肝脏类器官构建及其对小鼠部分肝切除术后肝再生的作用  

Construction of liver organoid and its effect on liver regeneration after partial hepatectomy in mice

作　　者：薛瑞丰 汤宁 潘晨燕[3] 董春龙 蔡佳晖 袁献温 王经琳[1,2,3] 任昊桢 施晓雷[1,2,3] XUE Rui-feng;TANG Ning;PAN Chen-yan;DONG Chun-long;CAI Jia-hui;YUAN Xianwen;WANG Jing-lin;REN Hao-zhen;SHI Xiao-lei(Department of General Surgery,the Affiliated Drum Tower Clinical Hospital of Nanjing Medical University,Nanjing 210005,China;Department of General Surgery,the Affiliated Drum Tower Hospital of Nanjing University Medical College,Nanjing 210029,China;Department of General Surgery,the Affiliated Drum Tower Clinical Hospital of Nanjing University of Chinese Medicine,Nanjing 210008,China)

机构地区：[1]南京医科大学附属鼓楼临床医学院普外科,江苏南京210005 [2]南京大学医学院附属鼓楼医院普外科,江苏南京210008 [3]南京中医药大学中西医结合鼓楼临床医学院普外科,江苏南京210029

出　　处：《肝胆胰外科杂志》2020年第5期290-296,共7页Journal of Hepatopancreatobiliary Surgery

基　　金：国家自然科学基金面上项目(81872359,81670566);中央高校基本科研业务费专项资金项目(021414380449);中国博士后科学基金面上项目(2018M642222);江苏省医学重点人才项目(ZDRCA2016066);江苏省自然科学基金青年项目(BK201901114)。

摘　　要：目的观察肝脏类器官对小鼠部分肝切除术后肝再生的作用。方法体外构建肝脏类器官,通过形态学、PCR和免疫荧光对类器官进行初步鉴定。C57BL/6小鼠随机等分为对照组和治疗组,每组18只。对照组进行肝左叶、中叶切除术+肝包膜注射200μL PBS;治疗组进行肝左叶、中叶切除术+肝包膜移植200μL类器官悬液。建模成功后分别于术后第1、4、7天收集标本。通过血清生化检测、免疫组化和Western blotting评估肝脏类器官对小鼠肝部分切除术后肝再生的作用。结果类器官体外培养3 d,从直径20μm的囊性结构扩增到约125μm的细胞团(P<0.05),直径扩增近6倍。肝脏干细胞标志基因EPCAM、SOX9和CK19明显上调(P<0.05),传代前后基因保持稳定。免疫荧光显示CK8、Desmin、AFP和PCNA呈阳性。HE显示术后第4天,治疗组肝细胞形态大小基本恢复正常,形态较清晰,无炎症细胞浸润,无脂肪或气球样变。对照组小鼠肝细胞核仁染色加深,仍有炎性细胞浸润,部分区域存在肝细胞坏死区。免疫组化Ki67和Western blotting对增殖水平进行检测,结果显示治疗组增殖能力是对照组的3倍。肝功能检测发现治疗组在术后第4天ALT、AST、TBIL和DBIL明显下降,ALB合成增加(P<0.05)。结论具有肝脏干细胞属性和增殖能力的肝脏类器官,能通过保护肝细胞、改善部分肝切除术后小鼠肝功能,发挥促进肝再生作用。Objective To observe the effect of liver organoid on liver regeneration after partial hepatectomy in mice.Methods Liver organoid was constructed in vitro and preliminarily identified by morphology,PCR and immunofluorescence.C57BL/6 mice were randomly divided into control group and treatment group with 18 in each group.The partial hepatectomized models were generated by performing consecutive liver lobe resections(left lateral lobe and median lobe).In addition,the control group were injected 200μL PBS into liver capsule,and the treatment group were injected 200μL liver organoid suspension into liver capsule.The specimens were collected on the 1st,4th,and 7th day after successful modeling.The effect of liver organoid on liver regeneration after partial hepatectomy in mice was estimated by serum biochemical detection,immunohistochemistry and Western boltting.Results The organoid was cultured in vitro for 3 days,and expanded from cystic structure with a diameter of 20μm to a cell mass of about 125μm(P<0.05),with the diameter amplification nearly 6 times.Signature gene of liver stem cell EPCAM,SOX9 and CK19 were significantly up-regulated(P<0.05),and the genes remained stable before and after passage.Immunofluorescence showed positive for CK8,Desmin,AFP and PCNA.HE showed that on the 4th day after operation,the hepatocyte morphology in the treatment group basically returned to normal,and the morphology was clear,no inflammatory cell infiltration,no fat or balloon-like changes.The nucleolus staining of hepatocytes in the control group was deepened,inflammatory cell infiltration still existed,and some areas had hepatocyte necrosis.Immunohistochemistry Ki67 and Western blotting were used to detect the proliferation level,showing that the proliferation ability in the treatment group was 3 times higher than that in the control group.Liver function test found that ALT,AST,TBIL,and DBIL in the treatment group decreased significantly on the 4th day after operation,and ALB synthesis increased(P<0.05).Conclusion Liver organ

关 键 词：肝脏类器官 肝部分切除术 肝再生 小鼠 

分 类 号：R458[医药卫生—治疗学] R657.3[医药卫生—临床医学]