茵陈水提物对多药耐药蛋白3基因突变致新生儿肠外营养相关性胆汁淤积的保护作用  

Protective effect of herba artemisiae scopariae aqueous extract on neonatal parenteral nutrition-associated cholestasis induced by multidrug resistance protein 3 gene mutation

作　　者：杨秀芳[1,2] 宾淑铭 李丹 梁慧英[3] 陈康 郑铠军[5] 丁俊彩[1] 朱侨伟[1] 施尚文[1] 陈桂灵 Yang Xiufang;Bin Shuming;Li Dan;Liang Huiying;Chen Kang;Zheng Kaijun;Ding Juncai;Zhu Qiaowei;Shi Shangwen;Chen Guiling(Department of Neonatology,Zhongshan City People's Hospital,Zhongshan 528403,Guangdong,China;Guangdong Medical University,Zhanjiang 524023,Guangdong,China;Department of Traditional Chinese Medicine,Zhongshan City People's Hospital,Zhongshan 528403,Guangdong,China;Inspection Centre,Zhongshan City People's Hospital,Zhongshan 528403,Guangdong,China;Department of Pediatrics,Zhongshan City People's Hospital,Zhongshan 528403,Guangdong,China;Department of Pediatrics,the First Dongguan Hospital,Affiliated Guangdong Medical University,Dongguan 523710,Guangdong,China)

机构地区：[1]中山市人民医院新生儿科,广东中山528403 [2]广东医科大学,广东湛江524023 [3]中山市人民医院中医科,广东中山528403 [4]中山市人民医院检验中心,广东中山528403 [5]中山市人民医院儿科,广东中山528403 [6]广东医科大学附属东莞第一医院儿科,广东东莞523710

出　　处：《中国中西医结合急救杂志》2024年第3期308-314,共7页Chinese Journal of Integrated Traditional and Western Medicine in Intensive and Critical Care

基　　金：广东省中医药局项目(20202257)。

摘　　要：目的探讨茵陈水提物对多药耐药蛋白3(MDR3)基因突变导致的新生儿肠外营养相关性胆汁淤积(PNAC)的保护作用及可能机制。方法①将人原代培养肝细胞应用体外细胞培养、CRISPR/Cas9慢病毒感染、MDR3突变基因导入等技术处理后,比较1%脂肪乳诱导处理前(0)和处理后16、32、48 h不同时间点肝细胞上清液中肝胆生化指标〔丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、总胆红素(TBil)、直接胆红素(DBil)、间接胆红素(IBil)、总胆汁酸(TBA)〕的水平,确定构建MDR3基因突变PNAC肝细胞模型脂肪酸诱导所需的时间。②将人原代培养肝细胞株按随机数字表法分为空白对照组、MDR3基因野生型组、MDR3基因突变组、茵陈水提物干预组。空白对照组只用培养液处理,MDR3基因野生型组应用慢病毒感染融入野生型MDR3基因和培养液培养,MDR3基因突变组应用慢病毒感染慢病毒导入MDR3(c.485T>A、c.2793insA、c.1031G>A、c.3347G>A)突变基因和培养液培养,茵陈水提物干预组应用慢病毒感染导入MDR3突变基因和培养液培养的基础上,加入100 g/L茵陈水提物预处理,然后将4组肝细胞分别加入1%脂肪乳诱导处理,处理时间为构建MDR3基因突变PNAC肝细胞模型脂肪酸诱导所需时间。采用酶联免疫吸附试验(ELISA)测定4组肝细胞上清液中肝胆生化(ALT、AST、TBil、DBil、IBil、TBA)水平,采用实时荧光定量聚合酶链反应(RT-PCR)检测4组肝细胞编码MDR3、胆盐输出泵(BSEP)、多药耐药相关蛋白(MRP)2~4和肿瘤坏死因子-α(TNF-α)的三磷酸腺苷结合盒蛋白(ABCB4、ABCB11、ABCC2、ABCC3、ABCC4)和TNF基因mRNA的表达丰度。结果与空白对照组和MDR3基因野生型组比较,MDR3基因突变组在经1%脂肪乳诱导处理前和处理后16 h肝细胞上清液中肝胆生化指标(ALT、AST、TBil、DBil、IBil、TBA)水平比较差异无统计学意义,经1%脂肪乳诱导处理32 h和48 h MDR3基因突变组肝细胞上清�Objective To investigate the protective effect of herba artemisiae scopariae extract on multidrug resistance protein 3(MDR3)gene mutation-induced neonatal parenteral nutrition-associated cholestasis(PNAC)and its possible mechanism.Methods①Human primary hepatocytes were treated with cell culture in vitro,CRISPR/Cas9 lentivirus infection and MDR3 mutant gene lead-in.The levels of hepatic and biliary biochemical indexes[alanine transaminase(ALT),aspartate transaminase(AST),total bilirubin(TBil),direct bilirubin(DBil),indirect bilirubin(IBil),total bile acid(TBA)]in the supernatant of hepatocytes before and after 16,32,48 hours were compared to determine the time required for fatty acid induction of PNAC hepatocyte model with MDR3 gene mutation.②Human primary hepatocytes were divided into blank control group,MDR3 gene wild type group,MDR3 gene mutation group,and herba artemisiae scopariae extract intervention group according to random number table method.The blank control group was treated with culture medium only,the MDR3 gene wild type group was infected with lentivirus and mixed with wild type MDR3 gene and culture medium,the MDR3 gene mutation group was infected with lentivirus and cultured in culture medium with the mutant genes lead-in of LV-MDR3KI(c.485T>A,c.2793insA,c.1031G>A,c.3347G>A)mutation,while the MDR3 mutant gene was lead-in by lentivirus infection and cultured in culture medium,and then pretreated with 100 g/L herba artemisiae scopariae extract in the herba artemisiae scopariae extract intervention group,then the four groups of hepatocytes were induced with 1%fat emulsion,and the treatment time was the time needed to construct the PNAC hepatocytes model with MDR3 gene mutation.The levels of ALT,AST,TBil,DBil,IBil and TBA in the supernatant of hepatocytes were measured by enzyme-linked immunosorbent assay(ELISA).The mRNA expression abundance of adenosine triphosphate binding cassette proteins(ABCB4,ABCB11,ABCC2,ABCC3,ABCC4)encoding MDR3,bile salt export pump(BSEP),multidrug resistance associated p

关 键 词：茵陈水提物 多药耐药蛋白3基因 肠外营养相关性胆汁淤积 新生儿 

分 类 号：R722.1[医药卫生—儿科]