The Experimental Research Of Rat BMSCs Transfected With Lentiviral Vector Carrying Beta-catenin In Vitro

BackgroundSmoke inhalation lung injury (SILI) is a special type of acute lung injure (ALI), which is the main cause of death in the fire burn patients surviving. It has a rapid onset, rapid development, in a bad way and other characteristics, mainly involving the respiratory tract, lungs, pathology showed trachea mucosa degeneration, cilia disappear, submucosal edema, inflammatory cell infiltration and alveolar rupture fusion, alveolar exudate, hemorrhage, pulmonary vascular endothelial necrosis, thrombosis, etc. Because of their potential of differentiae to all kinds of cells, mesenchymal stem cells receive significant attention. A number of studies reported that BMSCs could differentiate into alveolar epithelial cells in vitro and in vivo conditions, and repair of damaged alveolar epithelial cells. However, there were two questions:the undefined differentiation mechanism and the low differentiation efficiency. In addition to, our previous study found that the canonical Wnt signaling pathway in lung of rats had SILI was activated, and β-catenin was the core component of this signaling pathway, and this study aimed to observe β-catenin on proliferation of BMSCs based on this.ObjectiveConstruction of LV overexpressing β-catenin, then, transfected293T cells and packaged, purified virus transfected rat BMSCs, so that β-catenin overexpress in BMSCs, then, we can investigate the effect of β-catenin on the growth of BMSCs’proliferation.Method1. Primary rat BMSCs were isolated and cultured using of whole bone marrow cultured method, and were observed under an inverted microscope, the growth curve of BMSCs were charted by CCK-8way, and BMSCs were selected and induced to bone formation and fat formation, then the cells after osteogenic were detected by alizarin red staining, and cells after fat induction were identified by oil red O.2. Construction of LV overexpressing β-catenin, then, transfected293T cells and packaged, In order to clarify the transfection efficiency that the LV containing the β-catenin gene of BMSCs under various MOI, and explore the conditions that higher transfection efficiency and lower toxicity to cells, BMSCs in96well plates were transfected for pre-experiment after construction of LV overexpressing β-catenin. Then, cells were divided into five groups, A group:BMSCs that transfected with LV containing β-catenin gene, MOI=10; B groups:BMSCs that transfected with LV containing β-catenin gene but MOI=25; C group:BMSCs that transfected with negative control LV; D group: BMSCs with polybrene in the medium; E group:BMSCs. Detection of β-catenin expression in BMSCs by PCR, the proliferation activity of each group’s BMSCs were tested by CCK-8way.Result1. BMSCs adherent growth, elongated spindle-shaped, and similar to the shape of fibroblasts. Cell growth curve is S-shaped. After adding adipogenic medium72h, small lipid droplets appear intracellular, the number of lipid droplets increasing and cells merging about two weeks later, at the same time, cells changed from long spindle into circular or polygonal, and oil red O staining showed a large number of lipid precipitation. The cells increase in size, and like short spindle in shape after joining osteogenic solution for3days; then the cells were polygonal, and cell granules were increasing in the cytoplasm at the first seven days; the cytoplasmic of cells that grown in colonies were filled with granules, in addation,we can observe intercellular calcium deposition at the first14days; cells fusion gradually and lose the cell structure, at the same time, we observed calcium nodule significantly and red nodules by Alizarin red staining at the first21days.2. The titer of LV containing β-catenin gene was2.00E+8TU/ml. The infection efficiency of transfecting target gene in BMSCs after96h was around76%tested by flow cytometry when the MOI was25, and the β-catenin mRNA is overexpressed in BMSCs detected by PCR.3. The cell growth was slow relatively at the first1-2days after cell adherent in D/E groups, and it grow faster and into the logarithmic growth phase at the first3-5days, then the cells’ growth rate was slow down and stagnant gradually, and there were no significant difference between the two groups(P>0.05). Compared with cells in D/E group, the BMSCs transfected LV were inhibited, and its growth slowed significantly from the first two days(A/B/C groups compared with D groups namely, groups differences were statistical differences, and P<0.001). Group C compared with group A/B, its growth rate had a further decline (Group C compared with group A/B respectively, there were statistically significant from the beginning of the first three days, and P<0.05, there were significant statistical differences from the beginning of the first four days, and P<0.001). No significant difference in growth rate of BMSCs while group A compared with groups B.Conclusion1. We get BMSCs successfully by whole bone marrow adherent culture method.2. The overexpression of β-catenin can promote proliferation of BMSCs compared with BMSCs transferred negative control virus.