Med J,2015,4(4):205-207.
[18]Celebi Betül,Mantovani Diego,Pineault Nicolas.Irradiated mesenchymal stem cells improve the ex vivo expansion of hematopoietic progenitors by partly mimicking the bone marrow endosteal environment[J].J Immunol Methods,2011,370(1-2):93-103.
[19]Jing Duohui,Fonseca Ana-Violeta,Alakel Nael,et al.Hematopoietic stem cells in co-culture with mesenchymal stromal cells-modeling the niche compartments in vitro[J].Haematologica,2010,95(4):542-550.
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