摘要
目的探讨乙醛脱氢酶2(ALDH2)基因多态性与对乙酰氨基酚(APAP)药物所致肝功能异常的相关性。方法采用CRISPR/Cas9基因编辑技术构建ALDH2基因敲除小鼠模型,将获得的杂合子型小鼠与异性杂合子交配,提取子代小鼠鼠尾基因组DNA,采用聚合酶链反应(PCR)方法进行ALDH2基因型鉴定;进一步采用APAP在野生型和ALDH2基因敲除小鼠中诱导急性药物性肝损伤模型,采集小鼠血液和肝脏组织行肝功能指标检测及苏木素-伊红染色、F4/80免疫组织化学检测等。组间均数比较采用单因素方差分析、LSD-t检验方法进行两两比较。结果ALDH2基因敲除小鼠成功繁殖,子代小鼠基因型分别为野生型(ALDH2^(+/+))、杂合突变型(ALDH2^(+/-))、纯合突变型(ALDH2^(-/-))。APAP造模后的生化和组织学实验结果表明:空白对照组的丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、碱性磷酸酶(ALP)、总胆红素(TBil)水平无明显升高;与空白对照组相比,APAP实验组的ALT、AST、ALP和TBil水平均显著升高(P值均<0.05),其中突变组的ALT(P=0.004)、AST(P=0.002)、TBil(P=0.012)水平显著高于野生型组,且这些指标在纯合突变型的表达水平也显著高于杂合突变型(P值分别为0.003、0、0.006)。另外,杂合突变组小鼠的ALP水平高于纯合突变组(P=0.085)和野生型组小鼠,但只与野生型小鼠的比较差异具有统计学意义(P=0.002)。HE染色结果显示APAP实验组小鼠出现肝细胞变性、坏死,炎性细胞浸润增多,以突变型小鼠最明显;同时,F4/80免疫组织化学染色结果显示APAP实验组小鼠肝组织内可见棕褐色颗粒,其表达水平较空白对照组明显增高。结论APAP所致肝功能异常与ALDH2基因多态性相关,ALDH2突变型小鼠在APAP造模后的肝损伤症状有所加重,ALDH2基因缺陷可能在一定程度上加重APAP诱导的肝生物化学指标异常。
Objective To explore the association between aldehyde dehydrogenase 2(ALDH2)gene polymorphisms and abnormal liver function-induced by acetaminophen(APAP)drugs.Methods An ALDH2 gene knockout mouse model was constructed using CRISPR/Cas9 gene editing technology.The obtained heterozygous mice were mated with opposite sex of heterozygotes.Genomic DNA was extracted from the tail of the offspring mouse.The polymerase chain reaction(PCR)method was used to determine the ALDH2 genotype.APAP was further used to induce acute drug-induced liver injury models in wild-type and ALDH2 knockout mice.Blood and liver tissues of mice were collected for liver function index,HE staining,F4/80 immunohistochemistry,and other detections.The intergroup mean was compared using a one-way ANOVA.The LSD-t test was used for pairwise comparison.Results ALDH2 knockout mice were bred successfully.The genotyping of the offspring was segregated into the wild-type(ALDH2^(+/+)),heterozygous mutant(ALDH2^(+/-)),and homozygous mutant(ALDH2^(-/-)),respectively.Biochemical and histological results after APAP modeling showed that the level of alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP),and total bilirubin(TBil)was not significantly increased in the blank control group(P<0.05),while the ALT,AST,ALP,and TBil were all elevated in the APAP experimental group.The levels of ALT(P=0.004),AST(P=0.002),and TBil(P=0.012)were significantly elevated among the mutant group compared to those in the wild-type group,and the expression levels of these indicators were also significantly elevated among the homozygous mutant group compared to those in the heterozygous mutant group(P=0.003,0 and 0.006).In addition,the ALP levels were higher in the heterozygous mutation group than those in the homozygous mutant group(P=0.085)and wild-type group mice,but the difference was only statistically significant compared to wild-type mice(P=0.002).HE staining results showed that mice in the APAP experimental group had hepatocyte degeneration,necrosis,and increased inflammatory cell infiltration,which was mostly evident in mutant mice.Simultaneously,the F4/80 immunohistochemical staining results showed that brown granules were visible in the liver tissue of APAP experimental group mice,and its expression levels were significantly enhanced compared to the blank control group.Conclusion APAP-induced liver function abnormalities were associated with the ALDH2 gene polymorphism.The liver injury symptoms were increased in ALDH2 mutant mice following APAP modeling,and the ALDH2 gene defect may alleviate,to some extent,APAP-induced liver function abnormalities.
作者
陈凤
黎倩卉
吴英杰
吕丽媛
徐晓敏
王菲
Chen Feng;Li Qianhui;Wu Yingjie;Lyu Liyuan;Xu Xiaomin;Wang Fei(Digestive Medicine Center,the Seventh Affiliated Hospital,Sun Yat-sen University,Shenzhen 518107,China)
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2024年第2期133-139,共7页
Chinese Journal of Hepatology
基金
国家自然科学基金项目(82170605)
深圳市科技创新委员会项目(JCYJ20210324123212033)。
关键词
肝损伤
基因多态性
乙醛脱氢酶2
对乙酰氨基酚
Liver injury
Gene polymorphism
Acetaldehyde dehydrogenase 2
Acetaminophen
