谷氨酸诱导大鼠结肠Cajal间质细胞构建自噬模型

目的 通过谷氨酸诱导离体大鼠结肠Cajal间质细胞(ICC)构建细胞自噬模型.方法 使用不同浓度的谷氨酸不同时间点作用于原代培养的大鼠结肠ICC,免疫荧光鉴定大鼠结肠ICC细胞;CCK-8法检测谷氨酸对ICCs活力的影响;Western印迹检测不同浓度谷氨酸在不同作用时点对大鼠结肠ICC自噬蛋白微管相关蛋白轻链(LC)...

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

Objective To establish an autophagy model of glutamic acid-induced rat colonic interstitial cells of Cajal in vitro.Methods The primary cultured rat colonic Cajal interstitial cells were treated with different concentrations of glutamate at different time points,the rat colonic ICC cells were identified by immunofluorescence,and the effect of glutamate on ICCs activity was detected by CCK-8 assay.Western blot was used to detect the effects of different concentrations of glutamate on the expression of autophagy protein microtubule associated protein light chain(LC)3 in rat colonic Cajal interstitial cells.The changes of intracellular autophagy and ultrastructure after glutamate intervention were observed by transmission electron microscope.Results Compared with the control group,glutamate acid(5 mmol/L)for 24 hours significantly decreased the activity of colonic ICCs in rats(P<0.01).Glutamate(5 mmol/L)for 6 h and 24 h significantly increased the ratio of autophagy protein LC3-Ⅱ/LC3-Ⅰ(P<0.05),the ratio of LC3-Ⅱ/LC3-Ⅰwas the highest when treated with 5 mmol/L glutamate for 24 hours(P<0.01).The number of autophagy vacuoles and autophagy bodies in interstitial cells of rat colonic Cajal treated with 5 mmol/L glutamate for 24 hours was significantly increased by transmission electron microscope.Conclusions The optimal concentration and time of glutamate inducing autophagy of rat colonic Cajal interstitial cells are 5 mmol/L and 24 h.