吉林大学学报(医学版)  2018, Vol. 44 Issue (02): 275-280

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李胜男, 张海英, 孙千惠, 李艳茹, 葛鹤, 杜瑜, 王琳
LI Shengnan, ZHANG Haiying, SUN Qianhui, LI Yanru, GE He, DU Yu, WANG Lin
水流动力学注射对小鼠肝损伤及肝脏的自然修复作用
Liver injury induced by hydrodynamic injection and natural repair of liver in mice
吉林大学学报(医学版), 2018, 44(02): 275-280
Journal of Jilin University (Medicine Edition), 2018, 44(02): 275-280
10.13481/j.1671-587x.20180213

文章历史

收稿日期: 2017-08-12
水流动力学注射对小鼠肝损伤及肝脏的自然修复作用
李胜男 , 张海英 , 孙千惠 , 李艳茹 , 葛鹤 , 杜瑜 , 王琳     
吉林大学基础医学院病理学系 病理生物学教育部重点实验室, 吉林 长春 130021
[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: 水流动力学注射    肝损伤    肝再生    细胞因子    生长因子    
Liver injury induced by hydrodynamic injection and natural repair of liver in mice
LI Shengnan, ZHANG Haiying, SUN Qianhui, LI Yanru, GE He, DU Yu, WANG Lin     
Department of Pathology, School of Basic Medical Sciences, Jilin University, Key Laboratory of Pathobiology, Ministry of Education, Changchun 130021, China
[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: hydrodynamic injection     liver injury     liver regeneration     cytokine     growth factor    

水流动力学注射(hydrodynamic injection)是将大体积(占小鼠体质量的8%~10%)质粒DNA溶液经小鼠尾静脉在5~8 s内快速注射入小鼠体内,进而获得转基因表达的方法,通常转基因在肝脏表达最多[1]。除DNA外,已成功应用此方法将RNA、蛋白甚至病毒等转移到动物体内,广泛应用于基因功能测试、基因治疗等研究以及病毒性肝炎模型的制备等[2-3]。由于静脉注射的大体积质粒DNA溶液迅速进入体循环,使心脏负荷过重,导致大量的液体淤积在肝脏,肝内压力升高,迫使肝窦内皮细胞窗增大,肝细胞膜出现暂时性小孔,质粒DNA进入肝细胞,进而表达目的基因[4]。此方法产生的压力将基因送入肝细胞的同时,也可能引起肝窦内皮细胞和肝细胞损伤。但是关于肝脏的形态学改变的研究[5]极少,其修复过程尚不清楚。本研究通过水流动力学注射法制备小鼠肝损伤模型,观察肝脏在损伤及修复过程中的形态学变化,检测与修复有关的因子,为更好地利用此方法进行科学研究提供依据,为进一步探讨水流动力学注射性肝损伤的修复机制奠定基础。

1 材料与方法 1.1 实验动物和肝损伤模型制备

31只雌性Balb/c小鼠,体质量为18~20 g,购自吉林大学实验动物中心,动物许可证号:SCXK(吉)-2003-0001。用26 G针头将小鼠体质量10%的生理盐水(1.8~2.0 mL)在3 s内经尾静注射入小鼠体内。按照注射后时间点将小鼠分为5组:30 min组、8 h组、1 d组、3 d组和7 d组,每组5只。以6只未注射小鼠为对照组,记为0 min组。

1.2 各组小鼠血清丙氨酸氨基转移酶(alanine transaminase,ALT)测定

注射生理盐水后,于不同时间点经内眦静脉取血,按照ALT试剂盒(上海科华-东菱诊断用品公司)说明检测血清ALT水平。

1.3 肝脏形态学观察

分别于注射后不同时间点处死小鼠并称体质量。取肝脏观察大体改变,制备石蜡切片,经苏木精-伊红(HE)染色,显微镜下观察病理学改变。为了确定肝脏病理学改变的区域,根据参考文献[5]将围绕门静脉周围的肝细胞区域定为门静脉区,即1区;围绕在中央静脉周围的肝细胞区域定为中央静脉区,即3区;介于二者之间的肝细胞区域为2区。每只小鼠分别选取10个有代表性的区域,统计每高倍视野下的肝细胞数及双核细胞数。

1.4 免疫组织化学染色

上述制备的石蜡切片,利用免疫组织化学染色(SP法)观察增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的表达情况。一抗为鼠抗人PCNA单克隆抗体(福州迈新,1:300),二抗为羊抗鼠/兔IgG(福州迈新),DAB溶液显色,苏木精复染。以PBS代替一抗作为阴性对照。PCNA阳性表现为细胞核呈棕黄色。每只小鼠肝组织切片内分别选取10个视野,计算肝细胞及胆管细胞PCNA阳性细胞百分数,将其作为PCNA指数。

1.5 Real-time PCR法检测基因表达水平

按照RNAiso Plus(日本TaKaRa公司)说明书提取肝组织总RNA;根据Reverse Transcriptase M-MLV(RNase H-)(日本TaKaRa公司)说明书进行逆转录反应(RT),按照FastStart Universal SYBR Green Master (ROX) (瑞士Roche公司)说明书进行Real-time PCR法检测,利用ABI 7300实时荧光定量PCR仪(美国ABI公司)检测各组小鼠肝组织中肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白细胞介素6(interleukin-6,IL-6)、表皮生长因子(epidermal growth factor,EGF)、肝细胞生长因子(hepatocyte growth factor,HGF)、转化生长因子α(transforming growth factor-α,TGF-α)、Cyclin D1、血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)、Bax和Bcl-2 mRNA表达水平,基因相对表达水平以2-ΔΔCt表示,其中ΔΔCt=(实验组Ct目的基因-实验组Ct内参基因) -(对照组Ct目的基因-对照组Ct内参基因)。以m-Arbp和β-actin为内参,引物设计见表 1

表 1 引物序列 Table 1 Primer sequences
Gene Primer sequence
TNF-α F:5′-GAAACACAAGATGCTGGGACAGT-3′
R:5′-CATTCGAGGCTCCAGTGAATTC-3′
IL-6 F:5′-CCGGAGAGGAGACTTCACAG-3′
R:5'′-TCCACGATTTCCCAGAGAAC-3′
HGF F:5′-GGCTGAAAAGATTGGATCAGG-3′
R:5′-CCAGGAACAATGACACCAAGA-3′
EGF F:5′-ACAAACCAGGCTGATGATGGT-3′
R:5′-TGCAGCTGTGCAGCTATCTT-3′
TGF-α F:5′-CAGGGAGCAACACAAATGGA-3′
R:5′-AGCCTCCAGCAGACCAGAAA-3′
Cyclin D1 F:5′-TCCAAAATGCCAGAGGCGGATG-3′
R:5′-TACCATGGAGGGTGGGTTGG-3′
Bax F:5′-CGAGCTGATCAGAACCATCA-3′
R:5'-GGTCCCGAAGTAGGAGAGGA-3′
Bcl-2 F:5′-AGTACCTGAACCGGCATCTG-3′
R:5′-GCTGAGCAGGGTCTTCAGAG-3′
m-Arbp F:5′-CACTGGTCTAGGACCCGAGAAG-3′
R:5′-GGTGCCTCTGGAGATTTTCG-3′
β-actin F:5′-ACTGCGCTTCTTGCCGC-3′
R:5′-CATGACGCCCTGGTGTC-3′
1.6 统计学分析

采用Graph Pad Instat 5.0统计软件进行统计学分析。各组小鼠血清ALT水平、肝质量/初始体质量比、肝组织内细胞数量、PCNA指数和细胞因子mRNA表达水平均以x±s表示,计量资料结果符合正态分布,ALT水平、肝质量/初始体质量比、PCNA指数、每高倍视野下肝组织内细胞数量(包括肝细胞、双核细胞)以及细胞因子mRNA水平在多组间的比较采用单因素方差分析。以P < 0.05为差异有统计学意义。

2 结果 2.1 各组小鼠肝脏大体改变和血清ALT水平

水流动力学注射后,小鼠抽搐、呼吸急促,数秒钟后恢复正常;体质量增加约10%,8 h后体质量恢复。与对照组(0 min组)比较,30 min组小鼠肝质量/初始体质量比无明显变化,8 h组明显下降(P < 0.05),1 d组和7 d组差异无统计学意义(P>0.05)。见表 2。对照组小鼠肝脏呈灰白色,表面无出血点;30 min组小鼠肝脏呈灰红色;8 h组小鼠肝脏表面出现黑红色片状出血区;1 d组小鼠肝脏表面仍可见片状出血区;3 d组小鼠出血区面积减小;7 d组小鼠肝脏呈灰白色,与对照组相似。见图 1(插页四)。30 min组和8 h组小鼠血清ALT水平明显低于对照组(P < 0.01),1 d组明显高于对照组(P < 0.01),7 d组差异无统计学意义(P>0.05)。

表 2 各组小鼠肝质量/初始体质量比值和血清ALT水平 Table 2 Ratios of liver weights/original body weights and levels of serum ALT of mice in various groups
(n=5, x ±s)
Group Ratio of liver weight/original
body weight
ALT
[λB/(U·L-1)]
Control
 (0 min)
0.064±0.010 27.00±2.96
30 min 0.065±0.006 3.40±1.82**
8 h 0.049±0.005* 3.00±1.58**
1 d 0.064±0.005 250.50±62.69**
3 d 0.063±0.003 47.80±7.39
7 d 0.077±0.005 29.20±5.26
  * P < 0.05,** P < 0.01 vs control group.
A: 0 min group; B: 30 min group; C: 8 h group; D: 1 d group; E: 3 d group; F: 7 d group. 图 1 各组小鼠肝脏大体改变 Figure 1 Macroscopic changes of liversof mice in various groups
2.2 肝组织形态学表现

对照组(0 min组)小鼠肝小叶结构完整,肝细胞以中央静脉为中心,沿肝细胞索呈放射状排列,肝窦清晰。30 min组小鼠出现大量肿胀的肝细胞,胞质淡染,成群分布,主要位于1区和2、3区之间;此外,肿胀区域内可见小面积出血,肝细胞索被破坏;每高倍视野下肝细胞数明显少于对照组(q=4.760,P < 0.05)。见图 2(插页五)和表 3。8 h组小鼠肿胀细胞数量较30 min组明显减少,而出血更加明显,肝细胞、肝窦内皮细胞被破坏,可见红细胞进入肿胀的肝细胞内,在出血坏死灶周围未受损区域内可见双核肝细胞及核分裂象(图 2,见插页五);与30 min组比较,肝细胞数明显增多(q=7.310,P < 0.01),双核细胞数也明显增多(q=7.200,P < 0.01)。1 d组出血坏死灶面积减小(图 2,见插页一),肝细胞数明显少于8 h组(q=4.966,P < 0.05),双核细胞数也明显少于8 h组(q=6.596,P < 0.01),但是二者与对照组比较差异无统计学意义(P>0.05)。见表 3。3 d组除靠近肝脏表面处有个别出血灶外,肝实质内出血坏死灶基本消失,肝细胞数及双核细胞数与对照组比较差异无统计学意义(P>0.05)。7 d组肝组织结构与对照组相似,肝细胞、双核细胞数量与对照组比较差异无统计学意义(P>0.05)。所有时间点均未见胆管上皮细胞的破坏。

A: 0 min group; B: 30 min group; C: 8 h group; D: 1 d group; E: 3 d group; F: 7 d group."→"indicated the binuclear hepatocytes; "▲"indicated the mitotic hepatocytes. 图 2 各组小鼠肝组织形态学特点(HE, ×400) Figure 2 Morphological features of liver tissue of mice in various groups (HE, ×400)
表 3 每高倍视野下各组小鼠肝组织内细胞数量 Table 3 Number of hepatocytes and binuclear hepatocytes in liver tissue of mice in various groups per high power field
(n=5, x ±s)
Group No.of hepatocytes No.of binuclear hepatocytes
Control(0 min) 78.5±7.6 9.4±3.2
30 min 65.4±10.0* 7.6±2.6
8 h 88.0±9.7 15.2±3.3
1 d 71.4±2.0# 7.8±0.9##
3 d 78.0±3.7 6.7±0.6
7 d 77.4±3.3 7.6±0.7
  *P < 0.05 vs control group; P < 0.01 vs 30 min group; #P < 0.05, ##P < 0.01 vs 8 h group.
2.3 各组小鼠肝组织PCNA表达

免疫组织化学染色结果显示:PCNA阳性肝细胞主要位于2、3区受损区域周围。8 h组PCNA指数较对照组升高(t=4.458, P < 0.01),1 d组最高,且明显高于对照组(t=15.557, P < 0.01),7 d组与对照组比较差异无统计学意义(P>0.05)。30 min组汇管区胆管细胞PCNA指数明显高于对照组(t=3.985, P < 0.01),随后降低,7 d组与对照组比较差异无统计学意义(P>0.05)。见图 3

图 3 各组小鼠肝组织PCNA指数 Figure 3 PCNA indexes in liver tissue of mice in various groups
2.4 各组小鼠肝组织中多种因子的表达

30 min组小鼠肝组织中TNF-α mRNA水平明显高于8 h组(q=4.952,P < 0.05);8 h组仍高于7 d组(q=8.461,P < 0.01)。30 min组小鼠肝组织中IL-6 mRNA水平明显高于8 h组(q=14.750,P < 0.01),8 h组明显高于7 d组(q=6.923,P < 0.01)。30 min组各组小鼠肝组织中EGF mRNA水平明显高于8 h组(q=14.750,P < 0.01)。3 d组各组小鼠肝组织中HGF mRNA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TGF-α mRNA水平均明显高于30 min组(q=4.592,P < 0.05;q=8.137,P < 0.01)。8 h和1 d组Cyclin D1 mRNA水平均明显高于7 d组(q=4.736,P < 0.05;q=5.213,P < 0.05)。30 min组各组小鼠肝组织中VEGF mRNA水平明显高于8 h组(q=13.551,P < 0.01)。1 d组Bax/Bcl-2 mRNA比值明显高于30 min组(q=5.731,P < 0.01)。见表 4

表 4 各组小鼠肝组织中肝再生相关因子的mRNA相对表达水平 Table 4 Relative expression levels of liver regeneration-related factors in liver tissue of mice in various groups
(n=5, x ±s)
Group TNF-α IL-6 EGF HGF TGF-α Cyclin D1 VEGF Bax/Bcl-2
30 min 3.43±1.18 13.45±3.09 1.81±0.47 0.39±0.08 0.42±0.12 1.28±0.25 2.05±0.11 0.50±0.17
8 h 1.48±0.40* 1.36±0.32** 0.93±0.28** 0.42±0.24 0.88±0.27* 1.82±0.65 0.69±0.28** 0.94±0.42
1 d 0.68±0.14 0.95±0.41 1.00±0.08 0.48±0.18 0.82±0.28 2.02±0.85 1.00±0.23 1.36±0.38**
3 d 0.60±0.17 0.57±0.23 0.97±0.26 0.84±0.31*△ 1.52±0.75** 1.29±0.72# 1.37±0.19 0.80±0.28
7 d 0.40±0.15△△ 0.46±0.07△△ 0.88±0.44 0.74±0.10 0.79±0.17 0.77±0.37△# 1.17±0.14 0.68±0.32
  *P < 0.05, * * P < 0.01 vs 30 min group; P < 0.05, △△P < 0.01 vs 8 h group; #P < 0.01 vs 1 d group.
3 讨论

肝再生主要是由残存的健康成熟肝细胞通过增殖完成[6],除肝细胞外,胆管细胞能够通过TNF-α、IL-6和VEGF等因子的释放对组织损伤做出反应,并在上述因子的调节下,进行与胆管修复有关的增殖、凋亡以及血管生成[7]。另外,Kupffer细胞、肝星形细胞和肝窦内皮细胞可通过分泌多种细胞因子而参与肝再生,如TNF-α、IL-6以及VEGF等[8]。本实验中,注射后30 min,肝细胞数明显少于对照组,这是由于此时肝细胞肿胀、体积增大,导致单位面积内细胞数减少所致[5];注射后8 h肝细胞数以及双核细胞数明显多于30 min,肝细胞PCNA指数也明显高于30 min,这些增多的肝细胞可能是为了维持肝脏功能而产生。注射后8 h,出现弥漫的片状出血坏死灶,说明此时肝窦内皮细胞受到破坏,但是在注射后7 d,出血坏死消失,肝组织结构恢复正常。胆管细胞PCNA指数在注射后30 min高于其他时间点。以上结果说明:在水流动力学注射引起的肝损伤中,肝细胞、胆管细胞和肝窦内皮细胞均参与了肝组织修复。

本实验中,小鼠在水流动力学注射后30 min及8 h肝组织出现肝细胞肿胀、肝细胞坏死及出血,因肝细胞坏死导致8 h时肝质量/初始体质量比下降,ALT释放入血,但是因为体内注射了大量的液体,导致ALT浓度被稀释,使测得的30 min及8 h组血清ALT值明显低于对照组。注射后8 h,肝组织坏死的同时,肝细胞开始大量增殖,表现为肝细胞(含双核肝细胞)数量增加。1 d时,注射的液体已经被机体排出,1 d组小鼠血清ALT达到峰值,此时肝组织内出血、坏死灶明显减少,肝质量/初始体质量比也恢复到对照组水平,肝细胞数也与对照组相似。到注射后3 d,ALT水平、肝细胞数、PCNA指数均与对照组无差别,肝脏组织结构基本恢复正常。这与Budker等[5]的研究结果基本一致,即损伤快速出现、快速消退。

目前研究[9]认为:急性肝损伤时肝脏的再生是一个快速发生的过程,主要包括启动期、增殖期和终止期。在启动阶段,有多种细胞因子和生长因子参与调控,其中TNF-α和IL-6使肝细胞由G0期进入G1期。在四氯化碳(CCl4)诱导的肝损伤模型中,TNF-α和IL-6 mRNA表达水平在CCl4处理后1d最高;使用TNF-α抗体或者敲除TNF-α受体(TNFR1)导致TNF-α耗尽或缺失均会引起肝再生障碍,并且这与NF-κB和STAT3活化受阻从而不能诱导IL-6的表达有关[8]。肝切除后,TNF-α与非实质细胞,尤其是Kupffer细胞的受体结合,激活NF-κB信号通路产生IL-6,IL-6与肝细胞上的IL-6受体结合,激活肝细胞内STAT3以及ERK1/2信号通路,随后进行DNA复制,完成肝再生[10]。TNF-α和EGF联合可促进肝再生过程中的DNA复制。在增殖阶段,HGF、TGF-α和EGF等促有丝分裂原可通过Ras-MAPK和PI3K/AKT信号通路促进肝细胞DNA合成和细胞增殖[8]。EGF与EGFR结合后,通过促进Cyclin D1表达调节肝细胞增殖[11]。VEGF能够促进血管生成。在肝切除模型中,VEGF可以通过肝窦内皮细胞增殖,重建肝窦,促进肝细胞增殖。Bockhorn等[12]研究发现:肝切除后24 h,VEGF处理组肝细胞增殖极高,而VEGF抗体处理组肝细胞增殖几乎被完全抑制;VEGF通过与VEGFR结合刺激肝窦内皮细胞增殖,并释放IL-6和HGF,从而参与肝脏的修复。凋亡在肝脏的再生和终止阶段发挥作用[13-15]

本实验中,注射后30 min时TNF-α、IL-6、EGF和VEGF mRNA表达水平明显升高,而Bax/Bcl-2比值较低。8 h时TNF-α和IL-6仍很高,此时Cyclin D1也升高。1 d时Cyclin D1和Bax/Bcl-2比值较高,3 d时HGF和TGF-α水平较高。本文作者推测:在水流动力学注射造成肝损伤后,快速升高的TNF-α和IL-6启动了修复的过程,使肝细胞从G0期进入G1期;EGF通过Cyclin D1使肝细胞进入增殖阶段,VEGF促进肝窦内皮细胞增殖和肝窦的修复;而后期的修复停止,可能是由促凋亡的Bax基因以及HGF和TGF-α共同发挥作用完成的。

综上所述,水流动力学注射可引起急性肝损伤,主要表现为肝细胞肿胀并形成出血、坏死灶,肝损伤后可迅速启动修复过程,损伤可在1周左右通过细胞增殖自然修复;与肝再生有关的多种细胞因子和生长因子参与修复过程,但是具体机制仍有待于进一步研究。

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. DOI:10.1089/10430349950017734
[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. DOI:10.3389/fphar.2017.00591
[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. DOI:10.1002/hep.v66.1
[4] Zhang G, Gao X, Song YK, et al. Hydroporation as the mechanism of hydrodynamic delivery[J]. Gene Ther, 2004, 11(8): 675–682. DOI:10.1038/sj.gt.3302210
[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. DOI:10.1002/(ISSN)1521-2254
[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. DOI:10.1371/journal.pone.0030675
[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. DOI:10.1016/j.jhep.2012.10.011
[8] Mao SA, Glorioso JM, Nyberg SL. Liver regeneration[J]. Transl Res, 2014, 163(4): 352–362. DOI:10.1016/j.trsl.2014.01.005
[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. DOI:10.1007/s10620-017-4651-6
[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. DOI:10.1016/j.jss.2006.07.027
[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. DOI:10.1016/j.jss.2005.11.586
[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. DOI:10.11855/j.issn.0577-7402.2017.05.05