脂肪干细胞调控线粒体膜通透性减轻大鼠肝再灌注损伤的作用机制研究  

Mechanism of adipose derived stem cells in regulating mitochondrial membrane permeability to alleviate hepatic reperfusion injury in rats

作　　者：张雅卿 徐海涛 丁宇 代静雅 袁红斌 傅海龙 Zhang Yaqing;Xu Haitao;Ding Yu;Dai Jingya;Yuan Hongbin;Fu Hailong(Department of Anesthesiology,the Second Affiliated Hospital of Naval Medical University,Shanghai 200003,China)

机构地区：[1]海军军医大学第二附属医院麻醉科,上海200003

出　　处：《国际麻醉学与复苏杂志》2025年第1期17-25,共9页International Journal of Anesthesiology and Resuscitation

基　　金：上海市自然科学基金(22ZR1477200)。

摘　　要：目的阐明脂肪干细胞(ADSC)在大鼠肝缺血再灌注(IR)损伤时对肝线粒体膜通透性转换孔(MPTP)的影响及线粒体保护作用机制。方法SD雌性大鼠2只,分离提取ADSC并培养和鉴定。采用随机数字表法将24只SD大鼠分为假手术组(Sham组)、IR组、IR+ADSC组和IR+ADSC+MPTP开放剂[羧基苍术苷(CATR)]组,每组6只。Sham组大鼠开腹不做缺血处理,IR组和IR+ADSC组分别于缺血前即刻经门静脉注入50μl磷酸盐缓冲液(PBS)或50μl(5×105个/μl)ADSC悬液,IR+ADSC+CATR组在IR+ADSC组基础上于缺血前30 min门静脉注射5 mg/kg CATR,构建大鼠70%肝脏热IR损伤模型,缺血时间60 min,于再灌注6 h获取血清及肝组织。检测4组大鼠血清天冬氨酸转氨酶(AST)、丙氨酸转氨酶(ALT)水平变化;苏木精⁃伊红(H⁃E)染色和脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)计算肝脏坏死面积和肝细胞凋亡百分比;免疫组化染色和免疫印迹法(Western blot)检测肝组织胞外信号调节激酶1/2(ERK1/2)、磷酸化ERK1/2(p⁃ERK1/2)、糖原合成酶激酶3β(GSK⁃3β)、磷酸化GSK⁃3β(p⁃GSK⁃3β)表达情况和蛋白水平,检测肝组织活性氧(ROS)、超氧化物歧化酶(SOD)和丙二醛(MDA)水平及环磷酸腺苷(cAMP)浓度;分离肝线粒体,检测肝线粒体钙容纳力(CRC)。结果三代ADSC表面特征标记物CD90、CD73、CD44阳性表达率为96.9%、90.6%和93.3%,CD45、CD34和CD11b表达率均低于1%。ADSC的成脂、成骨和成软骨分化实验结果显示,获取的大鼠脂肪干细胞具有多向分化潜能,可被成功诱导分化为脂肪细胞、成骨细胞和软骨细胞。H⁃E染色结果显示,IR组大鼠可见肝组织局灶性坏死,严重空泡变性及大量炎症细胞浸润,而IR+ADSC组大鼠肝细胞空泡变性和坏死均明显减少。TUNEL染色结果显示,IR组大鼠凋亡肝细胞明显增加,而IR+ADSC组大鼠凋亡肝细胞减少。与Sham组比较:IR组、IR+ADSC组和IR+ADSC+CATR组大鼠肝脏坏死面积�Objective To elucidate the effect of adipose derived stem cell(ADSC)on mitochondrial membrane permeability transition pore(MPTP)in rats with hepatic ischemia⁃reperfusion(IR)injury and the possible mitochondrial protective mechanisms.Methods Two female SD rats were used for isolation,culture,and identification of ADSC.According to the random number table method,24 SD rats were divided into four groups(n=6):a sham operation(Sham)group,an IR group,an IR+ADSC group,and an IR+ADSC+MPTP opener[carboxyatractyloside(CATR)]group.In the Sham group,rats underwent laparotomy without ischemia treatment.In the IR and IR+ADSC groups,50μl phosphate buffer saline(PBS)or 50μl(5×10⁵cells/μl)ADSC suspension was injected via the portal vein immediately before ischemia,respectively.In the IR+ADSC+CATR group,rats were injected with 5 mg/kg CATR via the por⁃tal vein 30 min before ischemia,based on the treatment in the IR+ADSC group.A rat model of 70%hepatic warm IR injury was estab⁃lished through ischemia over 60 min,followed by reperfusion for 6 h and then serum and liver tissue samples were collected.The chang⁃es in serum aspartate aminotransferase(AST)and alanine aminotransferase(ALT)levels were measured.Liver necrosis areas and the per⁃centage of hepatocyte apoptosis were evaluated by hematoxylin⁃eosin(H⁃E)staining and terminal deoxynucleotidyl transferase⁃mediated dUTP⁃biotin nick end labeling assay(TUNEL).The expression of extracellular signal⁃regulated kinase 1/2(ERK1/2),phosphorylated ERK1/2(p⁃ERK1/2),glycogen synthase kinase⁃3β(GSK⁃3β),and phosphorylated GSK⁃3β(p⁃GSK⁃3β)was detected by immunohisto⁃chemistry and Western blot.The levels of reactive oxygen species(ROS),superoxide dismutase(SOD),malondialdehyde(MDA),and cyclic adenosine monophosphate(cAMP)in liver tissue were measured.The hepatic mitochondria were isolated to detect mitochondrial calcium retention capacity(CRC).Results The positive expression rates of surface markers CD90,CD73,and CD44 on the third⁃generation ADSC were

关 键 词：脂肪干细胞 缺血再灌注损伤 肝脏 线粒体膜通透性转换孔 信号转导 

分 类 号：R364[医药卫生—病理学]