| Currently,the biological characteristics of cell line are not consistent with clinical specimens,the original cells are complex with normal and abnormal cells.Previous study indicate that mantle cell lymphoma(MCL)is derived from the mature B cells of CD19 positive(CD19+),but some study suggests CD19 negative(CD19-)cells may be the origin cell,suggests the complex pathogenesis of malignant clone.The pathogenesis of MCL is unclear,which is a urgent problem to be solved in the basic and clinical researchs.The above disadvantages greatly restricted the research.Therefore,the establishment of a novel cell line which is highly consistent with the biological characteristics of the clinical specimen will break the bottleneck of the cell line,to further study the pathogenesis of malignant clone.Objective1 Establish a malignant clone cell line contains CD19+ and enriched CD19-early stage cells,and the biological characteristics are highly consistent with the clinical specimens.2 The research of pathogenesis in MCL malignant clone was based on the novel cell line,which is focus on whether MCL is a monoclonal or polyclonal tumor.To investgate the abnormal development stage,immunophenotype,gene expression,and tumor biological characteristics of MCL malignant clones.Methods1 Establishment of the JeKo-1-spheres cell line(1)Identification of spherical growth pattern: the expression of CD19 and the ratio of side-population(SP)in the spherical growth mode from JeKo-1-parental cells by flow cytometry(FCM).(2)Establishment of the JeKo-1-spheres by continuous cultivation to get natural purification cell line.2 The biological characteristics of the Jeko-1-spheres cell line.(1)Flow cytometry multicolor analysis the expression of CD34,CD38,TDT,CD22,CD5,CD10 and Ig M in CD19-cells and compared with normal controls to identify the immunophenotype.(2)Further analysis of Oct2,bcl-9,bcl-2,bcl-11 b,c-myc,CCND1,bcl-11 a,bmi-1,sox2,CD133 and nanog transcript level was carried out by qRT-PCR on RNA isolated from JeKo-1-parental and JeKo-1-spheres.(3)Jeko-1-parental and jeko-1-spheres were conducted clony formation assays.(4)Xenotransplantation assays.The brain,thymus,sternum,heart,lung,liver,spleen,Renal and adrenal gland,stomach,intestines and pancreas were all collected for H&E staining,and the spleen was used for immunohistochemistry.The results focused on the incidence of tumor,hemorrhage and extramedullary hematopoiesis of various organs.3 The pathogenesis of malignant clone(1)CD19-IgM-,CD19-IgM+ and CD19+IgM+ from Jeko-1-spheres,clinical and normal specimens,three fractions were isolated by fluorescence-activated cell sorting.Each flow cytometry-sorted population was conducted clony formation,the numbers of colonies were counted after two weeks.(2)Three subclones of the original cell and JeKo-1-spheres were isolated by fluorescence-activated cell sorting.Each subclone was conducted xenotransplantation assays in the NOD/SCID mice.Results1 The JeKo-1-spheres cell line was established,with the unique growth mode of multicellular spheres consistent with the original cells,enriched with SP and CD19-early stage cells.2 The pathogenesis of MCL involves the three abnormal development stages of pre-b,immature B and mature B cells,and the immunophenotype of the corresponding malignant clones are :CD19-/Ig M-,CD19-/Ig M+,and CD19+/Ig M+.3 The oncogenic potential of Jeko-1-spheres cell lines has obvious advantages in the expression of oncogenes and stem cell genes,the ability to colony formation and tumorigenic in vivo4 The three subclones were able to form colonies with abilities were as follows in the order: CD19-/IgM+ > CD19+/IgM+ > CD19-/IgM-.None of the four clinical specimens and JeKo-1-spheres had CD19+/ IgMsubgroups in healthy specimen.Any subclone retaining only cyclin D1(CCND1)specific high expression in spleen without other organ tumor formation.The CD19-/IgM+ subclone was more likely to have widespread infection and bleeding when compared to the other two subclones.Conclusion1 The growth of multicellular spheres is a unique way of CD19-early stage malignant clones in MCL.2 The Jeko-1-spheres cell line has a stronger oncogenic potential,and the biological characteristics are highly consistent with the clinical specimens,which is a good model for the research of malignant clones.3 The pathogenesis of MCL involves pre-b,immature B and mature B stages,and the immunophenotype of the corresponding malignant clones are :CD19-/Ig M-,CD19-/Ig M+,and CD19+/Ig M+.The immunophenotype of MCL was compared with the normal control without CD19+/ IgM-subgroup.MCL might be a polyclonal neoplasm with the advantage of CD19-/Ig M+ clone. |
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