The Construction Of BFGF And CTLA4-Ig Gene-modified RPE Cells And The Study On Its Expression Characteristics

ObjectiveTo construct retinal pigment epithelium (RPE) cells capable of stably and efficiently expressing basic fibroblast growth factors (bFGF) and cytotoxic T lymphocyte-asscciated antigen 4 immunoglobulin (CTLA4Ig) using a lentivirus systems, and detect its effect on expression characteristics of retinal pigment epithelial cellsMethods1. Construction of Retinal Pigment Epithelium Cells Stably Expressing bFGF:The genes of bFGF were cloned into a lentivirus expression vector, then, the recombinant lentivirus was packaged and harvested. RPE cells were infected, and the expression of the bFGF was detected by western blot.2. Construction of Retinal Pigment Epithelium Cells Stably Expressing CTLA4Ig:The total RNA was extracted from human peripheral blood mononuclear cells. The genes of the extracellular domain of CTLA4 and IgG constant region were amplified by RT-PCR, PCR production was cloned into plasmid pCDNA3, obtained CTLA4-Ig genes. The genes of CTLAa-Ig were cloned into a lentivirus expression vector, then, the recombinant lentivirus was packaged and harvested. RPE cells were infected, and the expression of the CTLA4-Ig was detected by western blot.3. Detection the proliferation of Gene-modified RPE cells:MTT assay: bFGF and CTLA4-Ig gene-modified RPE cells were seeded into a 96-well plate. After incubated 24 hours, added 5mg/ml MTT, and then added DMSO to dilute crystal. OD values were measured. Normal RPE cells served as control samples.Results 1. Gene sequencing showed that the lentiviral vectors contain bFGF gene were constructed successfully, the recombinant lentivirus titer was 5×10'IU/mL. After 1 week,4 weeks and 12 weeks when RPE cells were infected by the lentivirus, bFGF expression could be detected by Western Blot2. CTLA 4-Ig genes were obtained successfully. The lentiviral vectors contain CTLA4-Ig genes were constructed successfully. The recombinant lentivirus titer was 4×10'IU/mL. After 1 week,4 weeks and 12 weeks when RPE cells were infected by the lentivirus, CTLA4-Ig expression could be detected by Western Blot.3. Growth curve model:the curves were almost the same trend. OD values: there were no significant differences between gene-modified groups and normal cells group (P>0.05).ConclusionIn this study, we demonstrated that lentiviral vectors can efficiently transfer bFGF genes and CTLA4-Ig genes into RPE cells with stable long-term expression in vitro. Therefore, it is likely to culture a great number of human RPE cells in vitro to provide adequate and safe low-cost donor cells for RPE cells transplantation to treating retinitis pigmentosa and to solve immune rejection. A lower level of cytokines produced by gene-modified cells does not cause the abnormal proliferation of retinal pigment epithelial cells.