| Objective: BMSCs were successfully isolated and cultured, then modified by thioredoxin-1 gene(BMSCs-Trx-1). And models of hyperoxia induced cell injury were established to investigate the effect of hyperoxia exposure on the proliferation and apoptosis of Trx-1 gene modified BMSCs and its possible mechanism.Methods: BMSCs were successfully isolated and cultured, then BMSCs were identified. Construct lentivirus contain vector of Trx-1 gene and transfect BMSCs of passage 3, build Trx-1 modified BMSCs(BMSCs-Trx-1), at the same time, set non-transfected BMSCs and BMSCs modified by empty vector(BMSCs-p) as control group. After transfection, transfection efficiency was detected, Trx-1 m RNA expression levels in these three kind of cells were evaluated by real-time PCR, and the expression levels of Trx-1 protein were evaluated by western boltting. These three groups of cells were randomly classified into HO group and RA group. RA group processing conditions for: 21%O2, 5%CO2, HO group processing conditions as follows: 95%O2, 5%CO2, dealing with 0h, 12 h, 24 h and 48 h. In each subgroup,expression levels of Trx-1 protein were evaluated by western boltting, CCK-8 test assay was used to analyze cell proliferation, cell apoptosis rates were evaluate by flow cytometry. Trx-1 protein concentration in culture medium in each subgroup was deteced by ELISA, the expression level of ASK 1 protein in each group of BMSCs was detected by western blotting.Results: 1) Cell surface marker staining confirms that we successfully isolated,proliferated, and cultured rat BMSCs.2) Transfection efficiency of Trx-1 gene modified BMSCs was 87.3±5.7%, compared with BMSCs-p and BMSCs group, Trx-1 m RNA and Trx-1 protein expression in BMSCs-Trx-1 increased obviously(P<0.001).3) In RA group, no difference was observed in absorbtion among these three subgroups after treated for the same time(P>0.05). In HO group, the absorbtions of BMSCs-Trx-1 in every subgroup was higher than other two subgroups under the same condition, significant difference was observed after 12 hours(P<0.05). 4) In RA group, in terms of apoptosis rates, no difference was found among three subgroups(P > 0.05). And in HO group, the apoptosis rates of BMSCs-Trx-1subgroup were lower than other two subgroups, significant difference was observed after 12 hours(P<0.05).5) In RA and HO group, with the extension of processing time, Trx-1 peotein expression were still at a high level in BMSCs-Trx-1 subgroup than in other two subgroups(P<0.001).6) In RA and HO group, the concentration of Trx-1 protein in culture medium of BMSCs-Trx-1 subgroup were higher than other subgroups at every time point(P<0.001).7) In RA group, no difference was observed after treated in ASK 1 protein expression(P>0.05). In HO group, after treated for the same time, ASK 1 protein expression level in BMSCs-Trx-1 was lower than other two subgroups(P<0.001).Conclusions: 1. The methods of isolation and expansion of rat BMSCs were established.2. Successfully modified BMSCs by Trx-1 gene in vitro, and maintain stable high level expression in BMSCs.3. Compared with BMSCs and BMSCs-p, the proliferation of BMSCs-Trx-1 in hyperoxia was faster.4. Compared with BMSCs and BMSCs-p, the apoptosis rate of BMSCs-Trx-1 in hyperoxia was lower.5. The concentration of Trx-1 protein in culture medium of BMSCs-Trx-1 was higher than other two subgroups, and this maybe one of the possible mechanisms of overexpression of Trx-1 play a protective role to hyperoxia induced BMSCs injury.6. The ASK 1 protein expression level in BMSCs-Trx-1 in HO group was lower than other two subgroups, and this maybe another possible mechanism of overexpression of Trx-1 play a protective role to hyperoxia induced BMSCs injury. |
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