ABSTRACT
Objective To transduct with human telomerase reverse transcriptase(hTERT) to Schwann cells via electransfection technique to prolong the life span and enhance the proliferative capacity of Schwann cells.Methods The hTERT RNA was derived from esophagus cancer tissue and pcDNA3.1-hTERT vector was built.With electransfection technique, we tmnsfected vector into ADSCs.The PCR,TRAP-PCR, and PI-annexin V were tested to prove the expression of hTERT in ADSCs.Results After transduction the hTERT RNA in Schwann ceils, TRAP-PCR test, PI-annexin V dying and S100 dying was positive.Conclusion Telomerase catalytic subunit(hTERT) is transduct into Schwann cells, and it can to enhance the prolifertative capacity.
ABSTRACT
Objective To observe the morphologic changes of immature dendritic cells(imDCs) before and after the electransfection of human hepatic cancer cell RNA. Methods Monocytes were purified from human peripheral blood,and induced into imDCs.Then human hepatic cancer cell RNA was electransfected into monocyte-derived imDCs.ImDCs were identified by the immunocytochemical method with 7 specific antibodies before and after electransfection.These dendritic cells were observed by scanning electron microscopy. Results After electransfection of human hepatic cancer cell RNA there were few changes of molecule expressions in imDCs.ImDCs were in round,oval and irregular shapes before electransfection.Their sizes were not identical but all bigger than monocytes.There were many protrusions in different shapes which looked like dendrite,or/and bubble,veil cloud on the surface of these imDCs.Although there were some cell fusions and cell deaths after electransfection,most imDCs recovered from the damage.Electransfecting human hepatic cancer cell RNA into imDCs would make pores on cell membrane.Conclusions The pores on cell membrane make it possible that the exogenous material enters imDCs.This study can prove the possibility of electransfecting human hepatic cancer cell RNA into imDCs to make cancer vaccine,which provides a new way for tumor biologic therapy.
ABSTRACT
Objective To investigate the method and optimum conditions of electric transfection,and the major influential factors of electransfection efficiency and the survival rate of dendritic cells. Methods RNA was extracted from human hepatocarcinoma cell line(Bel 7402).Purified monocytes as precursor DC-s(pDC-s) were separated from human peripheral blood cells(PBMCs) by density gradient centrifugalization with lymphocyte gradation fluid and adherence method,pDCs were incubated in RPMI-1640 medium containing rhGM-CSF(8?10~5IU/L) and rIL-4(5?10~5IU/L) for 7 days and made them fully differentiate into immature DCs(imDCs).The total RNA human hepatocarcinoma cell and green fluorescent protein(GFP) were electransfected respectively into imDCs by electroporation apparatus with different electric voltages,times of impulse,cell concentrations,temperatures and electroporation buffers.Numbers of green fluorescence positive cells and the total cell number were counted respectively under fluorescent microscope,and visible light microscope.One day after the electric transfection,the cells were stained with 0.4% trypan blue,and electransfection efficiency and the cell survival rate were counted. Results Electransfection efficiency was increased to the highest value,up to about 49.7% when imDCs with the concentration of 5?10~6 cells/ml were mixed with 40?g-total RNA of human hepatocarcinoma cell,the electric voltage of electroporation apparatus was set at 300V,and the time of impulse was 500Us.Conclusion Electric transfection provides a technical possibility to make human hepatocarcinoma RNA into imDCs.The major influential factors of the electransfection efficiency were electric voltage and impulsing time.As receptor cells,the imDCs growing condition was also an important influential factor.