Research For The Lentiviral Vectors Carrying CD1d Transfected PANC-1Cells And Expression Of CD1d In Vitro

Objective Use of cell transfection,screening,RT-PCR and Western Blot detection methods to make human CD Id gene stability transfection pancreatic cancer cell lines PANC-1, Lay the foundation of studying CD1d-mediated immune escape for pancreatic cancer and immunotherapy of CD1d inhibitors at the cellular and molecular level.Methods Determine the concentration of the constructed lentiviral LP-S0249-LV201with human CD1d gene; MOI was determined by pre-test of lentiviral transfection. then infected it into pancreatic cancer cell lines PANC-1and selected the stably transfected cell lines by Puromycin,observed green fluorescent with the inverted fluorescent microscope at different time,Finally,used reverse transcriptase polymerase chain reaction (RT-PCR) and Western Blot to validate the expression of cell lines.Results The concentrated viral titer of lentiviral particles LP-S0249-LV201with CD1d gene was4.4×108copies/ml; Recombinant lentiviral particles LP-S0249-LV201successfully transfected pancreatic cancer cell line PANC-1.Selected the stably transfected cell lines with complete medium contained3ug/ml puromycin, then saw uniform green fluorescence with the inverted fluorescent microscope; the stably transfected cells with CD1d gene expressed significantly higher at the level of mRNA transcription and protein translation than the control group by RT-PCR and Western Blot analysis.Conclusion Lentiviral LP-S0249-LV201with CD1d gene was successfully transfected into PANC-1cells, which can effectively express CD1d gene at the RNA and protein level, indicating that we successfully established the PANC-1cell line which can steadily express CD1d.Lay the foundation for the further research of CD1d gene in the pancreatic cancer immuno-gene therapy.