高糖环境下成骨细胞增殖和分化与FoxO1及氧化应激的关系

Correlation of proliferation and differentiation of osteoblast cells with FoxO1 and oxidative stress under high glucose circumstance

目的:探讨Fox O1基因在高糖环境对成骨细胞增殖和分化能力影响中的作用,并观察其与氧化应激间的关系。方法:1将小鼠成骨细胞株MC3T3-E1铺板,约90%的细胞汇合后分为正常糖对照组、高糖组、高糖+抗氧化剂组3组;2靶向构建慢病毒载体,慢病毒侵染MC3T3-E1细胞后,将体外培养的MC3T3-E1分为正常细胞正常糖对照组(NG)、正常细胞高糖组(HG)、无关sh RNA转染对照组(NC)、Fox O1-sh RNA慢病毒转染组(Fox O1-sh RNA)4组。通过CCK-8检测成骨细胞增殖能力,测定碱性磷酸酶(ALP)活性并染色,检测茜素红染色,用实时PCR测定不同时间点成骨标志基因Runx2转录因子(runt-related transcription factor 2,Runx2)、骨钙素(ostcocalcin,OC)、ALP m RNA的表达及反映氧化应激相关超氧化物歧化酶、生长抑制及DNA损伤诱导基因45(growth arrest and DNA damage 45,Gadd45)的m RNA表达,并用蛋白印迹法检测Fox O1的蛋白表达水平。结果 :1与正常糖对照组相比,高糖组的成骨细胞增殖和分化能力及Runx2、ALP、OC基因m RNA表达水平显著降低,而加入抗氧化剂后上述能力则均有不同程度恢复,高糖组的超氧化物歧化酶和Gadd45 m RNA表达水平显著增高。2Fox O1-sh RNA组的Fox O1 m RNA及蛋白表达水平均明显低于高糖组,同时Fox O1-sh RNA组的Runx2、ALP m RNA表达水平亦显著低于高糖组。结论:高糖环境下,成骨细胞增殖、分化能力的下降与氧化应激密切相关,而Fox O1基因在氧化应激抑制成骨细胞的增殖、分化过程中起到一定的保护作用。

Objective： To investigate the role of FoxO1 in the event of high glucose affecting proliferation and differentiation of osteoblasts, and study its relation with oxidative stress. Methods： 1 MC3T3-E1 cells were divided into three groups： normal glucose group（Group A）, high glucose group（Group B）, high glucose＋ N-acetyl-L-cysteine group（Group C）.2 The lentivirus vector of sh RNA targeting FoxO1 was constructed in vitro. MC3T3-E1 cells cultured in vitro were divided into four groups： normal glucose group（NG）, high glucose group（HG）, transfected with sh RNA negative control group（NC）,Fox O1-sh RNA transfected group（FoxO1-sh RNA group）. Proliferation of osteoblasts was evaluated by CCK-8 assay. ALP activity and mineralized nodules were detected by ALP activity assay and staining, and alizarin red staining. The expressions of osteogenicmarkers such as Runx2, ALP and OC, as well as markers of oxidative stress such as SOD, Gadd45 m RNA at different time points were assayed with real-time PCR. The protein level of FoxO1 was measured by Western blotting.Results： 1Compared with normal glucose group（Group A）, the proliferation and differentiation of osteoblasts, as well as the m RNA levels of Runx2, ALP and OC, were significantly decreased in high glucose group（Group B）. On the other hand,the parameters mentioned above were increased in different extent after adding antioxidants（NAC）. The m RNA levels of SOD and Gadd45 were significantly increased in high glucose group（Group B）. 2 Compared with HG group, the mRNA levels of FoxO1, Runx2 and ALP, and the protein level of FoxO1 were decreased in Fox O1-sh RNA group. Conclusions：Decrease of cell proliferation and differentiation in osteoblasts induced by high glucose is closely related to oxidative stress.FoxO1 plays a protective role in oxidative stress-induced inhibiting proliferation and differentiation of osteoblasts.