新生期小鼠胰岛细胞自噬及其机制研究

Autophagy and its mechanism in pancreatic islet cells of neonatal mice

目的观察新生期小鼠胰岛细胞自噬的变化，分析白噬相关基因的表达差异并初步探讨相关调控机制。方法采用胶原酶消化法分离纯化第1、3和8周小鼠胰岛细胞，透射电镜观察白噬体显微结构。采用免疫荧光双染法检测胰岛中微管相关蛋白1轻链3（LC3）与胰岛素双阳性细胞比例的变化。采用实时定量PCR技术检测自噬相关基因（Atg）mRNA水平的表达。采用Western blotting法检测目的蛋白的表达。多组间数据比较采用单因素方差分析。结果（1）透射电镜结果显示，3周小鼠胰岛细胞出现内含胞浆或细胞器成分的具有双层膜结构的自噬体。免疫荧光结果显示，LC3在3周小鼠胰岛B细胞胞浆中着色较深，表达高于1周和8周（57．6±2．3比24．4±1．3、28．1±2．3，t=-21．44、-15．66，均P〈0．01）。（2）3周时，p53mRNA表达明显高于1周和8周，而哺乳动物雷帕霉素靶蛋白（mTOR）mRNA表达显著低于1周和8周（均P〈0．01）。新生期小鼠胰岛细胞Atg的表达呈动态变化。与1周和8周相比，3周时Atgl激酶同源蛋白（ULK—1）、Beclinl和Mapllc3b表达明显增高（P〈0．05）。与1周相比，3周时P13Kc3、Atg5、Mapllc3a和Atgl611表达显著增高（P〈0．05）；3周和8周时Atg4d的表达显著增高（P〈0．05）。Atg4c表达在3周时明显高于8周（P〈0．05）。（3）Western blotting显示，与1、8周相比，3周时p53、磷酸化腺苷酸活化蛋白激酶（pAMPK）、Beclinl和LC3-Ⅱ蛋白表达均显著增高（F=29．14～56．05，均P〈0．01），而mTOR蛋白表达明显低于1周和8周（0．61±0．06比1．00±0．02、0．89±0．09，F=29．41，P〈0．01）。结论p53／pAMPK／mTOR／Beclinl介导的自噬信号通路可能参与新生期小鼠胰岛细胞自噬的调控。

Objective To explore the changes and related mechanism of the autophagy of pancreatic islet cells during the neonatal period in mice. Methods Mice islets at 1-, 3-, 8-week after birth were isolated by collagenase. The morphological structure of autophagosome was observed by electron microscope. Double immunofluorescence staining was performed for insulin with microtubule-associ ated protein 1 light chain 3（LC3）. The expression of autophgy-related genes（Atg） were detected by real-time quantitative PCR. The expression of the targeted proteins was detected by Western blotting. One-way analysis of variance was used for data analysis. Results （1） The typical doubled-membrane autophagosome appeared at 3 w. Compared with 1 w and 8 w, the cytoplasmic fluorescence intensity of LC3 at 3 w was significantly increased （57.6±2.3 vs 24.4±1.3, 28.1±2.3, t=-21.44,-15.66, both P〈0.01）. （2） The mRNA level of p53 was higher at 3 w than that at 1 w and 8 w （P〈0.05）, while mammalian target of rapamycin （mTOR） was lower than that at 1 w and 8 w （P〈0.01）. The mRNA expression of the autophagy-related genes dynamically changed. Compared with 1 w and 8 w, the levels of ULK-1, Beclinl and Mapllc3b markedly increased at 3 w （P〈0.05）. Compared with 1 w, the levels of PI3Kc3, Atg5, Mapllc3a and Atgl611 increased at 3 w （P〈0.05）; the level of Atg4d was Significantly increased at 3 w and 8 w （P〈0.05）. At 3 w, the expression of Atg4c was higher than that at 8 w （P〈0.05）. （3） Western blotting showed that compared with 1 w and 8 w, the protein levels of p53, phosphorylated adenosine monophosphate-activated protein kinase（pAMPK）, Beelinl and LC3-II were markedly increased at 3 w （F=29.14-56.05, all P〈0.01）, while mTOR protein significantly decreased at 3 w （0.61 ±0.06 vs 1.00±0.02, 0.89±0.09, F=29.41, P〈0.01）. Conclusion The p53/pAMPK/mTOR/Beclinl mediated autophagy signaling pathway maybe involved in the regulation of autophagy in neonatal mice islet cells.