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目的观察低氧预处理对BMSCs葡萄糖代谢的影响,探讨其潜在机制,为干细胞疗法的优化提供理论依据。方法取1～3日龄SD大鼠骨髓,采用密度梯度离心法获得BMSCs,取第4代细胞用于实验。根据处理方法不同将细胞分为4组:A组采用常氧培养24 h;B组以1%低氧浓度培养24 h;C组于低氧预处理前用20μmol/L甲氧雌二醇处理24 h;D组于低氧预处理前用50μmol/L缺氧诱导因子1(hypoxia-inducible factor 1,HIF-1)特异的siRNA处理12 h。采用MTT法、生化分析仪及实时荧光定量PCR检测各组BMSCs活力、葡萄糖代谢以及HIF-1αmRNA及葡萄糖转运蛋白1(glucose transporter 1,Glut-1)mRNA的表达。结果 MTT检测示A、B、C、D组吸光度(A)值分别为387.67±58.92、322.50±50.60、297.00±53.00、286.00±41.00,各组间差异无统计学意义(P>0.05)。与A组相比,B组葡萄糖摄取量和乳酸生成量显著增加(P<0.05);C、D组略高于A组,但差异无统计学意义(P>0.05),但显著低于B组,差异有统计学意义(P<0.05);C、D组间比较差异无统计学意义(P>0.05)。与A组相比,B组HIF-1αmRNA和Glut-1 mRNA表达均显著上调(P<0.05);C、D组HIF-1αmRNA和Glut-1 mRNA表达较B组大幅度降低(P<0.05),但仍显著高于A组(P<0.05);C、D组间差异无统计学意义(P>0.05)。结论低氧预处理可上调大鼠BMSCs的葡萄糖摄取和代谢能力,其机制涉及HIF-1 mRNA及其下游基因Glut-1 mRNA的表达上调。
Objective To observe the effect of hypoxic preconditioning on glucose metabolism in BMSCs and to explore its potential mechanism to provide a theoretical basis for the optimization of stem cell therapy. Methods BMSCs from 1 to 3 days old SD rats were obtained. Density gradient centrifugation was used to obtain BMSCs. The 4th passage cells were used for the experiment. The cells were divided into 4 groups according to different treatment methods: group A was cultured in normoxia for 24 h; group B was cultured in 1% hypoxia for 24 h; group C was treated with 20 μmol / L methoxyestradiol before hypoxic preconditioning 24 h. Group D was treated with hypoxia-inducible factor 1 (HIF-1) -specific siRNA for 12 h before hypoxic preconditioning. The activity of BMSCs, glucose metabolism, HIF-1αmRNA and glucose transporter 1 (Glut-1) mRNA expression in each group were detected by MTT assay, biochemical analyzer and real-time fluorescence quantitative PCR. Results The MTT results showed that the absorbance values of A, B, C and D groups were 387.67 ± 58.92,322.50 ± 50.60, 297.00 ± 53.00 and 286.00 ± 41.00, respectively. There was no significant difference between groups (P> 0.05). Compared with group A, glucose uptake and lactate production in group B were significantly increased (P <0.05); group C and D were slightly higher than group A, but the difference was not statistically significant (P> 0.05), but significantly lower than group B Group, the difference was statistically significant (P <0.05); There was no significant difference between C and D groups (P> 0.05). Compared with group A, the expressions of HIF-1αmRNA and Glut-1 mRNA in group B were significantly increased (P <0.05); the expression of HIF-1αmRNA and Glut-1 mRNA in group C and group D were significantly lower than that in group B (P <0.05) , But still higher than that in group A (P <0.05). There was no significant difference between group C and D (P> 0.05). Conclusions Hypoxic preconditioning can up-regulate the glucose uptake and metabolism of BMSCs in rats, and its mechanism involves the up-regulation of HIF-1 mRNA and its downstream gene Glut-1 mRNA.