水流动力学注射对小鼠肝损伤及肝脏的自然修复作用

doi:10.13481/j.1671-587x.20180213

摘要/Abstract

摘要： 目的：观察水流动力学注射所致肝组织损伤的形态学特点，探讨肝脏修复的过程和机制。方法：25只雌性Balb/c小鼠在3 s内经尾静脉注射大体积（1.8~2.0 mL）生理盐水。根据注射后时间将小鼠分为30 min组、8 h组、1 d组、3 d组和7 d组，每组5只鼠。6只未注射雌性Balb/c小鼠作为对照组。小鼠内眦静脉取血，检测血清中丙氨酸氨基转移酶（ALT）水平。利用苏木精-伊红（HE）染色观察各组小鼠肝脏形态学，免疫组织化学染色检测各组小鼠肝细胞中增殖细胞核抗原（PCNA）蛋白表达情况。利用Real-time PCR法检测各组小鼠肝组织中肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）、表皮生长因子（EGF）、肝细胞生长因子（HGF）、转化生长因子α（TGF-α）、Cyclin D1、血管内皮细胞生长因子（VEGF）、Bax和Bcl-2的mRNA表达水平。结果：与对照组比较，8 h组小鼠肝质量/初始体质量比值明显下降（P<0.05），7 d组差异无统计学意义（P>0.05）。与对照组比较，1 d组小鼠血清中ALT水平明显升高（P<0.01），7 d组差异无统计学意义（P>0.05）。显微镜下，与对照组比较，30 min组小鼠出现肝细胞肿胀及少量出血，每高倍视野下肝细胞数量明显减少（q=4.760，P<0.05）。与30 min组比较，8 h组小鼠肿胀的肝细胞数量减少，出血坏死灶扩大，每高倍视野下的肝细胞数和双核细胞数均明显增多（q=7.310，P<0.01；q=7.200，P<0.01）。与8 h组比较，1 d组小鼠出血坏死灶减少，肝细胞数和双核细胞数均减少（q=4.966，P<0.05；q=6.596，P<0.01），但与对照组比较差异无统计学意义（P>0.05）。3 d组小鼠坏死灶基本消失，7 d组小鼠肝脏结构与对照组相似。与对照组比较，8 h组和1 d组小鼠肝细胞PCNA指数均明显升高（t=4.458，P<0.01；t=15.557，P<0.01），7 d组差异无统计学意义（P>0.05）。与对照组比较，30 min组小鼠胆管细胞PCNA指数明显升高（t=3.985，P<0.01），7 d组差异无统计学意义（P>0.05）。30 min组TNF-α、IL-6、EGF和VEGF mRNA表达水平明显高于8 h组（q=4.952，P<0.05；q=14.750，P<0.01；q=14.750，P<0.01；q=13.551，P<0.01）。3 d组小鼠肝组织中HGF mRNA表达水平明显高于30 min组和8 h组（q=5.031，P<0.05；q=4.631，P<0.05），8 h组和3 d组小鼠肝组织中TGF-α mRNA表达水平均明显高于30 min组（q=4.592，P<0.05；q=8.137，P<0.01）。8 h组和1 d组小鼠肝组织中Cyclin D1 mRNA表达水平均明显高于7 d组（q=4.736，P<0.05；q=5.213，P<0.05）。1 d组小鼠肝组织中Bax和Bcl-2 mRNA表达水平比值明显高于30 min组（q=5.731，P<0.01）。结论：水流动力学注射可引起急性肝损伤，表现为肝细胞肿胀并形成出血坏死灶。肝损伤在1周左右可自然修复，与肝再生有关的多种细胞因子和生长因子参与修复过程。

关键词: 肝损伤, 水流动力学注射, 肝再生, 细胞因子, 生长因子

Abstract: Objective:To observe the morphological features of liver injury induced by hydrodynamic injection,and to explore the procedure and mechanism of liver repair. Methods: Twenty-five female Balb/c mice were intravenously injected with large volume of saline solution (1.8-2.0 mL) in 3 s. The mice were divided into 30 min,8 h,1 d,3 d,and 7 d groups according to the post-injection time. Each group contained 5 mice. Six non-injected mice were used as control group. The blood samples from angular vein of the mice were collected to detect the levels of alanine transaminase (ALT).The morphological features of liver tissue of the mice in various groups were observed with HE staining.The proliferating cell nuclear antigen (PCNA) protein expressions in liver tissue of the mice in various groups were detected by immunohistochemical saining.The expression levels of tumor necrosis factor-α (TNF-α),interleukin-6 (IL-6),epidermal growth factor (EGF),hepatocyte growth factor (HGF),transforming growth factor-α (TGF-α),Cyclin D1,vascular endothelial growth factor (VEGF),Bax and Bcl-2 mRNA were determined by Real-time PCR. Results: Compared with control group,the ratio of liver weight/original body weight in 8 h group was significantly decreased (P<0.05),but there was no significant difference in 7 d group (P>0.05).Compared with control group,the serum ALT level in 1 d group was significantly elevated (P<0.01),but there was no significant difference in 7 d group (P>0.05).Compared with control group,the liver tissue of the mice in 30 min group showed hepatocyte swollen and small hemorrhagic area,and the number of hepatocytes per high power field was significantly declined (q=4.760,P<0.05) under microscope. Compared with 30 min group,the number of swollen cells was decreased in 8 h group,the hemorrhagic and necrotic area enlarged,both of the hepatocyte number and binuclear hepatocyte number per high power field were significantly increased (q=7.310,P<0.01;q=7.200,P<0.01).Compared with 8 h group,the hemorrhagic and necrotic areas in 1 d group were reduced,both of the hepatocyte number and binuclear hepatocyte number per high power field were significantly decreased (q=4.966,P<0.05;q=6.596,P<0.01); there were no significant differences compared with control group (P>0.05).The hemorrhagic and necrotic areas almost disappeared in 3 d group.The histological appearance of liver tissue of the mice in 7 d group was same as control group.Compared with control group,the PCNA indexes in hepatocytes in 8 h and 1 d groups were significantly increased (t=4.458,P<0.01; t=15.557,P<0.01); there was no significant difference in 7 d group (P>0.05).Compared with control group,the PCNA index in cholangiocytes in 30 min group was increased significantly (t=3.985,P<0.01); there was no significant difference in 7 d group (P>0.05).The mRNA expression levels of TNF-α,IL-6,EGF,and VEGF in liver tissue of the mice in 30 min group were significantly higher than those in 8 h group (q=4.952,P<0.05; q=14.750,P<0.01; q=14.750,P<0.01; q=13.551,P<0.01).The HGF mRNA expression level in liver tissue of the mice in 3 d group was significantly higher than those in 30 min group and 8 h group (q=5.031,P<0.05;q=4.631,P<0.05). The TGF-α mRNA expression levels in liver tissue of the mice in 8 h group and 1 d group were higher than that in 30 min group (q=4.592,P<0.05; q=8.137,P<0.01).The Cyclin D1 mRNA expression levels in liver tissue of the mice in 8 h group and 1 d group were higher than that in 7 d group (q=4.736,q=5.213,P<0.05). The Bax/Bcl-2 ratio of the mice in 1 d group was higher than that in 30 min group (q=5.731,P<0.01). Conclusion: Acute liver injury induced by hydrodynamic injection mainly shows the hepatocyte swelling and hemorrhagic necrosis.The injury could be repaired naturally in a week.The various cytokines and growth factors related with liver regeneration are involved in the repair procedure.

Key words: growth factor, liver injury, liver regeneration, hydrodynamic injection, cytokine

中图分类号:

R361.1

李胜男, 张海英, 孙千惠, 李艳茹, 葛鹤, 杜瑜, 王琳. 水流动力学注射对小鼠肝损伤及肝脏的自然修复作用[J]. 吉林大学学报(医学版), 2018, 44(02): 275-280.

LI Shengnan, ZHANG Haiying, SUN Qianhui, LI Yanru, GE He, DU Yu, WANG Lin. Liver injury induced by hydrodynamic injection and natural repair of liver in mice[J]. Journal of Jilin University Medicine Edition, 2018, 44(02): 275-280.

参考文献

[1] Zhang GF,Budker V,Wolff JA.High levels of foreign fene expression in hepatocytes after tail vein injections of naked plasmid DNA[J].Hum Gene Ther,1999,10(10):1735-1737.
[2] Huang M,Sun R,Huang Q,et al.Technical improvement and application of hydrodynamic gene delivery in study of liver diseases[J].Front Pharmacol,2017,8:591.
[3] Kosinska AD,Pishraft-Sabet L,WuW,et al.Low hepatitis B virus-specific T-cell response in males correlates with high regulatory T-cell numbers in murine models[J].Hepatology,2017,66(1):69-83.
[4] Zhang G,Gao X,Song YK,et al.Hydroporation as the mechanism of hydrodynamic delivery[J].Gene Ther,2004,11(8):675-682.
[5] Budker VG,Subbotin VM,Budker T,et al.Mechanism of plasmid delivery by hydrodynamic tail vein injection.Ⅱ.Morphological studies[J].J Gene Med,2006,8(7):874-888.
[6] Zou Y,Bao Q,Kumar S,et al.Four waves of hepatocyte proliferation linked with three waves of hepatic fat accumulation during partial hepatectomy-induced liver regeneration[J].PLoS One,2012,7(2):e30675.
[7] O'Hara SP,Tabibian JH,Splinter PL,et al.The dynamic biliary epithelia:molecules,pathways,and disease[J].J Hepatol,2013,58(3):575-582.
[8] Mao SA,Glorioso JM,Nyberg SL.Liver regeneration[J].Transl Res,2014,163(4):352-362.
[9] Tao Y,Wang M,Chen E,et al.Liver regeneration:analysis of the main relevant signaling molecules[J].Mediat Inflamm,2017.doi:10.1155/2017/4256352.
[10] Ogiso H,Ito H,Kanbe A,et al.Liver regeneration is impaired in mice with acute exposure to a very low carbohydrate diet[J].Dig Dis Sci,2017,62:2386-2396.
[11] Collin de L' hortet A,Gilgenkrantz H,Guidotti JE.EGFR:A master piece in G1/S phase transition of liver regeneration[J].Int J Hepatol,2012,2012:476910.
[12] Bockhorn M,Goralski M,Prokofiev D,et al.VEGF is important for early liver regeneration after partial hepatectomy[J].J Surg Res,2007,138(2):291-299.
[13] Takushi Y,Shiraishi M,Nozato E,et al.Expression of anti-apoptotic protein,Bcl-2,in liver regeneration after a partial hepatectomy[J].J Surg Res,2006,134(1):93-101.
[14] 高斯,赵相轩,卢再鸣,等.P53调控细胞凋亡在肝细胞癌治疗中的作用[J].临床肝胆病杂志,2017,33(7):1373-1376.
[15] 周娟娟,何文华,甘达凯,等.熊果酸对TGF-β1诱导肝细胞凋亡的抑制作用及其机制[J].解放军医学杂志,2017,42(5):383-388.