自噬对胃Cajal间质细胞钙离子浓度的影响

Effects of autophagy on the calcium concentration of gastric interstitial Cajal cells

目的研究自噬在大鼠胃Cajal间质细胞(ICCs)活性及钙离子稳态过程中的影响。方法选取SPF级健康大鼠30只,随机分为自噬诱导剂雷帕霉素组、自噬抑制剂羟氯喹及对照组,经灌胃构建不同自噬水平的动物模型,酶解法分离大鼠胃ICCs并进行c-kit特异性免疫荧光鉴定及逆转录聚合酶链式反应(RT-PCR)法检测LC3B表达水平,判断自噬模型是否成功构建。分离的ICCs体外培养并给予自噬诱导剂及抑制剂,用细胞计数法8(CCK8)法检测雷帕霉素组、羟氯喹组及对照组中ICCs的细胞活性,用Fluo-4/AM荧光染色分析雷帕霉素组、羟氯喹组及对照组中细胞内的钙离子浓度。结果成功分离并鉴定ICCs。雷帕霉素组、羟氯喹组ICCs的LC3B水平分别为4. 07±0. 45,0. 27±0. 05,与对照组(1. 00±0. 06)相比,差异有统计学意义(P <0. 05)。雷帕霉素组ICCs细胞活性和胞内钙离子浓度分别为(44. 32±5. 58)%,11. 68±1. 14,羟氯喹组分别为(154. 00±4. 86)%,0. 84±0. 02,与对照组的(100. 00±5. 73)%,2. 17±0. 11相比,差异均有统计学意义(均P <0. 05)。结论自噬诱导大鼠肠道内ICCs的细胞活性减少及功能异常,自噬激活的ICCs胞内游离钙离子升高,为靶向调控ICCs所致的胃肠动力障碍提供了新的靶点。

Objective To investigate the effects of autophagy on the activity and calcium homeostasis in rats’gastric cajal interstitial cells(ICCs).Methods Thirty healthy rats of SPF-grade were randomly divided into control group,autophagy inducer group(rapamycin)and autophagy inhibitor group(hydroxychloroquine).The animal experimental models were established by oral gavage.Rat ICCs was isolated by enzymolysis and identified by c-kit specific immunofluorescence staining.Reserve transcription polymerase chain reaction(RT-PCR)assay was used to detect the expression level of LC3B to determine whether the autophagy model was successfully constructed.For in vitro assays,isolated ICCs were treated with rapamycin or hydroxychloroquine and the cell counting kit-8(CCK8)was used to detect the cell viability of ICCs.Calcium concentration was analyzed by Fluo-4/AM staining.Results The ICCs were isolated and identified successfully.The LC3B expression level of ICCs in rapamycin and hydroxychloroquine group were 4.07±0.45,0.27±0.05,had significant difference with control group(1.00±0.06).The cell viability of ICCs and concentration of calciumions in rapamycin group were(44.32±5.58)%,11.68±1.14,which in hydroxychloroquine group were(154.00±4.86)%,0.84±0.02,all had significant difference with those in control group,which were(100.00±5.73)%,2.17±0.11(all P<0.05).Conclusion Activation of autophagy decreased cell viability and disordered the function of rat ICCs.The concentration of intracellular calcium of ICCs was increased by autophagy activator,which provided a new target for regulating gastrointestinal motility disorder caused by ICCs.