Effect Of Stem Cell Leukemia Gene Transfer Mediated By Lentivirus On Morphology Of Interstitial Cell Of Cajal In Diabetic Cystopathy Bladder

Objective 1.This study is to investigate the effect of stem cell leukemia(SCL)gene and green fluorescent protein(GFP)recombinant lentiviral vectors on the morphology of interstitial cells of Cajal(ICCs)in bladder under high glucose condition.2.To observe morphological changes of Cajal-like cells in guinea pig diabetic cystopathy(DCP)bladder by transurethral perfusion stem cell leukemia(SCL)gene and green fluorescent protein(GFP)gene recombinant lentiviral vectors.Methods 1.To construct SCL gene lentiviral expression vectors and determine virus titers;2.Bladder ICCs from guinea pigs were isolated,identified and were treated for 24 h with 5 mmol/L,15 mmol/L or 25 mmol/L glucose,respectively.Then,cells were transduced with Lenti-SCL,Lenti-control or left untreated for 2,3 and 5 days.The morphology and number of ICCs were analyzed by confocal fluorescence microscope observation.3.Establishment of guinea pig model of DCP;To observe the effect of ICC on the quantity,distribution and ultrastructure of DCP bladder tissue by transurethral perfusion SCL gene recombinant lentiviral vectors.Results 1.It was examined that virus titers was 5 × 108 TU / ml;2.Typical bladder ICCs were identified according to the morphology and specific c-Kit expression.High concentration of glucose concentration(15 mmol/L and 25 mmol/L)impaired the adherent morphology of ICCs.Lenti-SCL transduction successfully introduced SCL gene into ICCs.Lenti-SCL transduction gradually rescued the morphological damage induced by 15 mmol/L glucose.However,Lenti-SCL transduction didn’t recover the morphological damage caused by 25 mmol/L glucose.In contrast,damaged ICCs in untreated and Lenti-control transduction groups by high glucose were un-rescued.Additionally,Lenti-SCL transduction had no significant effect on cell number decrease induced by high glucose treatment.3.The successful rate of diabetic guinea pigs was 47.7%;Induced 27 DCP guinea pigs;In the experimental group,the 7days,14 days,28days time was prolonged,the number of ICC was gradually increased(P < 0.05),the distribution was gradually intensive,and the green fluorescence was gradually decreased,while in the blank control group and positive control group,the number of ICC gradually reduced(P < 0.05),the distribution is sparse,because no GFP expression,no green fluorescence by Laser scanning confocal microscopy;In the experimental group,the time of 7days,14 days and 28 days was prolonged,The number of organelles increased,the morphology became normal and the vacuoles decreased,both sides of the cells have significant convex branches increased and extend,The ultrastructure of damaged ICC is in constant recovery.On the contrary,in the blank control group and positive control group,The number of organelles was decreased,the morphology was irregular,the number of vacuoles increased,both sides of the cells have significant convex branches was scarce,and the ultrastructure of ICC was not improved under transmission electron microscope.Conclusion 1.Human SCL is expressed in glucose-treated ICCs by Lenti-SCL transduction,resulting in the partial recovery of damaged cell morphology caused by high glucose concentration.2.After SCL gene recombinant virus was transfected into guinea pig DCP bladder via urethra perfusion,Have certain effects on the recovery of ICC in morphology and quantity,to achieve a certain degree of treatment effect of DCP bladder,Can further provide ideas for the treatment of DCP by using SCL gene recombinant virus.