Preliminary Study On Lentiviruses Carrying Enhanced Green Fluorescent Protein Gene Labeling Rat Adipose Derived Stem Cells And Tracing In Vivo

Objective: Lentiviruses carrying EGFP infected rat adipose derived stem cells(ADSCs)for labeling.Transplanted into a normal allogeneic rat by local subcutaneous injection.The distribution,migration and colonization of the transplanted cells in the rat were detected by fluorescence microscopy and immunohistochemistry.Methods: ADSCs were extracted and cultured,cell morphology was observed by HE staining,multidirectional induction of osteogenesis,lipogenesis and chondrogenesis.The growth curve of adipose derived stem cells was drawn by CCK8 method.ADSCs were transfected with lentivirus carrying EGFP with MOI=0,1,5,10,25,and 50.The transfection rate was measured by flow cytometry to select the appropriate MOI.The proliferation of ADSCs was evaluated by CCK8 method.EGFP— ADSCs were induced into osteogenesis,lipogenesis and chondrogenesis.Results of induction were checked by alkaline phosphatase,oil red O and alxin blue staining.The labeled cells were injected into the subcutaneous layer of rats.After 7,14,28 day,the transplanted sites and main organs were taken for fluorescence observation and immunohistochemical staining to observe the expression of EGFP—ADSCs in vivo.Results: ADSCs were successfully extracted,and a small number of adherent cells were observed after 24 hours,cell morphology was irregular.After cell passage,the cell morphology was long spindle shape and the distribution direction was regular.The third generation of ADSCs were stained by HE.The nucleus was stained blue,and the cytoplasm was red.ADSCs were induced into osteogenesis,lipogenesis and chondrogenesis.The transfection rates of MOI=0,1,5,10,25 and 50 were 0.12%,3.62%,42.4%,66.6%,90.4%,and 98.0%,respectively.The proliferation of cells in the transfected group(MOI=25)and the untransfected group(MOI=0)was not significantly different by CCK8 method(P>0.05).After induction,EGFP—ADSCs still could differentiate into osteogenesis,lipogenesis and chondrogenesis.7 days,14 days,28 days after cell transplantation,EGFP expression was found at the transplant site,but EGFP expression was not found in the main organs.Conclusion:1.The optimal MOI of ADSCs with lentiviruses carrying enhanced green fluorescent protein gene was 25.The labeled ADSCs had good cell viability.The biological characteristics of the labeled ADSCs had no change.This method can be used for tracing of ADSCs.2.The expression of EGFP was observed in the transplantation site after 7,14 and 28 days of cell transplantation.With the extension of transplantation time,the number of transplanted cells decreased,and the distribution of transplanted cells was not detected in the main organs(heart,liver,spleen,lung,kidney,brain).