多房棘球蚴囊液对沉默DDIT4后小鼠肝细胞生物学功能的影响.pdf
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1、195Yin FengjeRu20232023年JOURNALOFCHINESEHIGHTITUDEMEDICINEANDBIOLOGYNo.Vol.44第3 期第44卷杂志国高原医生物多房棘球坳囊液对沉默DDIT4后小鼠肝细胞生物学功能的影响尹凤娇2,徐凯2,陈佳昕2,仁增卓嘎2,七茹-2冶廣搏12,庞明泉12,樊海宁12,曹得萍3,王海久1.2*,王志鑫12(1.青海大学附属医院肝胆胰外科,西宁8 10 0 0 1;2.青海省包虫病研究重点实验室,西宁8 10 0 0 1;3.桂林医学院基础医学院基础医学教研室,桂林54119 9)摘要目的探讨多房棘球坳囊液(Hydatid cyst fl
2、uid,HCF)对沉默DNA损伤诱导转录因子4(DNAdamage-inducible transcript4,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(WestermBlot)、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WesternBlot法检测Beclin-1、Ca s p a s e-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
3、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-RNAi1、LV-Ddit4-RNAi2、LV-D d i t 4-RNA i 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-RNAi3组尤为显著(P0.01)。2.LV-D d i t 4-RNA i 0.4、0.8 m g/m L组细胞OD值明显大于LV-Ddit4-NC0.4、0.8 m g/m L组(P0.01);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-Dd
4、it4-NC组明显为高。3.与LV-Ddit4-NC con组比较,LV-Ddit4-NC 0.4mg/mL HCF组和LV-Ddit4-NC 0.8mg/mL HCF组的LC3-II、Be c l i n-1、Ca s p a s e-3蛋白表达水平显著增高(P0.01);分别与LV-Ddit4-NC0.4、0.8 mg/mLHCF组比较,LV-Ddit4-RNAi0.4、0.8 m g/m LH CF组的LC3-II、Be c l i n-1、Ca s p a s e-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
5、水平低;LV-Ddit4-RNAi0.4、0.8 m g/m L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-NC0.4、0.8 m g/m L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。关键词多房棘球坳;囊液;沉默DNA损伤诱导转录因子4;细胞;凋亡;自噬中图分类号R383.3文献标志码AD0I10.13452/ki.jqmc.2023.03.007Effects of Echinococcus mu
6、ltilocularis cyst fluid on thebiological function of mouse hepatocytes after silencing DDIT4*iaol-,Xu Kail-2,Chen Jiaxin-2,Renzeng zhuoga2,Ni收稿日期:2 0 2 2-12-6:青海省科技厅重点实验室项目(2 0 2 0-ZJ-Y01):第二批青海省“高端创新人才千人计划(培养拔尖人才)项目;中科院“西部之光(青年学者)项目。*通信作者,主任医师,教授,硕士生导师,尹凤娇(19 9 6-),女,汉族,青海籍,在读硕士研究生196Ye Gengbo-2,Pa
7、ng Mingquan-2,Fan Haining*,Cao Deping,Wang Hajiu-2*,Wang Zhixin-(1.Department of Hepatopancreatobiliary Surgery,Qinghai University Affiliated Hospital,Xining 810001,China;2.Qinghai Provincial Key Laboratory of Hydatid Disease Research,Xining 810001,China;3.Department of Basic Medicine,School of Basi
8、c Medicine,Guilin Medical University,Guilin 541199,China)Abstract Objective To investigate the effects of hydatid cyst fluid(HCF)on the biological function of mousehepatocytes after silencing DNA damage-inducible transcript 4(DDIT 4).Methods 1.Western Blot and RT-qPCRwere performed to detect the DDI
9、T4 protein expression in mouse hepatocytes which were treated with HCF to detectthe transfection efficiency.2.Normal mouse hepatocytes DDIT4 suitable for lentivirus transfection were selected forHCF treatment.Cell counting reagent 8(CCK-8)was used to detect the cell activity of mouse hepatocytes in
10、eachgroup.3.The levels of Beclin-1,Caspase-3,LC3 and other autophagy-related proteins were detected by WesternBlot.4.The effects of HCF on the ultrastructure of mouse hepatocytes was observed by transmission electron micros-copy.Results 1.The detection results of DDIT4 gene and protein expression le
11、vel of lentivirus transfected mousehepatocytes after HCF treatment showed that compared with LV-Ddit4-NC group,DDIT4 protein content of LV-Ddit4-RNAi 1,LV-Ddit4-RNAi 2,LV-Ddit4-RNAi 3 decreased significantly,especially in LV-Ddit4-RNAi 3group(P0.01).2.The cell OD value of LV-DDit4-Rnai 0.4 and 0.8 m
12、g/mL groups was significantly higher thanthat of LV-Ddit4-NC 0.4 and 0.8 mg/ml groups(P0.01).After co-culture with HCF for 48 hours,the morphol-ogy of cells in the silencing DDIT4 group was close to that of normal AML-12 cells,mostly oval or round.The cellgrowth density was significantly higher than
13、 that in the LV-Ddit4-NC group.3.Compared with LV-Ddit4-NC congroup,LC3-II,Beclin-1 and Caspase-3 protein expression levels in LV-Ddit4-NC 0.4 mg/mL HCF group andLV-Ddit4-NC 0.8 mg/mL HCF group were significantly increased(P0.01);Compared with LV-Ddit4-NC 0.4and 0.8mg/mL HCF groups,LC3-II,Beclin-1 a
14、nd Caspase-3 protein expression levels in LV-Ddit4-RNAi 0.4and O.8 mg/mL HCF groups were significantly decreased with statistical significance.4.There was no obvious dam-age to the cytoplasm of the cells in the two control groups.The mitochondrial matrix was relatively uniform.Therough endoplasmic r
15、eticulum was not significantly expanded and the autophagy level was low.In the LV-Ddit4-RNAi 0.4 and 0.8 mg/mL groups,the cell damage was light.No rough endoplasmic reticulum expansion was ob-served.There were more lipid droplets in the cytoplasm,and the number of autophagy was increased.In LV-Ddit4
16、-NC 0.4 and 0.8 mg/mL groups,the cells were severely damaged and the rough endoplasmic reticulum was severelyexpanded.There were a large number of lipid droplets in the cytoplasm,and a large number of autophagy structurescould be seen.Conclusion The effects of HCF on the biological function of mouse
17、 hepatocytes after silencing DDIT4are as follows:1.The number of AML-12 cells increased;2.The number of apoptosis and autophagy of AML-12cells decreased.KeywordsEchinococcus multilocularis;cyst fluid;silencing DNA damage-inducible transcription factor 4(DDIT 4);cell;apoptosis;autophagyDNA损伤诱导转录因子4(D
18、NAdamage-induc-ibletranscript4,DDIT4)是一种与代谢、免疫等相关的蛋白,在损伤等应激条件下高表达。在前列腺癌、卵巢癌等众多恶性肿瘤的表达中有显著性差异,且在抗肿瘤治疗中起着“保护伞作用-3。肝多房棘球坳病病灶边界不清楚,囊液持续外渗,刺激周围肝细胞,导致正常肝细胞损伤。徐凯4 等研究发现,正常小鼠肝细胞经多房棘球坳囊液(HCF)干预后,细胞活性明显受到抑制,且DDIT4在调亡、自噬相关蛋白中高表达。本研究观测HCF对沉默DDIT4后小鼠肝细胞生物学功能的影响1材料与方法1.1材料1.1.1实验细胞系正常小鼠肝细胞株(AML-12)购自中国科学院上海生命科学研究
19、所,保存于青海大学附属医院中1970.4、0.8 m g/m L的HCF,分别与细胞培养48 h。10个/孔),2 4h后更换培养基并加人浓度为0.0、心实验室。1.1.2小鼠BALB/c小鼠购自南京市江宁区青龙山动物繁殖场,动物合格证号:No.201930977。1.1.3主要试剂和仪器RIPA裂解液、0.2 5%胰蛋白酶消化液(不含EDTA)、嘌呤霉素(puromycin)均购自北京索莱宝科技有限公司,DMEM/F12培养基购自武汉普诺赛生命科技有限公司,CCK-8试剂盒、BCA蛋白浓度测定试剂盒、BCA蛋白定量试剂盒购自美国Thermo公司,DDIT4一抗、Beclin-1一抗购自美国A
20、bcam公司,Caspase-3一抗、LC3A/B一抗购自美国Cell SignalingTechnology公司,-actin抗体、HRP山羊抗兔二抗购自武汉爱博泰克生物科技有限公司,RNASimple总RNA提取试剂盒、逆转录试剂盒购自天根生化科技有限公司,DDIT4引物、18S引物购自华大基因公司,LV-Ddit4-RNAi、阴性对照病毒购自吉凯基因公司。生物倒置显微镜系统购自日本OLYMPUS公司,荧光化学发光成像系统购自美国CE公司,酶标仪购自德国Tecan公司,电泳仪购自BIORAD公司,PCR基因扩增仪购自美国ABI公司,普通高速离心机购自Sigma公司,Cytation5细胞成
21、像微孔板检测系统购自美国BioTek公司,实时荧光PCR仪购自Roche公司,透射电子显微镜购自HITACHI公司。1.2方法1.2.1囊液收集及处理将造模小鼠采用颈椎脱白法处死,以无菌方式打开腹腔并小心分离多房棘球坳囊泡(用PBS反复多次清洗),并将囊泡放至2 0 mL无菌注射器内,以挤压方式分离囊液并离心(32 0 0 r/min,7 m i n),用滤器(0.2 2 m)过滤离心囊液后分装,并存于-8 0 冰箱备用。1.2.2慢病毒LV-Ddit4-RNAi转染小鼠肝细胞按规定密度(410 4个/mL)接种于六孔板中。2 4h后当细胞覆盖率为2 0%30%时进行慢病毒转染。根据慢病毒使用
22、操作手册要求,每组细胞分别加人LV-Ddit4-RNAi 1、LV-D d i t 4-RNA i2、LV-D d i t 4-RNA i 3和LV-Ddit4-NC病毒,以及相应的转染增强液置培养箱(37,5%CO,)培养。用嘌呤霉素筛选稳定细胞株,并进行RT-qPCR及WesternBlot鉴定,检测各组DDIT4基因mRNA和蛋白表达量,并确定合适的慢病毒进行转染1.2.3小鼠肝细胞DDIT4蛋白的表达量检测分别收集经HCF作用后的LV-Ddit4-RNAi1、LV-Ddit4-RNAi 2、LV-D d it4-RNA i 3和LV-Ddit4-NC细胞,细胞用含有PMSF蛋白酶抑制剂
23、的Ripa缓冲液做冰上裂解(30 min)、离心(4,12 0 0 0 r/min,10min)后取上清液后用5蛋白上样缓冲液稀释并水浴(9 5,10 min),通过BCA蛋白定量试剂盒测定蛋白样品浓度。取30 g总蛋白进行SDS-PACE电泳。结束后转移至0.2 mPVDF膜上,用5%脱脂奶粉封闭(室温,1h)。将膜与DDIT4(1:10 0 0)、-actin(1:1500)一抗一同孵育(4,过夜)。第二天对稀释(1:50 0 0)的用HRP标记的二抗进行孵育(室温,1h)。使用增强型ECL化学发光系统进行印记检测(以-actin为内参)。使用ImageJ软件对蛋白条带进行灰度分析。1.2
24、.4小鼠肝细胞DDIT4的表达量检测直接提取细胞总RNA,将适量的RNA逆转录成cDNA,采用RT-qPCR法检测4组DDIT4的mR-NA表达量,筛选慢病毒。将4个样品用DDIT4基因和内参基因引物扩增,见表1。每个反应重复3次。采用2 0 L体系建立扩增体系。采用两步法PCR反应程序进行实验。PCR结果分析由ABIQ5PCR仪完成。1.2.2慢病毒LV-Ddit4-RNAi转染表1DDIT4、18 S 引物序列Table1Primer sequences of DDIT4,18S基因Forward primerReverse primerDDIT4CAAGGCAAGAGCTGCCATAGC
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