Empirical Study Of Lentiviral-S100A1Transfection Umbilical Cord Wharton’s Jelly-derived MSCs

Part I Modified the mathod of ligating coronary for improving the survival rate of myocardial infarction model ratsObjective To explore a better way for improving the survival rate of myocardial infarction model rats.Methods The myocardial infarction model was produced produced by ligating left anterior descending branch coronary artery through left thoracotomy after injection of narcotic into abdominal cavity and trachea intubation via mouth.The rats were killed by injecting overdose of narcotic at week4after operation. The heart was taken out to observe the shape, infarct size and the pathological morphology by HE staining.Results The successful rate of model was100%, and96.7%of rats survived after operation. After operation for4weeks, the left ventricle became larger, the ischemic region became thinner and the endocardium tissus of the survival rats were fibrotic.Conclusion This modified method could improve the survival rate of animal model.and be used to make a stable model for the basic research of mayocardial infarction,and reduce the bias in the experiment due to the unstable animal models. Part Ⅱ Empirical study of Lentiviral S100A1Gene Transfection Umbilical cord wharton’s jelly-derived MSCsObjective To investigate the transfection efficiency of human UW-MSCs with lentiviral vector carrying S100A1at different multiplicities of infection (MOI), different time points and its influences on cell growth. Meanwhile, the safety of transfected UW-MSCs transplantation was also investigated.Methods UW-MSCs cultured in vitro were infected by Lentiviral-S100A1-enhan-ced green fluorescent protein (Lenti-S100A1-EGFP) reporter gene with different MOIs (0,5,10,50). The expression of EGFP was observed under the fluorescent microscopy at2,7and10days after the infection. At the same time points, the viabili-ty, proliferation multiplicity and differentiation status of the daughter cells were measured to show the effects of the lentivirus on the survival, proliferation and diff-erentiation of UW-MSCs. The mRNA transcription and content of S100A1protein was detected by Real-time PCR and Western blot at10days.Flow cytometry was employed to assess the transfection efficiency and the fluorescence index (FI) number. The transfected UW-MSCs were transplanted into the infarcted myocardium of a murine MI model, then the vital signs were monitored and outcome of the rats were assessed. The rats were sacrificed after4weeks, and hearts were sliced and observed under a fluorescent microscope to determine the expression of EGFP.Conclusion The UW-MSCs showed green fluorescence36hours after the lentiviral infection, with uneven fluorescence intensity, which intensifies with time and reached a stable level after7days. At all time points, there are no obvious differences of viability, proliferation multiplicity.and differentiation status among cells treated with different MOIs of the virus (P>0.05). At the same MOI. The proliferation multiplicity increased progressively in a time-dependent manner(P<0.05). At the same MOI value. at10days.the results of PCR and WB show that the CT index of the study group was higher than the control group at MOI=5(P=0.015), MOI=10(P=0.005) and MOI=50(P=0.02). and the optic density of the study group was significantly higher than the control group(P=0.003).Flow cytometry showed that the transfection efficiency and the FI increased in various extent with increase of MOI and the incubation time (P<0.05). At4weeks after the transplantation, the rats didn’t show any deterioration of the vital signs or death; pathological observation showed green fluorescence within the myocardium of the UW-MSCs transplanted rats.Discussion The present study showed that the UW-MSCs can be efficiently transfected with the Lenti-S100A1-EGFP vector. Within a short time, the transfection efficiency and the FI increased obviously with the increase of MOI and incubation time. The viability and proliferation of the UW-MSCs were not affected by the infection of Lenti-S100A1-EGFP, showing that lentivirus can be a safe and efficient transferring vector for engineering UW-MSCs. The present study also investigated the safety and persistence of the gene expression, which confirmed the safety and showed persistent gene expression within the period of observation. However, due in part to the relatively short period of observation, we are not sure the longest duration that the gene signal may persist in vivo, which may be further elucidated in subsequent studies.