摘要
目的分析盲肠结扎穿孔术(CLP)诱导的脓毒症对肠上皮细胞增殖和分化的影响。方法①动物实验:将16只雄性C57BL/6小鼠按随机数字表法分为假手术组(Sham组)和CLP致脓毒症模型组(CLP组),每组8只。术后5 d取空肠组织,用聚合酶链反应(PCR)检测富亮氨酸重复序列G蛋白耦联受体5(LGR5)和肠型碱性磷酸酶(IAP)的转录表达水平;用蛋白质免疫印迹试验(Western blotting)检测LGR5的翻译水平;用免疫组化法分析增殖细胞核抗原(Ki67)的表达;用改良钙钴染色法和比色法检测组织IAP水平;免疫荧光法检测肠道潘氏细胞标记分子溶菌酶1(LYZ1)和杯状细胞标记分子黏蛋白2(MUC2)的表达。②细胞实验:取对数生长期的大鼠小肠隐窝细胞(IEC6细胞),并分为空白对照组和脂多糖(LPS)组(LPS 5μg/mL)。24 h后分别用PCR和Western blotting检测LGR5的转录和翻译水平;用5-乙炔基-2'-脱氧尿苷(EdU)染色检测IEC6细胞增殖情况;分别用PCR和比色法检测IAP的转录和翻译水平。结果①动物实验:免疫组化结果显示,CLP组小鼠空肠组织Ki67染色阳性率低于Sham组〔(41.7±2.5)%比(48.7±1.4)%,P=0.01〕。PCR和Western blotting结果显示,CLP组与Sham组小鼠空肠组织LGR5表达差异均无统计学意义(Lgr5 mRNA:0.7±0.1比1.0±0.2,P=0.11;LGR5/β-actin:0.83±0.17比0.68±0.19,P=0.24);CLP组小鼠空肠组织IAP在转录水平(0.4±0.1比1.0±0.1,P<0.01)和蛋白水平(U/g:47.3±6.0比73.1±15.3,P<0.01)均低于Sham组。免疫荧光显示,CLP组小鼠空肠组织LYZ1、MUC2的表达水平均低于Sham组。②细胞实验:PCR和Western blotting结果显示,LPS组与空白对照组IEC6细胞LGR5表达水平差异无统计学意义(Lgr5 mRNA:0.9±0.1比1.0±0.2,P=0.33;LGR5/β-actin:0.71±0.18比0.69±0.04,P=0.81)。LPS组IEC6细胞增殖率低于空白对照组,但差异无统计学意义〔EdU阳性率:(40.5±3.8)%比(46.5±3.6)%,P=0.11〕。LPS组IAP转录水平(0.5±0.1比1.0±0.2,P<0.01)和蛋白水平(U/g:15.0±4.0比41.2±
Objective To analyze the impact of cecal ligation and puncture(CLP)-induced sepsis on the proliferation and differentiation of intestinal epithelial cells.Methods①Animal experiment:sixteen male C57BL/6 mice were divided into sham operation group(Sham group)and CLP-induced sepsis model group(CLP group)by random number table method,with 8 mice in each group.After 5 days of operation,the jejunal tissues were taken for determination of leucine-rich-repeat-containing G-protein-coupled receptor 5(LGR5)and intestinal alkaline phosphatase(IAP)by polymerase chain reaction(PCR).The translation of LGR5 was detected by Western blotting.The expression of proliferating cell nuclear antigen(Ki67)was analyzed by immunohistochemistry.IAP level was detected by modified calcium cobalt staining and colorimetry.Immunofluorescence staining was used to detect the expression of Paneth cell marker molecule lysozyme 1(LYZ1)and goblet cell marker molecule mucin 2(MUC2).②Cell experiment:IEC6 cells in logarithmic growth stage were divided into blank control group and lipopolysaccharide(LPS)group(LPS 5μg/mL).Twenty-four hours after treatment,PCR and Western blotting were used to analyze the transcription and translation of LGR5.The proliferation of IEC6 cells were detected by 5-ethynyl-2'-deoxyuridine(EdU)staining.The transcription and translation of IAP were detected by PCR and colorimetric method respectively.Results①Animal experiment:the immunohistochemical results showed that the positive rate of Ki67 staining in the jejunal tissue of CLP group was lower than that of Sham group[(41.7±2.5)%vs.(48.7±1.4)%,P=0.01].PCR and Western blotting results showed that there were no statistical differences in the mRNA and protein expressions of LGR5 in the jejunal tissue between the CLP group and Sham group(Lgr5 mRNA:0.7±0.1 vs.1.0±0.2,P=0.11;LGR5/β-actin:0.83±0.17 vs.0.68±0.19,P=0.24).The mRNA(0.4±0.1 vs.1.0±0.1,P<0.01)and protein(U/g:47.3±6.0 vs.73.1±15.3,P<0.01)levels of IAP in the jejunal tissue were lower in CLP group.Immunofluor
作者
张学鹏
付建垒
刘茂霞
张赓
邱桐
周江元
张梓欣
龚雪
付沁怡
吉毅
陈思源
Zhang Xuepeng;Fu Jianlei;Liu Maoxia;Zhang Geng;Qiu Tong;Zhou Jiangyuan;Zhang Zixin;Gong Xue;Fu Qinyi;Ji Yi;Chen Siyuan(Department of Critical Care Medicine,West China Hospital,Sichuan University,Chengdu 610041,Sichuan,China;Department of Pediatric Surgery,West China Hospital,Sichuan University,Chengdu 610041,Sichuan,China)
出处
《中华危重病急救医学》
CAS
CSCD
北大核心
2024年第5期496-502,共7页
Chinese Critical Care Medicine
基金
四川省科技计划重点研发项目(2022YFS0225)。
关键词
脓毒症
盲肠结扎穿孔术
肠上皮
增殖
分化
Sepsis
Cecal ligation and puncture
Intestinal epithelium
Proliferation
Differentiation
