仙连解毒方对结直肠癌湿热瘀毒证模型小鼠肠道细胞类型的影响  

Effect of Xianlian Jiedu Formula (仙连解毒方) on Intestinal Cell Types in Colorectal Cancer Model Mice with Damp-heat Stasis Toxin Pattern

作　　者：张钦畅 程海波[1,2] 余成涛 沈卫星 徐长亮[1,2] 谭佳妮 赖岳阳[1,2] 范旻旻 李柳 ZHANG Qinchang;CHENG Haibo;YU Chengtao;SHEN Weixing;XU Changliang;TAN Jiani;LAI Yueyang;FAN Minmin;LI Liu(First School of Clinical Medicine,Nanjing University of Chinese Medicine,Nanjing,210023;Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine in Prevention and Treatment of Tumor)

机构地区：[1]南京中医药大学第一临床医学院,江苏省210023 [2]江苏省中医药防治肿瘤协同创新中心

出　　处：《中医杂志》2022年第5期461-467,共7页Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金(81930117)。

摘　　要：目的采用单细胞转录组测序技术探讨仙连解毒方治疗结直肠癌湿热瘀毒证的作用机制。方法30只雄性C57BL/6N小鼠随机分为对照组、模型组、仙连解毒方组,每组10只。模型组和仙连解毒方组小鼠采用高脂饮食与氧化偶氮甲烷(AOM)/葡聚糖硫酸钠(DSS)诱导建立结直肠癌湿热瘀毒证模型,对照组给予正常饮食。3个循环的DSS饮水结束后,仙连解毒方组开始给予仙连解毒方12.9 g/kg灌胃,对照组及模型组给予等体积的生理盐水,每天1次,连续灌胃6天后停1天,连续给药16周。末次给药结束4 h后,取各组小鼠结直肠组织,HE染色观察小鼠结直肠的组织病理形态。取距肛门6 cm的远端结肠段2 cm,解离组织样本,分离单细胞悬液,提取RNA行qPCR扩增,进行单细胞转录组测序。采用CellRanger-v5.0.0进行数据质控,Seurat-v3.1.1软件包进行降维聚类分析、细胞类型鉴定,对差异基因行GO和KEGG富集分析。结果HE染色结果显示,空白组小鼠结肠腺体排列规则,细胞核位于基底部,呈扁平或立方状;模型组上皮细胞极性消失,呈巢状、筛孔状,中央见坏死,细胞核浆比增大,核染色深,核仁明显,核分裂现象显著;仙连解毒方组上述情况得到明显改善。单细胞转录组测序共捕获3组小鼠肠道细胞45480个,降维聚类将所有细胞归为20个细胞群,鉴定各细胞群标记基因,分析得出小鼠肠道细胞主要包括9种细胞类型:肠上皮细胞、B细胞、自然杀伤T细胞、天然淋巴细胞、干细胞、巨噬细胞、中性粒细胞、肥大细胞、成纤维细胞。仙连解毒方组较模型组小鼠肠道组织中肠上皮细胞占比上升,B细胞、自然杀伤T细胞、巨噬细胞、中性粒细胞占比下降。GO与KEGG富集分析显示,仙连解毒方对结直肠癌湿热瘀毒证的作用主要富集在免疫系统进程、抗原处理和呈递、适应性免疫反应、转运ATP合酶活性旋转机制等方面,信号通路主要涉及免疫�Objective To explore the mechanism of Xianlian Jiedu Formula(仙连解毒方,XJF)in treating colorectal cancer mice with damp-heat stasis toxin pattern by using single-cell transcriptome sequencing.Methods Thirty male C57 BL/6 N mice were randomly divided into control group,model group and XJF group,with 10 mice in each group.Mice in model group and XJF group were induced by high fat diet and azomethane oxide(AOM)/dextran sulfate sodium(DSS)to establish the model of colorectal cancer with damp-heat stasis toxin pattern,while those in the control group were given normal diet.After three cycles of DSS drinking water,XJF group was given 12.9 g/kg by gavage,while the control group and model group were given equal volume of normal saline,once daily for six consecutive days a week,with one day off,for consecutive 16 weeks.Four hours after the last administration,the colorectal tissues of mice in each group were collected,and the histopathological morphology of the mice was observed by hematoxylin-eosin staining(HE).A 2 cm-long distal colon segment located 6 cm away from the anus was taken,and tissue samples were dissociated;single-cell suspension was isolated,and RNA was extracted for qPCR amplification.Single-cell transcriptome sequencing was performed.CellRanger was used for data quality control.Seurat-v3.1.1 software package was used to perform dimensionality reduction cluster analysis,cell type identification,and gene ontology(GO)and kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis of differential genes.Results HE staining showed that the colon glands of the mice in the control group were arranged regularly,and the nuclei were located at the base,which were flat or cubic.Epithelial cells in the model group lost their polarity,and were nest-like and cribriform-like,with necrosis in the center,having increased nucleocytoplasmic ratio,deep nuclear staining,obvious nucleoli,and significant mitotic phenom;the above conditions were improved in the XJF group.Totally,45480 intestinal cells were captured in

关 键 词：结直肠癌 湿热瘀毒证 仙连解毒方 肠道细胞 单细胞转录组测序 

分 类 号：R285.5[医药卫生—中药学]