谷氨酸诱导大鼠胃Cajal间质细胞自噬模型的建立
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摘要
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(P<0.01)。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(P<0.01),其中以中浓度谷氨酸作用3 h的LC3-II/LC3-I比值最高(P<0.01)。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(P<0.01)。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。
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 at3h,6h and 24h were significantly increased as compared with control group and low concentration group (P < 0. 01),and that in medium concentration group at 3h 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 3h.
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 at3h,6h and 24h were significantly increased as compared with control group and low concentration group (P < 0. 01),and that in medium concentration group at 3h 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 3h.
引文
[1]陈健海,仲婕,王凡,等.胃肠道Cajal间质细胞起搏功能的研究进展[J].中国病理生理杂志,2017,33(1):184-188.
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[3]Zhang LM,Zeng LJ,Deng J,et al.Investigation of autophagy and differentiation of myenteric interstitial cells of Cajal in the pathogenesis of gastric motility disorders in rats with functional dyspepsia[J].Biotechnol Appl Biochem,2017 Dec 23.[Epub ahead of print]
[4]陈兆耀,陈茂刚,卢婷婷,等.谷氨酸致原代培养皮层神经元损伤中自噬的激活[J].中国病理生理杂志,2011,27(1):62-66.
[5]宁海恩,凌江红,梁纲,等.柴胡疏肝散对大鼠胃Cajal间质细胞活性及细胞内钙离子浓度的影响[J].中国中西医结合杂志,2016,36(9):1091-1096.
[6]张智,凌江红,宁海恩,等.柴胡疏肝散含药血清对大鼠胃Cajal间质细胞增殖及细胞周期的影响[J].世界华人消化杂志,2015,23(30):4792-4799.
[7]Oudenhove LV,Aziz Q.The role of psychosocial factors and psychiatric disorders in functional dyspepsia[J].Nat Rev Gastroenterol Hepatol,2013,10(3):158-167.
[8]孟晶,丁小燕,朱晓波,等.银杏内酯B对体外培养的视网膜神经细胞内钙离子浓度和线粒体功能的影响[J].中国病理生理杂志,2009,25(11):2192-2196.
[9]Hyer-Hansen M,Bastholm L,Szyniarowski P,et al.Control of macroautophagy by calcium,calmodulin-dependent kinase kinase-β,and Bcl-2[J].Mol Cell,2007,25(26):193-205.
[10]Sou Y,Waguri S,Iwata J,et al.The Atg8 conjugation system is indispensable for proper development of autophagic isolation membranes in mice[J].Mol Biol Cell,2008,19(11):4762-4775.
[11]Kimura S,Fujita N,Noda T,et al.Monitoring autophagy in mammalian cultured cells through the dynamics of LC3[J].Methods Enzymol,2009,452:1-12.
[12]徐树莹,乔录新,张玉林,等.自噬功能研究进展[J].临床荟萃,2012,27(13):1181-1184.
[2]曾丽君,凌江红,邓静,等.柴胡疏肝散对功能性消化不良大鼠胃窦肌间Cajal间质细胞自噬的影响[J].时珍国医国药,2017,28(5):1041-1044.
[3]Zhang LM,Zeng LJ,Deng J,et al.Investigation of autophagy and differentiation of myenteric interstitial cells of Cajal in the pathogenesis of gastric motility disorders in rats with functional dyspepsia[J].Biotechnol Appl Biochem,2017 Dec 23.[Epub ahead of print]
[4]陈兆耀,陈茂刚,卢婷婷,等.谷氨酸致原代培养皮层神经元损伤中自噬的激活[J].中国病理生理杂志,2011,27(1):62-66.
[5]宁海恩,凌江红,梁纲,等.柴胡疏肝散对大鼠胃Cajal间质细胞活性及细胞内钙离子浓度的影响[J].中国中西医结合杂志,2016,36(9):1091-1096.
[6]张智,凌江红,宁海恩,等.柴胡疏肝散含药血清对大鼠胃Cajal间质细胞增殖及细胞周期的影响[J].世界华人消化杂志,2015,23(30):4792-4799.
[7]Oudenhove LV,Aziz Q.The role of psychosocial factors and psychiatric disorders in functional dyspepsia[J].Nat Rev Gastroenterol Hepatol,2013,10(3):158-167.
[8]孟晶,丁小燕,朱晓波,等.银杏内酯B对体外培养的视网膜神经细胞内钙离子浓度和线粒体功能的影响[J].中国病理生理杂志,2009,25(11):2192-2196.
[9]Hyer-Hansen M,Bastholm L,Szyniarowski P,et al.Control of macroautophagy by calcium,calmodulin-dependent kinase kinase-β,and Bcl-2[J].Mol Cell,2007,25(26):193-205.
[10]Sou Y,Waguri S,Iwata J,et al.The Atg8 conjugation system is indispensable for proper development of autophagic isolation membranes in mice[J].Mol Biol Cell,2008,19(11):4762-4775.
[11]Kimura S,Fujita N,Noda T,et al.Monitoring autophagy in mammalian cultured cells through the dynamics of LC3[J].Methods Enzymol,2009,452:1-12.
[12]徐树莹,乔录新,张玉林,等.自噬功能研究进展[J].临床荟萃,2012,27(13):1181-1184.
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