谷氨酸诱导大鼠胃Cajal间质细胞自噬模型的建立

目的:通过谷氨酸诱导离体大鼠胃Cajal间质细胞(ICCs)建立细胞自噬模型。方法:使用高、中和低浓度(分别为10 mmol/L、5 mmol/L和2.5 mmol/L)的谷氨酸作用于原代培养的ICCs不同时间(3h、6h和24h),然后通过CCK-8法检测不同浓度谷氨酸对ICCs活力的影响;Western blot检测不同浓度谷氨酸在不同作用时点对ICCs自噬蛋白微管相关蛋白1轻链3(LC3)表达水平的影响;透射电镜观察谷氨酸干预后细胞内自噬体和超微结构的变化;免疫荧光检测谷氨酸诱导后自噬蛋白LC3的荧光表达。结果:与空白对照组相比,谷氨酸显著降低了大鼠胃ICCs活力(P0.01)。中、高浓度谷氨酸作用不同时间均可显著提高自噬蛋白LC3-II/LC3-I的比值(P0.01),其中以中浓度谷氨酸作用3 h的LC3-II/LC3-I比值最高(P0.01)。透射电镜检测中浓度谷氨酸干预3 h发现,细胞内自噬体数量明显增加,线粒体和内质网数量及结构遭到破坏。免疫荧光结果提示,与空白对照组比较,谷氨酸中浓度组平均每个细胞内自噬蛋白LC3荧光表达显著增强(P0.01)。结论:应用谷氨酸可成功诱导大鼠胃ICCs自噬,谷氨酸的最佳诱导条件为5 mmol/L作用3h。

Establishment of autophagy model of rat gastric interstitial cells of Cajal induced by glutamic acid

AIM: To establish an autophagy model of glutamic acid-induced rat gastric interstitial cells of Cajal (ICCs) in vitro. METHODS: Glutamic acid was added to the medium of cultured ICCs in vitro at different concentrations (high, medium and low concentrations were 10 mmol/L, 5 mmol/L and 2.5 mmol/L, respectively) for different periods (3 h, 6 h and 24 h). The cell viability was measured by CCK-8 assay. The protein expression of microtubule-associated protein 1 light chain 3 (LC3) in the ICCs after cultured with glutamic acid at different concentrations for different periods was determined by Western blot. The ultrastructure and autophagic vacuoles of ICCs were observed under electron microscope, and immunofluorescence technique was used to measure the fluorescence intensity of autophagic protein LC3 after co-cultured with glutamic acid at medium concentration. RESULTS: Compared with control group, glutamic acid significantly inhibited the viability of ICCs (P<0.01). The ratios of LC3-Ⅱ/LC3-I in medium and high concentration groups at 3 h, 6 h and 24 h were significantly increased as compared with control group and low concentration group (P<0.01), and that in medium concentration group at 3 h was the best to increased the ratio of LC3-Ⅱ/LC3-I (P<0.01). Under electron microscope, the ICCs in glutamic acid group showed obvious autophagic vacuoles and cytoplasmic vacuoles. The fluorescence intensity of autophagic protein LC3 in the ICCs of glutamic acid group was significantly enhanced (P<0.01). CONCLUSION: Glutamic acid successfully induces autophagy in ICCs. The optimum condition of glutamic acid was 5 mmol/L for 3 h.