摘要
目的从在体和离体两个层面评价右美托咪定对肝细胞肝癌肿瘤学行为的影响及核转录因子红系2相关因子2(Nrf2)在其中的作用。方法在体层面:将雄性C57BL/6J小鼠随机分为对照(Ctrl)组、肝细胞肝癌(HCC)组、HCC+右美托咪定(HCC+Dex)组。小鼠经N-亚硝基二乙胺(DEN)/四氯化碳(CCl 4)联合诱导形成HCC,随后2周每日腹腔注射右美托咪定10μg/kg,继续饲养小鼠1个月后,检测小鼠肝脏肿瘤的数量及最长径,利用Ki67免疫组化评估肝癌的增殖能力,通过免疫荧光检测肿瘤组织中Nrf2蛋白的表达水平。离体层面:在Hepa1-6细胞中,给予不同浓度的右美托咪定(0.1、1.0、5.0 nmol/L)孵育48 h,分别采用四甲基偶氮唑盐比色法(MTT法)、Transwell法检测细胞的增殖、侵袭和迁移能力,使用Western blot和免疫荧光检测肝癌细胞中Nrf2蛋白的表达水平。经si-RNA转染敲低Nrf2,继以1 nmol/L右美托咪定孵育48 h后,分别使用MTT、Transwell法检测细胞的增殖、侵袭和迁移能力。结果与HCC组比较,解剖学检查结果显示HCC+Dex组小鼠肝脏肿瘤数量增多、最长径增长(P<0.05);Ki67免疫组化结果显示,HCC+Dex组肝癌组织Ki67阳性细胞数增加(P<0.01);免疫荧光结果显示,HCC+Dex组Nrf2表达水平上调(P<0.01)。MTT结果显示1 nmol/L的右美托咪定增强了Hepa1-6细胞的细胞活力(P<0.05);Transwell法结果显示0.1、1和5 nmol/L的右美托咪定增强了Hepa1-6细胞的侵袭能力,0.1、1 nmol/L的右美托咪定增强了Hepa1-6细胞的迁移能力(P<0.05);Western blot和免疫荧光结果显示,使用1 nmol/L的右美托咪定处理后,细胞的Nrf2表达水平上调(P<0.01);使用si-RNA敲低细胞的Nrf2表达水平,再继以1 nmol/L的右美托咪定处理后,MTT、Transwell检测结果显示,Hepa1-6细胞的活力降低,侵袭和迁移的能力降低(P<0.01)。结论右美托咪定可能通过提高Nrf2的表达水平来促进肝癌的增殖、侵袭和迁移能力。
Objective To investigate the impact of dexmedetomidine on the oncological behavior of hepatocellular carcinoma and explore the role of NF-E2-related factor 2(Nrf2)at both in vitro and in vivo levels.Methods In vivo experiment,Male C57BL/6J mice were randomly divided into a control group(Ctrl group),a hepatocellular carcinoma group(HCC group),and a hepatocellular carcinoma+dexmedetomidine group(HCC+Dex group).Hepatocellular carcinoma was induced in mice by combining N-Nitrosodiethylamine(DEN)/carbon tetrachloride(CCl 4),followed by daily intraperitoneal injection of 10%dexmedetomidine for two weeks.After feeding the mice for one month,the mice were assessed for the quantity and size of liver tumors.The proliferation ability of liver cancer was evaluated using Ki67 immunohistochemistry.Additionally,the expression level of Nrf2 protein in tumor tissue was measured through immunofluorescence.In vitro experiment,Hepa1-6 cells were incubated with different concentrations of dexmedetomidine(0.1,1,5 nmol/L)for 48 hours to examine their effects.The proliferation,migration and invasion abilities of Hepa1-6 cells were evaluated using the MTT and Transwell methods.The expression level of Nrf2 protein in the Hepa1-6 cells was measured using Western blot and immunofluorescence.Additionally,the proliferation,migration and invasion abilities of cells were assessed after Nrf2 knockdown via si-RNA transfection,in combination with incubation with 1 nmol/L dexmedetomidine for 48 hours.Results Compared to the HCC group,the anatomical examination results revealed an increase in the number of liver tumors and the longest diameter in the HCC+Dex group(P<0.05).Ki67 immunohistochemistry results indicated the number of Ki67 positive cells in liver cancer tissue increased in the HCC+Dex group(P<0.01).The immunofluorescence assay demonstrated an upregulation of Nrf2 expression level in the HCC+Dex group(P<0.05).MTT results showed that 1 nmol/L of dexmedetomidine increased the cell viability of Hepa1-6 cells(P<0.05).Transwell results indicated that 0.1,1,and 5 nmol/L of dexmedetomidine enhanced the invasive ability of Hepa1-6 cells,while 0.1 and 1 nmol/L of dexmedetomidine enhanced the migration ability(P<0.05).Western blot and immunofluorescence results showed an upregulation of Nrf2 expression level in cells after treatment with 1 nmol/L dexmedetomidine(P<0.01).The Nrf2 expression level of cells was reduced using si-RNA,followed by treatment with 1 nmol/L dexmedetomidine.The results from MTT and Transwell assays revealed a decrease in the viability,invasion and migration ability of Hepa1-6 cells(P<0.01).Conclusion Dexmedetomidine may enhance the proliferation,invasion and migration capacity of hepatocellular carcinoma by upregulating the expression of Nrf2.
作者
吴瑞欣
周大臣
唐赛兰
黄春霞
Wu Ruixin;Zhou Dachen;Tang Sailan;Huang Chunxia(Dept of Anesthesiology and Perioperative Medicine,The Second Affiliated Hospital of Anhui Medical University,Hefei 230601;Key Laboratory of Anesthesiology and Perioperative Medicine,Hefei 230601;Dept of Hepatobiliary Surgery,The Second Affiliated Hospital of Anhui Medical University,Hefei 230601)
出处
《安徽医科大学学报》
CAS
北大核心
2024年第1期15-22,共8页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:81801050)
安徽医科大学基础与临床合作研究提升计划(编号:2019xkjT026)。
