Study Of The Inhibited Effects Of HSV-TK/GCV System Mediated By RAAV2 On Lens Epithelial Cells

Chapter One Recombinant Adeno-associated Virus-mediated EfficiencyExpression of Enhanced Green Fluorescent Protein Gene into LensEpithelial Cells in vitroObjective To investigate the efficiency of recombinant adeno-associated virus type 2(rAAV2)-mediated enhanced green fluorescentprotein(EGFP) gene and the effect on the proliferation of rabbit lensepithelial cells(N/N1003A) in vitro, and the possibility of rAAV2 as avector in the gene therapy of posterior capsular opacification(PCO).Methods According to various multiplicity of infection(MOI)(MOI=10⁴, 10⁵, 2×10⁵, 10⁶), N/N1003A cells were transfected withrAAV2-EGFP, and contrast phase microscope observation was performedto assess the changes of morphology of the cells transfected. After thecells were transfected for 1, 3, 5, 7, 9days, the expression of EGFP wereobserved by inverted fluorecent microscope and laser confocalmicroscope. The percentage of positive EGFP in random 200 cells werecalculated. After the cells were transfected for2, 7 days, the transfecionefficency is detected by flow cytometry. The effect of rAAV2 vector onN/N1003A cells was determined by MTT colorimetric assay.Results EGFP expression was observed in N/N1003A cells aftertransfection in 1 day. The transfecion efficiency of the rAAV2 was increased with the increasing of MOI and the prolongation of transfectiontime, reached the fastigium at 7 days. At that time, the transfectionefficiency was 71.52%±0.50% (MOI=10⁶). The transfected cells had noobviously changes of morphology, and MTT methods showed that theOD records between transfected groups (with various MOI) anduntransfected group had no statistical difference (P＞0.05).Conclusions Recombinant AAV2 vector encoding the EGFP genecan be transfected into lens epithelial cells stably and efficiently, whichproves AAV is a good vector in the gene therapy of posterior capsularopacification.Chapter Two Construction of Recombinant Adeno-associated VirusVector Containing HSV-TK Gene and its ExpressingObjective To construct a recombinant adeno- associatedvirus(AAV) vector plasmid pSNAV2.0-TK containing HSV-TK gene,produce recombinant adeno- associated virus rAAV2/HSV-TK, and detectthe integration and expression of HSV-TK gene in lens epithelial cellstransfected with rAAV2/HSV-TK, which provides the foundation for genetherapy for posterior capsular opacification(PCO). Methods By using recombinant technique, TK gene was excisedfrom plasmid pDC316-TK by SalI+EcoRI, inserted into the plasmidpSNAV2.0 to construct recombinant plasmid pSNAV2.0-TK. The newplasmid was verified by polymerase chain reaction(PCR) and restrictonendonuclease. The cell strain containing pSNAV2.0-TK were screened byG418 after the plasmid pSNAV2.0-TK transfected BHK-21 cells, withhelper virus HSV1-rc/AUL2 to produced the recombinant virusrAAV2/HSV-TK. The purity of rAAV2/HSV-TK was detected bySDS-PAGE and HPLC methods. The titre of rAAV2/HSV-TK wasobserved by DNA dot blot hybridization. The TK gene in lens epithelialcells transfected with rAAV2/HSV-TK was investigated by PCR andreverse transcription-polymerase chain raction(RT-PCR).Results The recombinant plasmid pSNAV2.0-TK was verified byPCR and restricton endonuclease. The coat protein of AAV was detectedby SDS-PAGE method, the purity of rAAV2/HSV-TK was＞98% byHPLC method and the titre of rAAV2/HSV-TK was 1×10¹²v.g./ml byDNA dot blot hybridization. The expression of HSV-TK gene wasobserved in lens epithelial cells transfected with rAAV2/HSV-TK.Conclusions A recombinant adeno- associated virus vectorplasmid containing HSV-TK gene (pSNAV2.0-TK)was successfullyconstructed, and high titer recombinant adeno- associated virus(rAAV2/HSV-TK) were obtained. The integration and expression of HSV-TK gene in lens epithelial cells were observed after transfected withrAAV2/HSV-TK.Chapter Three Effects of Recombinant Adeno-associated Virus-mediated HSV-TK/GCV system on lens epithelial cellsObjective To study the efficiency of recombinant adeno-associated virus-mediated herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) suicide gene therapy system on rabbit lensepithelial cells(N/N1003A)in vitro and investigated the mechanism of celldeath.Methods N/N1003A cells were infected in vitro by therecombinant adeno-associated virus type 2(rAAV2) Vector containingHSV-TK and followed by the treatment of GCV, and the uninfectedN/N1003A cells were used as the control. The percentages of survivalcells in various dose of GCV cells were determined at indicated timerespectively by MTT assay. The infected cells were cultured togetherwith the uninfected cells in different proportion to detect bystander effect.Recombinant virus rAAV2/HSV-TK infected N/N1003A cells in variousmultiplicity of infection(MOI)to analyse the effects of HSV-TK/GCV system. Apoptosis and necrosis were observed by electron microscopeand Hoechst33258 staining methods. The apoptotic rate and cell cyclewere detected by flow cytometry.Results The percentages of survival cells in various dose GCVbetween the study group and control group had statistical difference(P＜0.01). The IC₅₀ of GCV in the study group was 2mg/L but 524mg/L incontrol group and the therapeutic index was 262. The percentages ofsurvival cells in N/N1003A-TK cells decreased with the prolongation ofaction time of GCV. The percentages of survival cells in mixing cells wasonly 51.47%±1.44% when the N/N1003A-TK cells is 10%. Thecytotoxicity of GCV was increased with the increasing MOI. Thephenomenon of apoptosis was observed in N/N1003A-TK cells followedby GCV, and the percentages of apoptotic cells was significantly higherthan control group(t=3.83, P＜0.01). The percentages of N/N1003A-TKcells in the S phase of the cell cycle were 11.86%±3.71%, which wasstatistically higher than control group(P＜0.01). Whereas the percentagesof the G₀/G₁ phase was significantly lower than control group(P＜0.01).Conclusions GCV can kill efficiently the N/N1003A cells infectedby recombinant virus rAAV2/HSV-TK, and there is strong bystandereffect. The mechanism of cell death caused by HSV-TK/GCV mayinvolve in apoptosis and necrosis. Therefore, recombinantadeno-associated virus-mediated HSV-TK/GCV suicide gene system may provide an effective approach to the treatment of posterior rcapsularopacification(PCO).