The Optimization Of The Methods And The Influencing Factors For Immortalizing Human B Lymphoblastoid Cell Lines

B cells are lymphocytes that play a critical role in the humoral immune response(HIR). In this case, the HIR is a process that B cells recognition of antigen undergo activation, proliferation and differentiation. The study of B cell characters and immunoglobulin secretion has significant clinical value for immune diseases pathogenesis and medical therapy. To detect the cell surface markers in different stages of B cell activation and proliferation, could be helpful for the comprehension of correlations between B cells and diseases and clinical treatments for deseases.Epstein-Barr virus (EBV), a prominent member ofγ-herpesviruses, causes many human malignant diseases, especially tumors derived from epithelial or lymphocytic origin. It could infect primary resting B lymphocytes and transform B cells into EBV-immortalized LCLs. As a result, EBV is thought to be an effective method for cell immortalization with stable chromosome and original genetic character. B-LCLs are convenient sources of antibody-secreting cells and antigen-presenting cells. In recent studies, scientists used B-LCLs as an important mean to produce humanized antibodies. CpG motifs could activate human immune cells and induce Th1 type immune response. It has been reported that CpG motif-containing oligodeoxynucleotides could activeate human B lymphocytes, lead to cytokine secretion and cell surface molecules expression. B cells can avoid apoptosis and improve its living ability for the important moleculear CD40 could binds with CD40mAb. We use CpG DNA motifs and agonist CD40mAb(5C11) to raise EBV transfection efficiency. Optimization of transfected human B lymphoblastoid cell lines by EBV makes great sense to study B cells and its application in vitro. The optimization of transfected human B lymphoblastoid cell lines by EBV in vitroAIM: Investigation of optimized methods and the influencing factors for the establishment of B lymphoblastoid cell lines (LCLs) derived from human PBMC in vitro. METHODS: The peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and incubated with EB virus (EBV) while agonist CD40mAb(5C11), CpG DNA motifs for stimulating B lymphocytes and cyclospofin A (CyA) for inhibiting T lymphocytes were added at the same time. Morphological characteristics of immortalized B-lymphocytes were observed under optical microscope. Flow cytometry analysis was used to identify the expression of surface molecule CD19 on LCLs. RESULTS: PBMC incubated with EBV for 3 weeks were transformed into immortalized B lymphoblastoid cell lines. These immortalized cells accumulated into groups and were able to be cultured in the long term proliferation. The agonist CD40mAb and CpG DNA motifs markedly promoted the EBV transformation process. EBV-immortalized LCLs maintained the biological features of mature B lymphocytes. CONCLUSION: The combination of CD40 signal activation and CpG DNA motifs could effectively promote the EB virus transformation into human B lymphocytes and cell line establishment.The establishment of human B lymphoblastoid cell lines in vitro and its biological functionAIM: Establish B lymphoblastoid cell lines (LCLs) derived from healthy human PBMC in vitro and study its biological function. METHODS: Optical microscope was used to observe B lymphoblastoid cells stained by Giemsa technique. We extracted total RNA from LCLs and detected the expression of EpsteinBarr virus (EBV) encoded latent membrane protein 1(LMP1) gene. Flow cytometry analysis identified the expression of surface molecule CD19 and CD40 on LCLs. RESULTS: PBMC incubated with EBV for 3 weeks were transformed into immortalized B lymphoblastoid cell lines. These immortalized cells accumulated into groups and were able to be cultured in the long term proliferation. EBV-immortalized LCLs expressing LMP1 maintained the biological features of mature B lymphocytes. CONCLUSION: LCLs was successfully established by EBV transformation method. The transfected B lymphoblastoid cells not only continue to proliferate but also keep the biological features as mature B cells.