Influence of double gene recombinant vector transfected alcohol-induced rabbit stem cells on expressions of adipogenic and osteogenic genes

Abstract

Objective To investigate the influence of transfection with double gene recombinant vector on the expression of adipogenic and osteogenic genes in alcohol-induced rabbit stem cells. Methods The rabbit bone marrow stem cells (BMSCs) of third generation were randomly divided into 6 groups: (1) normal group (BMSCs given no special treatment); (2) model group (MBSCs given 0.09 mol/L ethanol, no gene transfection); (3) unrelated sequence group (BMSCs givne 10 μl unrelated sequence transfection); (4) siPPARγ group (BMSCs given 10 μl siPPARγ gene vector transfection); (5) exCGRP group [BMSCs given 10 μl calcitonin gene-related peptide (CGRP) gene transfection]; (6) double gene group (BMSCs given 10 μl double gene recombinant vector transfection). Except the normal group, the final mass concentration of 0.09 mol/L ethanol was added to the remaining groups of the cells after transfection and every time the liquid was changed. The expression of PPARγ, CGRP, runt related transcription factor-2 (Runx2) and Osteocalcin mRNA and their proteins was detected at 7th and 14th day of the experiment. Results At 7th day, the expression levels of PPARγ mRNA and its protein in groups of double gene, siPPARγ and normal were low (0.329±0.037 and 0.162±0.013, 0.413±0.045 and 0.174±0.023, 0.376±0.042 and 0.169±0.021), significantly lower than those in model group (0.672±0.079 and 0.871±0.096), unrelated sequence group (0.674±0.083 and 0.823±0.089), and exCGRP group (0.683±0.079 and 0.839±0.091), and the differences were statistically significant (P=0.000). The expression levels of CGRP mRNA and its protein in groups of double gene and exCGRP were high, significantly higher than those in model group, unrelated sequence group, siPPARγ group and normal group, and the differences were statistically significant (P=0.000). The expression levels of Runx2 mRNA, Osteocalcin mRNA and theirs proteins in groups of double gene, exCGRP, siPPARγ and normal were high, those in double gene group were significantly higher than those in model group and unrelated sequence group, and higher than those in groups of exCGRP, siPPARγ and normal, and the differences were statistically significant (P=0.000). At 14th day, the expression levels in all groups were consistent with those at the 7th day. Conclusion Double gene recombinant vector transfected alcohol-induced rabbit stem cells can effectively inhibit the expression of adipogenic gene and up-regulate the expression of osteogenic genes in rabbit stem cells, which is more significant than single gene action. Key words: Double gene recombinant vector; Transfect; Alcohol; Stem cells; Adipogenic genes; Osteogenic genes; Gene expression