Effect of double gene recombinant vector on the proliferation and osteogenic activity of rabbit stem cells induced by ethanol

Abstract

Objective To investigate the effects of double gene recombinant vector on the proliferation and osteogenesis of alcohol-induced rabbit stem cells. Methods The rabbit bone marrow mesenchymal stem cells (BMSCs) at the third-generation were randomly divided into 6 groups: (1) normal group [bone marrow stem cells (BMSCs) without special treatment]; (2)model group (BMSCs without gene transfection); (3) unrelated sequence group (10 μl unrelated sequence transfected BMSCs); (4) siPPARγ group (10 μl siPPARγ gene vector transfected BMSCs); (5) exCGRP group (10 μl CGRP gene vector transfected BMSCs); (6) double gene group (10 μl double gene recombinant vector transfected BMSCs). The final mass concentration of 0.09 mol/L alcohol was added to all groups except the normal group. BMSCs proliferation was detected by methyl thiazol tetrazolium (MTT) method. Alkaline phosphatase (ALP) activity, laminin content, collagen type I content, and osteocalcin content in medium and ALP staining were detected at 14th day later. Results The proliferation in double gene group was obvious, and the proliferation curve was close to the normal group. The ALP activity, laminin content, collagen type I content and osteocalcin content in medium of the cells in the double gene group were the highest [(18.593±2.350) U/L, (24.422±3.110) ng/ml, (5.951±0.650) μg/L, (5.836±0.630) ng/ml respectively], which were significantly higher than those in model group [(6.528±0.830) U/L, (9.422±1.250) ng/ml, (1.733±0.230) μg/L, (2.016±0.240) ng/ml], unrelated sequence group [(7.011±0.850) U/L, (9.693±1.260) ng/ml, (1.663±0.220) μg/L, (1.913±0.230) ng/ml], obviously higher than those in siPPARγ group [(13.536±1.710) U/L, (17.103±2.230) ng/ml, (3.486±0.410) μg/L, (3.686±0.420) ng/ml], exCGRP group [(13.692±1.760) U/L, (17.185±2.240) ng/ml, (3.525±0.420) μg/L, (3.833±0.430) ng/ml], normal group [(15.056±1.910) U/L, (18.528±2.430) ng/ml, (4.035±0.460) μg/L, (4.012±0.460) ng/ml], and the difference was statistically significant (all P=0.000). The contents in model group and unrelated sequence group were significantly lower than those in normal group (all P=0.000). The contents in siPPARγ and exCGRP groups were slightly lower than those in normal group: for ALP activity (P=0.222, 0.274), for laminin (P=0.362, 0.390), for collagen type I (P=0.082, 0.105), and for osteocalcin (P=0.276, 0.543). The contents in double gene group were significantly higher than those in normal group: for ALP activity (P=0.031), for laminin (P=0.010), for collagen type I and osteocalcin (all P=0.001). The contents in double gene group were significantly higher than those in siPPARγ and exCGRP groups: for ALP activity (P=0.005, 0.006), for laminin (all P=0.003), for collagen type I and osteocalcin (all P=0.000). ALP staining showed that the cytoplasm in a large number of cells in double gene group was dyed dark blue and more than that in normal group. Conclusion The double gene recombinant vector can maintain the normal proliferation of rabbit stem cells induced by ethanol, and significantly promote osteogenic activity, which is superior to the single gene effect. Key words: Double gene recombinant vector; Ethanol; Stem cells; Proliferation; Osteogenic activity; Rabbit