高糖环境对小鼠成骨前体细胞及脂肪干细胞成骨分化的影响

Effects of high glucose on osteoblastic differentiation of primary osteoblasts and adiposederived stem cells in mice

目的研究不同葡萄糖浓度对小鼠成骨前体细胞(primary osteoblasts,POBs)和脂肪干细胞(adipose-derived stem cells,ASCs)成骨分化的影响。方法分离培养小鼠成骨前体细胞、棕色脂肪干细胞(brown adipose-derived stem cells,B-ASCs)、白色脂肪干细胞(white adipose-derived stem cells,W-ASCs),复苏MC3T3-E1细胞系,分别给予不同浓度葡萄糖(5.5 mmol/L、25 mmol/L和55 mmol/L)刺激,常规成骨诱导分化7d,采用碱性磷酸酶(alkaline phosphatase,ALP)染色观察不同细胞成骨分化程度。取成骨前体细胞分别成骨诱导分化3d、7d,采用定量PCR 检测各组细胞Runx2、碱性磷酸酶、Osterix 及骨桥蛋白(osteopontin,OPN)等成骨分化标记物的表达水平。采用Western blot检测成骨相关蛋白的表达。结果在不同浓度葡萄糖刺激下,MC3T3-E1、POBs和W-ASCs的成骨分化能力随着糖浓度的升高而升高,而B-ASCs的成骨分化能力则受到抑制。对POBs成骨诱导培养3d和7d后,25mmol/L、55mmol/L组成骨分化程度显著高于5.5mmol/L组。Real-timePCR结果显示3d和7d时,25mmol/L、55mmol/L组Runx2、ALP、Osterix和OPN的表达也显著高于5.5mmol/L组。Westernblot检测结果证明高糖在成骨诱导过程中可激活骨钙素(osteocalcin,OCN)的表达。结论高糖对成骨前体细胞的成骨分化起促进作用。

Objective To study the effects of different glucose concentrations on osteogenic differentiation of primary osteoblasts and adipose-derived stem cells in mice. Methods Mouse primary osteoblasts(POBs), brown adipose-derived stem cells(B-ASCs) and whiteadipose stem cells(W-ASCs) were isolated and cultured. MC3 T3-E1 cell line cells were resuscitated. And then they were all stimulated with glucose at different concentrations(5.5 mmol/L, 25 mmol/L and 55 mmol/L). The osteogenic differentiation of cells was observed by alkaline phosphatase(ALP) staining at 7 d after osteogenic differentiation. Mouse primary osteoblasts were taken and osteoblastic differentiation was induced for 3 and 7 days respectively. The expression levels of Runx2, ALP, Osterix, OCN, OPN and other markers of osteoblastic differentiation in each group were detected by quantitative PCR. Western blot was used to detect the expression of osteogenic related proteins. Results The osteogenic differentiation ability increased with the increase of glucose concentration in MC3 T3-E1,POBs and W-ASCs, but it was inhibited in B-ASCs. At 3 d and 7 d after osteoblastic induction culture of POBs, the osteogenic differentiation of 25 mmol/L group and 55 mmol/L group was significantly higher than that of 5.5 mmol/L group. RealtimePCR results showed that the expressions of Runx2, ALP, Osterix and OPN in the 25 and 55 mmol/L concentration groups were also significantly higher than those in the 5.5 mmol/L group. Western blot results showed that high glucose could activate OCN expression during osteogenic induction. Conclusion High glucose promotes the osteogenic differentiation of primary osteoblasts.