Effects of adenovirus transfection and small molecular hydrogels-mediated hepatocyte growth factor gene modification on biological characteristics of bone marrow mesenchymal stem cells in rats

Abstract

Objective To investigate the effects of adenovirus transfection and small molecular hydrogels (SMHs) mediated HGF on the biological characteristics of bone marrow mesenchymal stem cells (MSCs) in rats.Methods The MSCs of the 9th passage were assigned to be treated with common culture media (group MSC),Ad-HGF transfection followed by culture in common media (group Ad-HGF+ MSC),incubation with SMHs alone (group SMH + MSC),and Ad-HGF transfection followed by incubation with SMHs (group SMH+Ad-HGF+MSC).MSCs were transfected with Ad-HGF and the assessment of transfection rate was carried out at 48 h by using flow cytometry.Expressions of HGF mRNA in groups MSC,Ad-HGF+ MSC and SMH+Ad-HGF+MSC were examined by RT-PCR.The supernatant HGF protein of MSCs in groups MSC,Ad-EGFP+MSC,Ad-HGF+MSC and SMH+Ad-HGF+MSC were detected by ELISA till day 23 following transfection.The phase-contrast microscope was employed to compare the MSCs morphology of groups MSC,Ad-HGF+MSC and SMH+Ad-HGF+MSC.MTT approach was adopted to determine the growth of cells in all groups at days 1 to 7 for depiction of growth plots,and flow cytometry was used to assay the cellular surface markers and cell cycles at week 2 following transfection.After 24-h incubation with 5-aza,MSCs of 4 groups were cultured for 3 weeks,and assessment of the capacity of differentiation to cardiac cells was carried out by immune cell fluorescence assay.Results Incubation of MSCs for 48 h yielded a transfection rate of 74.7％.At 48 h after transfection,Ct value was 14.99 in group MSC,8.38 in group SMH+ Ad-HGF+MSC,8.51 in group Ad-HGF+MSC.There was conclusive HGF gene expression in two transfection groups.Apart from group SMH +Ad-HGF+ MSC,supematant HGF protein was noted in group Ad-HGF+ MSC,in which the level peaked at 48 h (121 μg/L) following transfection and sustained to day 23.The supernatant HGF protein peaked at day 3 (118 μg/L) following transfection and sustained to day 23 in group SMH+ Ad-HGF+ MSC.The cells in groups MSC and Ad-HGF+ MSC grew mimicking fibroblasts,whilst those in groups SMH+ Ad-HGF+ MSC and SMH+MSC appeared mostly rod-or partly spherical-shaped,with tight junction and clustered proliferation.Groups MSC and Ad-HGF+ MSC were characterized by similar growth plots,in which the light absorption at each time point did not differ statistically (all P＞0.05),suggesting a close pattern of cellular growth.These two groups,when compared with groups SMH+MSC and SMH+Ad-HGF+MSC,showed similar cellular growth trends without marked difference in absorption at all time points,and had no significant difference in the absorption at days 1 to 4 (all P＞0.05) but had considerably elevated level thereafter (all P＜0.05).At week 2,groups MSC,SMH+MSC,Ad-HGF+ MSC and SMH+Ad-HGF+MSC all had higher expressions of CD44,CD90 and CD49,and lower expressions of CD45,CD34 and CD31.There were no significant differences of uniform antigens in all the 4 groups.The differences in the distribution of Go-G1,S stage and G2-M were unremarkable among all the 4 groups.At week 3 following incubation of MSC with 5-aza for 24 h,groups SMH+Ad-HGF+MSC,Ad-HGF+MSC and SMH+ MSC had increased cellular cTnT expression compared with group MSC [(27.07±6.49)％,(21.43±6.75)％and (28.12±7.26)％ vs (20.09±4.17)％,all P＜0.05].Group SMH+Ad-HGF+MSC had a higher rate of cTnT expression than groups Ad-HGF+MSC and MSC,but was similar to group SMH+MSC.Conclusions The SMH may act as a three-dimensional culture medium to promote cellular proliferation.This is evidenced by the fact that the addition of SMH to Ad-HGF modified MSCs does not alter the cellular surface markers nor the cell cycles but may facilitate the differentiation of MSCs induced by 5-aza. Key words: Small molecular hydrogel; Adenovirus; Hepatocyte growth factor; Bone marrow; Mesenchymal stem cells