Effects Of Insulin-like Growth Factor-1Genetic Modification On The Bioactivity Of Adipose Tissue-derived Mesenchymal Stem Cell

Background:Stem cell have the ability of self-renewal and multi-differentiation, which werewidely used in basic and clinical study. It represent a significant area of interest in thefield of cell therapy. adipose tissue-derived mesenchymal stem cell（ADMSCs） werewidely accepted due to that they can be easily harvested by a simple, minimallyinvasive method and also easily cultured. But it still face lots of problems, low rate oftransplantation survival and retention is one of the issue. There are various methodscan be used to improve the activity of transplantation of stem cells, includingpretreatment, choose different delivery route or through genetic modification. Geneticmodification has generated considerable attention in recent years through promotion orsuppression of gene expression, which may allow further improvement of therapeuticpotential of stem cells. Nowadays, several gene and vector can be used, so it isnecessary to select the proper gene and vector for ADMSC modificationHere, we intended to generate a lentiviral vector for expression of insulin-likegrowth factor-I (IGF-1) and investigated the impact of IGF-1transduction on theproperties of cultured ADMSCs and furthermore studied the mechanism of apoptosis,which aims to promote the activity of transplated stem cells and provide theoreticalevidence for clinical application.Methods:Rat ADMSCs were isolated and cultured in vitro, then flow cytometry was carriedout to characterize the cells. After that, lentivirus-mediated expression of insulin-likegrowth factor-1transduced into ADMSCs. The study was divided into three groups,ADMSCs，ADMSCs-GFP and ADMSCs-IGF. Stable secretion of IGF-1in ADMSCs was confirmed by enzyme-linked immunosorbent assay (ELISA) and expression ofAkt and P-Akt were confirmed by Western blotting analysis. MTT assay wasperformed to detect the proliferation and IGF-induced anti-apoptosis was evaluated byflow cytometry.Result:We successfully isolated a mesenchymal cell population from rat adipose tissue andthird passaged ADMSCs were assessed by flow cytometric analysis, indicatingADMSCs were positive for CD90and CD29, but negative for CD31, CD34and CD45.After lentivirus-mediated expression of insulin-like growth factor-1tranduced intoADMSCs for48h, the transduced cells can show green fluorescence confirmed byimmunofluorescence staining. The control group without treatment with lentivirusshowed slightly apoptosis. Compared with ADMSCs-GFP, ADMSCs-IGF shows lowerapoptosis. The MTT result shows that at the five and six days, ADMSCs-IGF promotethe proliferation of ADMSCs. ADMSCs can secrete IGF-1by paracrine and there is nosignificant difference of IGF secretion between the control group and ADMSCs-GFPgroup. ADMSCs treated with lenti-IGF secrete higher level of IGF-1compared tocontrol and ADMSCs-GFP, especially on the third days. All of the three group inducedAkt activation. Notably, it shows that while the addition of IGF-1to the cells causes adramatic increase in the expression of p-AKT.Conclusion:1.ADMSCs was succesfully isolated and cultured from rat adipose tissue and wasmodified by IGF-1.2.IGF-1genetic modification of ADMSCs allow permanent and high expression ofIGF-1in ADMSCs and ADMSCs can secrete IGF-1by paracrine.3.IGF-1genetic modification of ADMSCs can promote the proliferation and inhibit theapoptosis of stem cells.4.The anti-apoptosis effect of IGF-1on ADMSCs may though activate the pathway ofPI3K/Akt.