高剂量葡萄糖对小鼠肌管细胞自噬表达的影响

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摘要目的：探讨高剂量葡萄糖对肌管细胞自噬表达的影响。方法：使用分化培养基诱导小鼠成肌细胞分化成肌管细胞，分正常剂量（5.5 mmol/L）和高剂量（17 mmol/L和30 mmol/L）葡萄糖浓度的3组培养基培养肌管细胞，于6、12、24、36、48、60、72 h后分别收集细胞，检测自噬相关蛋白和mRNA表达量，并用串联荧光标记LC3（mRFP-GFP-LC3）慢病毒转染小鼠成肌细胞，动态监测自噬流。结果：高剂量葡萄糖条件下肌管细胞自噬表达存在2个阶段，30 mmol/L组培养24 h自噬表达下降，LC3和Beclin-1蛋白表达水平比5.5 mmol/L组低（P＜0.05）；而在30 mmol/L葡萄糖条件下培养48、60和72 h后自噬水平增强，LC3和Beclin-1的基因表达均明显升高（P＜0.05）。肌管细胞GFP和RFP荧光斑点的定位与数量分析也证明30 mmol/L葡萄糖浓度的培养基培养48、60和72 h后可显著增加自噬体和自噬溶酶体的合成。结论：高剂量葡萄糖对肌管细胞自噬表达有重要影响，不同时间自噬表达存在差异，长期高血糖状态可诱导自噬过度活化。

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	罗胜男
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关键词 ：自噬, C2C12细胞, 糖尿病, LC3, Beclin-1

Abstract：Objective: To observe the effect of high glucose on the expression of autophagy in myotubes, and to investigate the expression of autophagy in myotubes cultured with high glucose for different time periods. Methods: Murine C2C12 myoblasts were induced into myotubes using differentiation medium, and myotubes were cultured with normal (5.5 mmol/L) and high dose (17 mmol/L or 30 mmol/L) of glucose, and cells were collected at 6, 12, 24, 36, 48, 60, and 72 h to detect autophagy-related proteins and mRNA expression. Additionally, murine myoblasts were transfected with tandem fluorescently labeled LC3 (mRFP-GFP-LC3) lentiviral vector. The red and green fluorescent spots in the cells were observed by laser confocal microscopy to monitor autophagic flux. Results: The autophagic flux in myotubes exposed to high glucose was divided into two phases. When myotubes incubated for 24 h with high glucose, the protein expression of LC3 and Beclin-1 was lower than that of the normal group (P<0.05); When C2C12 myotubes were cultured with 30 mmol/L of glucose for 48, 60 and 72 h, respectively, the levels of LC3 and Beclin-1 mRNA were significantly increased (P<0.05). In addition, localization and quantitative analysis of GFP and RFP fluorescent puncta in myotubes also demonstrated a significant increase in the synthesis of autophagosomes and autolysosomes after 48, 60 and 72 h of 30 mmol/L glucose culture, respectively. Conclusion: High glucose plays an important effect on autophagy in myotubes. Long-term hyperglycemia may eventually induce excessive autophagy.

Key words： autophagy C2C12 cells diabetes LC3 Beclin-1

收稿日期: 2018-12-20

基金资助:国家自然科学基金资助项目（81671403）；浙江省自然科学基金资助项目（LY15H270017）。

通讯作者: 苏震，主任医师，教授，Email：cnsuzhen@wmu.edu.cn。

作者简介: 罗胜男（1992-），女，浙江杭州人，硕士生。

链接本文:

https://xb.wmu.edu.cn/CN/ 或 https://xb.wmu.edu.cn/CN/Y2019/V49/I5/313

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