Effect of nano-selenium loaded with lycium barbarum polysaccharide on the proliferation of lens epithelial cells after UVB damage in vitro

AIM:To investigate the effect of nano-selenium loaded with different concentrations of lycium barbarum polysaccharide(LBP-Se NPs)on the proliferation of human lens epithelial cells(HLECs)from UV irradiation.METHODS:LBP-Se NPs were prepared and their particle size was detected.HLECs(SRA01/04)were irradiated with UVB for different time(0,10,20,30,40,50,60 min)to construct a damaged model,the survival rate of cells was determined by methylthiazol tetrazolium(MTT)assay.The 4’,6-Diamidine-2’-phenylindole dihydrochloride(DAPI)staining was used to observe the status of cell nucleus and drug entering cytoplasm through cell membrane in SRA01/04 cells after adding LBP-SENPS loaded with coumarin fluorescence agent 24 h under fluorescence microscope.SRA01/04 normal and UVB-damaged cells were treated with different amounts of LBP-Se NPs at different concentrations,cells proliferation were observed.RESULTS:The particle size of LBP-Se NPs was stable in the range of 150-200 nm.The survival rate changes with time after UVB irradiation were statistically significant.The 10 min of UVB exposure as the time was chosen to construct the cell damage model.With DAPI staining,LBPSe NPs were observed to enter the cytoplasm through the cell membrane under fluorescence inverted microscope.Cytotoxicity of SRA01/04 at different concentrations of LBPSe NPs were measured.Cell survival rate was statistically different compared with the control group.The higher the loading concentration of LBP in nano-Se drugs was,the higher the cell proliferation rate was(P<0.05).The lower the concentration of LBP-Se NPs,the higher the cell proliferation rate,showing a negative growth trend(P<0.05).The group with the highest average cell proliferation rate was 0.5μmol/L 2.0 mg/m L LBP-Se NPs(128.80%).When the 2.0 mg/m L LBP-Se NPs group was selected for cell photography,the cell density was higher at 0.5μmol/L.With the increase of concentration,SRA01/04 cells appeared more cytoplasm dehydration,cell shrinkage and apoptotic bodies,and cell density decreased.CONCLUSION:LBP-Se NPs has moderate particle size and good stability.LBP-Se NPs can protect HLECs(SRA01/04)from UVB-induced damage,and the cell proliferation rate is further increased with increasing the amount of loaded LBP and decreasing nano-selenium concentration.

Construction of adenovirus vectors encoding the lumican gene by gateway recombinant cloning technology

AIM： To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. METHODS： Gateway recombinant cloning technology was used to construct adenovirus vectors. The wild-type （wt） and mutant （mut） forms of the lumican gene were synthesized and amplified by polymerase chain reaction （PCR）. The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDown- multiple cloning site （MCS）-/internal ribozyme entry site （IRES）/enhanced green fluorescent protein （EGFP）. Then the desired DNA fragments were integrated into the destination vector pAV.Desld yielding the final expression constructs pAV.Exld-CMV〉wt-lumican/IRES/ EGFP and pAV.Exld-cytomegalovirus （CMV） 〉mutlumican/IRES/EGFP, respectively.RESULTS： The adenovirus plasmids pAV.Exld-CMV〉 wt-lumican/IRES/EGFP and pAV.Exld-CMV 〉mutlumican/IRESlEGFP were successfully constructed by gateway recombinant cloning technology. Positive clones identified by PCR and sequencing were selected and packaged into recombinant adenovirus in HEK293 cells. CONCLUSION： We construct adenovirus vectors containing the lumican gene by gateway recombinant cloning technology, which provides a basis for investigating the role of lumicangene in the pathogenesis of high myopia.