| Background and objectivesFor people with diabetes, 15% of them also developed DFUs(Diabetic foot ulcers) which would do great damage to the patients and is hard to be cured with conventional treatment. Thus, medical specialists around the world have been actively seeking new methods to cure diabetic patients with peripheral vascular disease. In recent years, autologous stem cell transplantation has been approved and gradually gaining attention in the treatment of ischemic disease. Among the autologous stem cells, MSCs(mesenchymal stem cells) can be easily collected and enjoy higher security of in viro expansion, thus, MSCs transplantaion is the top candidate in the treatment of diabetic lower limb ischemia. However, due to factors such as hypoxia, lacking of nutrition, inflammation, etc, large quantities of stem cells would die after transplantation, which is the key problem confronted in the application of this therapy.According to the preliminary studies conducted by this research group, it was found that PGC-1α(peroxisome proliferator-activated receptor-γ coactivator-1α) could activate the Bcl-2 of the rat MSCs to achieve anti-apoptosis, thus equipping MSCs with apoptosis resistance. However, the upstream regulatory mechanism of Bcl-2 protein is still unclear and more explorations are required in this aspect.ERRα(estrogen-related receptor α) was one of the earliest orphan nuclear receptors being discovered in the world and it plays an important role in the activation of energy metabolism, but there are few explorations on the role of ERRα in the cell apoptosis. In fact, both PPARγ and ERRα could affect cell apoptosis and in this paper, the author tries to explore the role of ERRα in the MSCs apoptosis and in the Bcl-2 gene expression.As the specific antagonist of ERRα, XCT790 was employed to reverse the effects of ERRα. Then the relationship between ERRα and MSCs apoptosis can be explored by observing the performance of MSCs apoptosis under the condition of hypoxia, lacking of nutrition and high glucose.Experimental method and result1.The purchased 57BL/6 bone marrow mesenchymal stem cells were induced in the inducing medium to differentiate into adipogenesis and osteoblast and both the oil red O staining and alizarin red staining were found to be positive. Then FCM(flow cytometry) was employed to detect MSCs surface antigen markers and it was found that Sca-1 and CD44 were positive while CD34 and CD45 were negative.2.For all MSCs under normal culture conditions, they were divided into five groups: a blank control group and other four intervention groups with different concentrations of XCT790(with 0.25% DMSO+0μmol/L XCT790, 0.25% DMSO+5μmol/L XCT790, 0.25% DMSO+10μmol/L XCT790 and 0.25% DMSO+20μmol/L XCT790 for each culture respectively). For all those five groups, there were three complex holes in each group and all the MSCs were cultured in the complete medium DMEM/F12 which contained 10% FBS. 24 hours later, Becton Dickinson FACS Calibur was employed to detect apoptosis rate and it was found that there was no significant difference among the five groups in terms of apoptosis rate(P>0.05).3.For all MSCs undergoing apoptosis-inducing experiments, they were divided into five groups: a control group which was cultured in the medium containing 0.25% DMSO+10% FBS under normal oxygen partial pressure, and other four intervention groups with different concentrations of XCT790(with 0.25% DMSO+0μmol/L XCT790,0.25% DMSO+5μmol/L XCT790, 0.25% DMSO+10μmol/L XCT790 and 0.25% DMSO+20μmol/L XCT790 for each culture respectively). Then MSCs received apoptosis-inducing under the condition where there was 3% O2, no FBS and glucose with a concentration of 25 mmol/L. 12 hours and 24 hours later, the apoptosis rate was detected respectively and it was found that 24 hours later, for intervention groups with XCT790 concentration of 0 μmol/L, 5 μmol/L, 10 μmol/L and 20 μmol/L, the apoptosis rates were 24.60%±1.37%, 32.73%±3.01%, 42.64%±4.46% and 61.35%±6.78% respectively. It was also found that the apoptosis rates in invention groups with XCT790 concentration of 5 μmol/L, 10 μmol/L and 20 μmol/L were significantly higher than that in the invention group with XCT790 concentration of 0 μmol/L. Besides, dose-dependence was also discovered in invention groups with XCT790 concentration of 5 μmol/L, 10 μmol/L and 20 μmol/L(P<0.05, P<0.01).4.24 hours later after MSCs apoptosis-inducing, Real-Time PCR was employed to detect Bcl-2 gene, Bax gene and the protein expression level and it was found that Bcl-2 m RNA level and the ratio between Bcl-2 and Bax in invention groups with XCT790 concentration of 5 μmol/L, 10 μmol/L and 20 μmol/L were significantly lower than that in the invention group with XCT790 concentration of 0 μmol/L(P<0.05) and the ratio between Bcl-2 and Bax was also found to be dose-dependent(P<0.05). 24 hours later, the Bcl-2 and Bax protein levels were also detected and it was found that the Bcl-2 protein level and the ratio between Bcl-2 and Bax in invention groups with XCT790 concentration of 5 μmol/L, 10 μmol/L and 20 μmol/L were significantly lower than those in the invention group with XCT790 concentration of 0 μmol/L(P<0.05) while Bax protein level in the invention group with XCT790 concentration of 0 μmol/L was higher than other intervention groups(P<0.05). It was also found that the ratio between Bcl-2 and Bax protein levels was dose-dependent(P<0.05).Conclusion1.Through the present experiment, it was found that the MSCs employed could differentiate adipogenesis and osteoblast and they could express Sca-1 and CD44 markers but not hematopoietic stem cell markers such as CD34 and CD45, which are in accordance with the identification features of rat MSCs.2.Under normal culture conditions, the antagonism of ERRα didn’t cause obvious MSCs apoptosis in the next 24 hours.3. Under the condition of hypoxia, lacking of nutrition and high glucose, the antagonism of ERRα would cause high rate of MSCs apoptosis, an increase of Bax protein expression level, a sharp decline of Bcl-2 gene and protein expression level and a sharp decline of the ratio between Bcl-2 and Bax, which suggests that ERRα could affect apoptosis through the effects of Bcl-2 protein. |
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