摘要
目的探讨多房棘球蚴囊液(Hydatid cyst fluid,HCF)对沉默DNA损伤诱导转录因子4(DNA damage-inducible transcript 4,DDIT4)后小鼠肝细胞生物学功能的影响。方法1.采用免疫蛋白印迹法(Western Blot)、实时荧光定量法(RT-qPCR)检测经HCF处理的小鼠肝细胞中DDIT4蛋白的表达量,以检测转染效率;2.选用慢病毒转染小鼠正常肝细胞DDIT4后做HCF处理,采用细胞计数试剂8法(CCK-8)检测各组小鼠肝细胞的细胞活性;3.用Western Blot法检测Beclin-1、Caspase-3、LC3等凋亡自噬相关蛋白水平;4.用透射电镜观察HCF对小鼠肝细胞超微结构的影响。结果1.经HCF作用后慢病毒转染小鼠肝细胞的DDIT4基因及蛋白表达水平检测结果显示,LV-Ddit4-RNAi 1、LV-Ddit4-RNAi 2、LV-Ddit4-RNAi 3组分别与LV-Ddit4-NC组比较,前三组DDIT4蛋白含量显著下降,LV-Ddit4-RNAi 3组尤为显著(P<0.01)。2.LV-Ddit4-RNAi 0.4、0.8 mg/mL组细胞OD值明显大于LV-Ddit4-NC 0.4、0.8 mg/mL组(P<0.01);与HCF共培养48 h后,沉默DDIT4组细胞形态接近正常AML-12细胞的形态,多呈椭圆形或者圆形,且细胞生长密度较LV-Ddit4-NC组明显为高。3.与LV-Ddit4-NC con组比较,LV-Ddit4-NC 0.4 mg/mL HCF组和LV-Ddit4-NC 0.8 mg/mL HCF组的LC3-II、Beclin-1、Caspase-3蛋白表达水平显著增高(P<0.01);分别与LV-Ddit4-NC 0.4、0.8 mg/mL HCF组比较,LV-Ddit4-RNAi 0.4、0.8 mg/mL HCF组的LC3-II、Beclin-1、Caspase-3蛋白表达水平显著降低,均具有统计学意义。4.两对照组细胞胞质未见明显损伤,线粒体基质较均匀,粗面内质网未见明显扩张,自噬水平低;LV-Ddit4-RNAi 0.4、0.8 mg/mL组细胞损伤较轻,未见粗面内质网扩张,胞质内存在较多脂滴,自噬数量增多;LV-Ddit4-NC 0.4、0.8 mg/mL组细胞损伤严重,粗面内质网严重扩张,胞质内存在大量脂滴,可见大量自噬结构。结论HCF对沉默DDIT4后小鼠肝细胞生物学功能的影响:1.AML-12细胞增殖;2.AML-12细胞凋亡、自噬数量减少。
Objective To investigate the effects of hydatid cyst fluid(HCF)on the biological function of mouse hepatocytes after silencing DNA damage-inducible transcript 4(DDIT 4).Methods 1.Western Blot and RT-qPCR were performed to detect the DDIT4 protein expression in mouse hepatocytes which were treated with HCF to detect the transfection efficiency.2.Normal mouse hepatocytes DDIT4 suitable for lentivirus transfection were selected for HCF treatment.Cell counting reagent 8(CCK-8)was used to detect the cell activity of mouse hepatocytes in each group.3.The levels of Beclin-1,Caspase-3,LC3 and other autophagy-related proteins were detected by Western Blot.4.The effects of HCF on the ultrastructure of mouse hepatocytes was observed by transmission electron microscopy.Results 1.The detection results of DDIT4 gene and protein expression level of lentivirus transfected mouse hepatocytes after HCF treatment showed that compared with LV-Ddit4-NC group,DDIT4 protein content of LVDdit4-RNAi 1,LV-Ddit4-RNAi 2,LV-Ddit4-RNAi 3 decreased significantly,especially in LV-Ddit4-RNAi 3 group(P<0.01).2.The cell OD value of LV-DDit4-Rnai 0.4 and 0.8 mg/mL groups was significantly higher than that of LV-Ddit4-NC 0.4 and 0.8 mg/ml groups(P<0.01).After co-culture with HCF for 48 hours,the morphology of cells in the silencing DDIT4 group was close to that of normal AML-12 cells,mostly oval or round.The cell growth density was significantly higher than that in the LV-Ddit4-NC group.3.Compared with LV-Ddit4-NC con group,LC3-II,Beclin-1 and Caspase-3 protein expression levels in LV-Ddit4-NC 0.4 mg/mL HCF group and LV-Ddit4-NC 0.8 mg/mL HCF group were significantly increased(P<0.01);Compared with LV-Ddit4-NC 0.4 and 0.8mg/mL HCF groups,LC3-II,Beclin-1 and Caspase-3 protein expression levels in LV-Ddit4-RNAi 0.4 and O.8 mg/mL HCF groups were significantly decreased with statistical significance.4.There was no obvious damage to the cytoplasm of the cells in the two control groups.The mitochondrial matrix was relatively uniform.The rough endoplasmic r
作者
尹凤娇
徐凯
陈佳昕
仁增卓嘎
乜茹
冶赓搏
庞明泉
樊海宁
曹得萍
王海久
王志鑫
Yin Fengjiao;Xu Kai;Chen Jiaxin;Renzeng zhuoga;Nie Ru;Ye Gengbo;Pang Mingquan;Fan Haining;Cao Deping;Wang Hajiu;Wang Zhixin(Department of Hepatopancreatobiliary Surgery,Qinghai University Affiliated Hospital,Xining 810001,China;Qinghai Provincial Key Laboratory of Hydatid Disease Research,Xining 810001,China;Department of Basic Medicine,School of Basic Medicine,Guilin Medical University,Guilin 541199,China)
出处
《中国高原医学与生物学杂志》
CAS
2023年第3期195-202,共8页
Journal of Chinese High Altitude Medicine & Biology
基金
青海省科技厅重点实验室项目(2020-ZJ-Y01)
中科院“西部之光”(青年学者)项目。
关键词
多房棘球蚴
囊液
沉默DNA损伤诱导转录因子4
细胞
凋亡
自噬
Echinococcus multilocularis
cyst fluid
silencing DNA damage-inducible transcription factor 4(DDIT 4)
cell
apoptosis
autophagy
