胶原凝胶复合骨髓间充质干细胞对小鼠急性肝衰竭的疗效与机制研究  

Effect and mechanism of collagen gel combined with bone marrow mesenchymal stem cells on acute liver failure in mice

作　　者：陈笑艳 王经琳 施晓雷[1] CHEN Xiao-yan;WANG Jing-lin;SHI Xiao-lei(Drum Tower Clinical Medical College,Nanjing Medical University,Nanjing 210008,China;Department of General Surgery,Southeast University Hospital,Nanjing 210005,China;Hepatobiliary Institute,Nanjing Drum Tower Hospital,Nanjing 210008,China)

机构地区：[1]南京医科大学鼓楼临床医学院,江苏南京210005 [2]东南大学医院普通外科,江苏南京210008 [3]南京鼓楼医院肝胆研究所,江苏南京210008

出　　处：《肝胆胰外科杂志》2019年第10期609-615,共7页Journal of Hepatopancreatobiliary Surgery

基　　金：国家自然科学基金面上项目（81872359;81670566）

摘　　要：目的探讨移植胶原凝胶复合骨髓间充质干细胞(bone marrow mesenchymal stem cells,MSCs)对小鼠急性肝功能衰竭(acute liver failure,ALF)的治疗效果与机制。方法 105只雄性C57BL/6 小鼠随机分为5组:对照组、ALF组、ALF+胶原凝胶组、MSCs组和胶原凝胶-MSCs组,每组21只。ALF组、ALF+胶原凝胶组、MSCs组和胶原凝胶-MSCs组采用腹腔注射D-氨基半乳糖苷(D-Gal)12 h后,分别于肝脏多点注射200 μL PBS、200 μL胶原凝胶、1×10^6 MSCs和1×10^6 胶原凝胶-MSCs。记录各组小鼠7 d存活率,全自动生化仪检测肝功能水平,HE染色检测肝脏组织病理学改变,免疫组化检测肝细胞凋亡和增殖情况,利用实时荧光定量PCR(RT-qPCR)检测肝脏炎症因子水平,Western blotting检测PI3K-AKT信号通路表达。结果扫描电镜示胶原凝胶与MSCs具有较好的相容性,动物活体成像检测显示胶原凝胶-MSCs移植的信号强于单独MSCs移植的ALF小鼠。D-Gal处理后,胶原凝胶-MSCs组和MSCs组的小鼠生存率明显提高(P<0.05),小鼠血清AST、ALT在胶原凝胶-MSCs组和MSCs组的小鼠明显优于ALF组和ALF+胶原凝胶组(P<0.05),且肝功能指标明显改善,胶原凝胶-MSCs组要更优于MSCs组(P<0.05)。HE染色提示胶原凝胶-MSCs组肝脏的病理改变(肝细胞坏死部位和范围)轻于MSCs组,Western blotting、Ki-67 和cleaved-Caspase3 免疫组化结果显示,胶原凝胶-MSCs组肝细胞增殖能力强于MSCs组,同时肝细胞凋亡程度要低于MSCs组,相应的胶原凝胶-MSCs组肝脏内IL-1β、IL-6、TNF-α、iNOS和CCL2 等炎性因子水平也低于MSCs组。且胶原凝胶-MSCs移植能明显激活肝脏内的PI3K-AKT通路。结论胶原凝胶复合MSCs移植能增强MSCs的肝脏定植能力,通过抑制细胞凋亡和促进细胞增殖减轻肝损伤,并调控AKT信号通路改善肝细胞功能,因此胶原凝胶复合MSCs移植优于单纯MSCs移植治疗ALF。Objective To investigate the therapeutic potential of collagen gel combined with bone marrow mesenchymal stem cells (MSCs) on acute liver failure (ALF). Methods 105 male C57BL/6 mice were divided in to five groups (21 mice in each group) randomly: control group, ALF group, ALF+collagen gel group, MSCs group and collagen gel-MSCs group. Except for the control group, mice in the other four groups revived D-galactosamine (D-Gal) by intraperitoneal injection. After 12 hours, 200 μL PBS, 200 μL collagen gel, 1×10^6 MSCs, and 1×10^6 collagen gel-MSCs were directly injected to left lobe of liver respectively. The 7-day survival rate of mice in each group was recorded. The level of liver function was detected by automatic biochemical instrument. The pathological changes of liver tissue were detected by HE staining. The apoptosis and proliferation of hepatocytes were detected by immunohistochemistry. The levels of liver inflammatory factors were detected by real-time fluorescence quantitative PCR (RT-qPCR). The expression of PI3K-AKT signal pathway was detected by Western blotting. Results Scanning electron microscope showed that collagen gel had good compatibility with MSCs, and in vivo imaging showed that the signal of collagen gel-MSCs transplantation was stronger than that of ALF mice transplanted with MSCs alone. After D-Gal treatment, the survival rate of mice in collagen gel-MSCs group and MSCs group was significantly improved (P<0.05). Serum AST and ALT of mice in collagen gel-MSCs group and MSCs group were superior to those in ALF group and ALF+collagen gel group (P< 0.05), and liver function indexes were significantly improved, especially in collagen gel-MSCs group, the liver function of which group was better than that in MSCs group (P<0.05). HE staining showed that the pathological changes of liver (the location and extent of hepatocyte necrosis) in collagen gel-MSCs group were smaller than those in MSCs group. Western bloting, Ki-67 and cleaved-Caspase3 immunohistochemical results showed that proliferatio

关 键 词：急性肝功能衰竭 胶原凝胶复合骨髓间充质干细胞 干细胞移植 小鼠 

分 类 号：R575.3[医药卫生—消化系统] Q813.1[医药卫生—内科学]