鼠暴发性肝衰竭中Fas表达与肝细胞凋亡的关系

摘要

目的: 研究Fas表达及肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)在暴发性肝衰竭(fulminant hepatic failure, FHF)中与肝细胞凋亡的关系.

方法: 采用脂多糖(LPS)和D-氨基半乳糖(D-GalN)联合用药制备FHF小鼠模型; 采用免疫组化方法检测肝组织Fas表达, 分别采用ELISA方法和RT-PCR法检测血清TNF-α水平及肝组织TNF-αmRNA表达; 采用肝组织DNA琼脂糖凝胶电泳和TUNEL法检测肝细胞凋亡; 在用药后2, 4, 8, 12 h的不同时期动态观察Fas表达、血清TNF-α水平及肝组织TNF-αmRNA表达及肝细胞凋亡的变化, 并对模型鼠给予TNF-αmAb, 动态观察上述指标的变化.

结果: 在FHF模型小鼠中, 用药后2 h开始Fas有少量表达, 至8 h和12 h表达均很多, 二者比较无显著差异, 与2 h组比较P<0.01,与4 h组比较P<0.05. 用药后2-4 h肝组织TNF-αmRNA表达显著增加(0.91±0.75, 正常值为0.32±0.10), 伴血清TNF-α水平升高(320±87 ng/L, 正常值为17±7 ng/L), 8 h可出现典型的肝细胞凋亡表现, 血清ALT和TBil水平显著增加(分别为9 352±1 000 nkat/L和163.7±34.5 mmoL/L, 正常值分别为393±134 nkat/L和14.9±4.8 mmoL/L), 12 h肝细胞坏死和凋亡同时存在, 血清ALT和TBil水平达最高峰(分别为11141±1312 nkat /L 和 203.2±19.9 mmoL/L). 给予TNF-αmAb后LPS/D-GalN介导的肝细胞凋亡和损伤被阻断, Fas表达亦被阻断.

结论: 在FHF中, TNF-α对肝细胞凋亡及肝损伤起重要的作用, 肝细胞凋亡的发生与Fas的表达增加有关.

关键词: N/A

Relationship between hepatocytic apoptosis and Fas expression in mouse fulminant hepatic failure

Yu-Mei Wang, Guo-He Feng, Fen Huang, Li-Lan Shi, Ying Li, Zhan-Ying Wang

Yu-Mei Wang, Guo-He Feng, Fen Huang, Li-Lan Shi, Ying Li, Zhan-Ying Wang, Department of Infectious Diseases, Second Affiliated Hospital, China Medical University, Shenyang 110004, Liaoning Province, China

Correspondence to: Yu-Mei Wang, Department of Infectious Diseases,Second Affiliated Hospital, China Medical University, Shenyang 110004, Liaoning Province, China. wangym7002@yahoo.com

Received: November 18, 2003
Revised: January 9, 2004
Accepted: February 1, 2004
Published online: May 15, 2004

Abstract

AIM: To study the relationship among tumor necrosis factor-a, Fas expression and hepatocyte apoptosis in an experimental model of fulminant hepatic failure (FHF).

METHODS: A mouse model of FHF was established by LPS and D-GalN. The expression of Fas in liver tissue was detected by immunohistochemical method. Serum TNF-α level and TNF-α mRNA expression in liver were analyzed by ELISA and RT-PCR method respectively. Hepatocytic apoptosis was examined by DNA agarose gel electrophoresis and TUNEL method. TNF-α, Fas and hepatocytic apoptosis were observed in the different stage after drug administr-ation. In addition, changes of the above items were observed after pretreatment with anti-TNF-αIgG1.

RESULTS: There was a little expression of Fas at 2 h in model group. The expression of Fas increased distinctly at 8 h and 12 h and there was no statistical difference between them. The expression of Fas at 8 h and 12 h was higher than that at 2 h and 4 h (P < 0.01 and P < 0.05, respectively). TNF-αmRNA expression in liver increased statistically (0.91±0.75) and the data of normal control was (0.32±0.10) in 2 hours to 4 hours after administration of LPS and D-GalN. The level of serum TNF-αincreased (320±87 ng/L, the data of normal control was 17±7 ng/L). There was typical manifestation of hepatocytic apoptosis at 8 h after the drug administration. The level of serum ALT and TBil obviously increased (9 352±1 000 nkat/L and 163.7±34.5mmoL/L, respectively, the data of normal control was 393±134 nkat/L and 14.9±4.8 mmoL/L, respectively). and there were hepatocytic apoptosis and necrosis at 12 hour after drug administration, at the same time, the level of serum ALT and TBil reached the peak (11 141±1 312 nkat /L and 203.2±19.9 mmol/L, respectively). Hepatocytic apoptosis and liver injury and the expression of Fas could be blocked after antagonized with TNF-α.

CONCLUSION: TNF-α plays an important role on hepatocytic apoptosis and liver injury in fulminant hepatic failure. The hepatocytic apoptosis induced by TNF-α is correlated with the expression of Fas.

Key Words: N/A

0 引言

近年研究发现, 暴发性肝衰竭(fulminant hepatic failure,FHF)中肝细胞凋亡与坏死密切相关, 是肝细胞坏死的早期过程. 在肝脏疾病中, 肝细胞的凋亡主要是通过Fas/FasL途径介导所致[12,18-20], 但在FHF动物模型中对Fas表达与肝细胞凋亡关系的研究却很少见, 我们对FHF模型鼠肝细胞凋亡、肝组织Fas及TNF-α表达的检测, 探讨在暴发性肝衰竭(fulminant hepatic failure, FHF)中肝组织Fas及TNF-α表达与肝细胞凋亡的关系.

1 材料和方法

1.1 材料

LPS购自Sigma公司, D-GalN购自重庆医科大学, 抗TNF-αIgG1(Endogen公司产品)、TNF-α酶标试剂盒(Golden bridge公司产品)购自深圳晶美生物公司, ALT试剂盒购自上海复星长征医学科学有限公司, TBil试剂盒购自日本纺绩株式会社, TNF-αmRNA引物购自北京奥科生物公司, cDNA合成试剂盒购自宝生物工程有限公司(TaKaRa产品), TUNEL试剂盒及Fas免疫组化试剂盒均购自武汉博士德生物公司. 雄性昆明小鼠(中国医科大学附属二院动物实验室提供)76只, 清洁级, 质量20-25 g, 随机分成12组, 取4组(每组7只)给予LPS 10 mg/kg sc, D-GalN 800 mg/kg ip, 另取4组(每组7只)除注射LPS和D-GalN外, 同时sc抗TNF-αIgG1mAb 100 mg, 余4组动物(每组5只)注射同样体积的生理盐水, 对上述小鼠分别于注药后2, 4, 8, 12 h处死, 每个时间点为1组动物, 留取血清及肝脏标本, 将血清于-20 ℃冻存待检, 肝组织一部分于40 g/L中性甲醛固定, 一部分于-80 ℃冻存待检.注射LPS+D-GalN后 2, 4, 8, 12 h分别编号为A1, A2, A3, A4组, 注射生理盐水后2, 4, 8, 12 h分别编号为B1, B2, B3, B4组, 注射LPS+D-GalN+抗TNF-αIgG1单抗后2, 4, 8, 12 h分别编号为C1, C2, C3, C4组.

1.2 方法

于日立7170全自动生化分析仪进行血清ALT, TBil水平检测. TNF-α水平检测按试剂盒说明书进行, 抗原抗体反应后显色, 在酶标仪492 nm处读取A值, 并以标准曲线为标准计算样品的含量. TNF-αmRNA的相对含量的检测采用异硫氰酸胍-酚-氯仿方法, 提取肝组织内RNA, 采用TNF-α-2(antisense primer), 在30-65 ℃, 35 min条件下逆转录合成TNF-αmRNA cDNA后进行PCR反应, 引物序列如下: TNF-α-1 5'-ACCAGAGCGGCAAGAAGAACCAT-3'(sense primer); TNF-α-2 5'-CATCAGACATCGGAGGCAGGAAG-3'anti-sense primer)(产物长329 bp), 同时设内参照, 引物序列: b-ctin-1 5'-TGTATGCCTCTGGTCGTACCAC-3'(sense primer);b-ctin-2 5'-ACAGAGTACTTGCGCTCAGGAG-3'(anti-sense primer)(产物长592 bp). PCR反应条件: 94 ℃变性3 min, 然后94 ℃ 30 s, 57 ℃ 1 min, 72 ℃ 1 min进行32个循环, 72 ℃延伸5 min. 将PCR产物在20 g/L琼脂糖凝胶上电泳, 用Kodak 1D型凝胶成像分析系统分析检测各扩增带的产物含量, 用下列公式表示: TNF-αmRNA的相对含量 = TNF-αmRNA/bactin100. Fas免疫组化检测按试剂盒说明书进行, 对石蜡包埋的肝组织切片进行抗原抗体反应后DAB显色, 染色后细胞质呈棕褐色染色的为阳性细胞, 细胞质无棕褐色染色的为阴性细胞. 阳性细胞记数分3级: 低倍镜视野阳性细胞数小于1/3为(+), 1/3-2/3为(++), 大于2/3为(+++). 采用酚、氯仿提取肝组织DNA, 于7 g/L琼脂糖凝胶电泳(100 V, 1.5 h)观察DNA梯形带的变化. 缺口原位末端标记技术按试剂盒说明书进行检测, 对石蜡包埋的肝组织切片进行缺口原位末端标记后DAB显色, 染色后细胞核呈棕褐色染色的为阳性细胞, 细胞核无棕褐色染色的为阴性细胞. 阳性细胞记数分3级: 低倍镜视野阳性细胞数小于1/3为(+), 1/3-2/3为(++), 大于2/3为(+++). 肝组织石蜡切片用HE染色于光镜下观察肝组织学变化.

统计学处理 采用mean±SD表示血清ALT, TBil, TNF-α水平及TNF-αmRNA相对含量, 使用SAS软件包作方差分析, 并进行两两比较, 采用χ²检验进行Fas阳性表达率及TUNEL法检测肝细胞凋亡的比较.

2 结果

2.1 血清ALT, TBil, TNF-α和肝组织TNF-αmRNA表达

与对照组相比, 应用LPS+D-GalN后2, 4, 8 h血清TNF-α水平、肝组织TNF-αmRNA的相对含量及阳性表达率均显著升高(P<0.01), 其中, 尤以2 h组升高最为显著, 以后逐渐下降. 从LPS+D-GalN应用后4 h开始, ALT, TBil明显升高, 与各时间点对照组相比有显著差异(P<0.01), 其中8, 12 h升高更明显, 与4 h组相比P<0.01. 阻断TNF-α后各时间点ALT, TBil水平均降至正常. 经阻断TNF-α后, 血清TNF-α水平及肝组织TNF-αmRNA的相对含量及阳性表达率均降至正常水平(表1).

表1 鼠血清ALT, TBil和TNF-α水平变化及肝组织TNF-αmRNA表达的变化(mean±SD).

分组	ALT(nkat/L)	TBil(mmol/L)	TNF-α(ng/L)	TNF-αmRNA相对含量
A1	624±67	15.8±3.0	320±87b	0.91±0.75b
A2	1 238±180a	38.9±5.9a	282±53b	0.82±0.08b
A3	9 352±1 000b	163.7±34.5b	145±29b	0.70±0.06b
A4	11 140±1 312bc	203.2±19.9bc	70±17ac	0.44±0.09c
B1	386±136	13.3±6.3	17±7	0.32±0.10
B2	401±154	13.8±4.7	16±6	0.30±0.11
B3	393±134	14.9±4.8	17±4	0.31±0.10
B4	381±120△	15.1±6.6△	17±5△	0.30±0.09△
C1	523±122	13.5±4.0	17±4	0.33±0.09
C2	558±115	12.5±4.36	17±5	0.32±0.08
C3	416±191	12.7±4.5	17±5	0.34±0.08
C4	429±152	13.0±4.3	17±5	0.33±0.09

^aP<0.05,

^bP<0.01, vs 对照组;

^cP<0.05, vs B4, C4.

2.2 肝细胞凋亡的检测结果

LPS+D-GalN应用后8 h可见典型的DNA梯形带, LPS+D-GalN应用后12 h则出现坏死带, 其余时间点均无DNA梯形带或坏死带的出现, 经阻断TNF-α后, 各时间点均无DNA梯形带或坏死带的出现(图1). 肝组织TUNEL检测结果显示, LPS+D-GalN应用后2 h, 肝组织可出现少量阳性细胞, 4 h时阳性细胞有所增加, 8 h时阳性细胞明显增加, 12 h时亦存在凋亡细胞, 且肝组织出现明显的坏死表现. 经阻断TNF-α后, 各时间点阳性细胞均显著减少(图2).

图1 鼠肝组织DNA琼脂糖凝胶电泳结果.

图2 鼠肝组织TUNEL检测结果.

2.3 肝组织Fas表达的检测结果

在LPS+D-GalN应用后2 h Fas有少量表达, 4 h表达较多, 8 h和12 h时表达均很多, 8 h和12 h组比较无显著差异, 与2 h组比较P<0.01,与4 h组比较P<0.05. 阻断TNF-α后Fas表达显著减少, 与未阻断前12, 8 h比较P<0.01, 与4 h组比较P<0.05(表2).

表2 鼠肝组织Fas检测结果%(n/n).

分组	表达(+++)	表达(++)	表达(+)	表达(-)
A1	0	0.14 (1/7)	0.43 (3/7)	0.43 (3/7)bd
A2	0.14 (1/7)	0.14 (1/7)	0.57 (4/7)	0.14 (1/7)bd
A3	0.86 (6/7)	0.14 (1/7)	0	0b
A4	0.57 (4/7)	0.29 (2/7)	0.14 (1/7)	0b
B1	0	0	0.20 (1/5)	0.80 (4/5)
B2	0	0	0.20 (1/5)	0.80 (4/5)
B3	0	0	0	1.00 (5/5)
B4	0	0	0	1.00 (5/5)
C1	0	0	0.43 (3/7)	0.57 (4/7)
C2	0	0	0.43 (3/7)	0.57 (4/7)e
C3	0	0	0.57 (4/7)	0.43 (3/7)d
C4	0	0	0.29 (2/7)	0.71 (5/7)f

^bP<0.01, vs 对照组;

^dP<0.01, vs A3;

^eP<0.05, vs A2;

^fP<0.01, vs A4.

2.4 肝组织学改变

LPS+D-GalN 应用后2 h组肝组织基本上无异常所见, 4 h组可见肝细胞胞质疏松化, 呈气球样变, 并可见散在的嗜酸变性, 8 h组可见点片状肝细胞坏死, 其中有较多的嗜酸变性和坏死, 12 h组则见肝细胞呈大片坏死, 核碎裂或溶解, 呈现大块状出血坏死. LPS+D-GalN+抗-TNF-α组肝组织HE染色基本上无异常所见.

3 讨论

暴发性肝衰竭是病毒性肝炎和许多肝病死亡的主要原因, 死亡率高达70%以上, 其发病机制复杂, 至今尚未完全阐明. 许多学者采用多种方法进行不同种动物FHF的体内和体外实验[1-7], 对其发病机制及治疗等方面进行研究, 取得了一定的进展. 我们应用LPS10 mg/kg +D-GalN 800 mg/kg联合用药建立FHF小鼠模型, 从动物的临床表现、生化检查和肝脏病理学检查所示的各种变化均符合FHF, 与既往的研究一致[3,8-9]. TNF-α对肝脏具有明显的损伤, 可引起肝细胞坏死[10], 近年研究还发现, TNF-α可通过诱导肝细胞凋亡参与肝脏疾病的发病过程, Leist et al的研究表明, 同时给小鼠注射LPS和D-GalN, 早期可出现肝细胞凋亡的病理及生化改变, 表明在LPS+D-GalN的FHF模型中, LPS的毒性作用实际上是体现为严重的凋亡性肝损伤和完全的破坏, TNF-α是LPS诱导肝细胞凋亡的重要递质. 已证实细胞内蛋白激酶的激活是TNF-α诱导细胞凋亡的一种早期过程, 在TNF-α诱导肝衰竭的实验研究中肝细胞凋亡与肝细胞坏死有着非常密切的联系[1-2,10-17]. 本结果表明, 给予LPS和D-GalN后2 h TNF-α产生最多, 以后逐渐下降, 至12 h时降至正常, 且肝功及肝组织学变化显著, 于给药后8 h出现肝细胞凋亡, 12 h出现肝细胞坏死, 给予TNF-αmAb后TNF-α水平降至正常, 同时亦阻断了肝细胞凋亡及肝生化及组织学变化, 提示TNF-α在FHF早期肝细胞凋亡的过程中起重要作用. Fas/Apo-1系统与其mAb结合后Ca²⁺ 内流, 胞质内Ca²⁺浓度升高可直接激活依赖Ca²⁺/Mg²⁺内源性核酸酶从而导致DNA断裂, 染色质固缩而使细胞凋亡, 在肝脏疾病中, 肝细胞的凋亡主要是通过Fas/FasL途径介导所致[1,5,11-15,18-20,28-29]. 在病毒性肝病中, 肝细胞的死亡主要是通过Fas抗原介导的, 对暴发性肝衰竭模型小鼠腹腔内注射Fas 受体的抗体可加重肝细胞凋亡和肝功能衰竭, 在重型肝炎、慢性肝病患者的肝细胞表面均有Fas抗原的表达[21-25]. 本结果表明, 在LPS+GalN用药后Fas表达增加, 且其表达水平的变化与肝细胞凋亡的检测结果相一致, 提示在此种FHF模型中肝细胞凋亡的发生与Fas的表达增加有关. caspase-3的激活在肝细胞凋亡的发生中起重要的作用, Fas系统参与caspase-3细胞凋亡作用的发挥, 在Fas介导细胞凋亡的过程中, caspase-3是其死亡信号途径下游的一个关键效应组分, 他可被水解成活性酶形式, 进而酶切PARP, 导致DNA片段化、细胞凋亡[27]. TNF-α能激活caspase-3蛋白酶活性并能诱发肝细胞凋亡[26-27,30]. 本研究结果表明, 在LPS+GalN所致的FHF中, TNF-α水平及TNF-αmRNA的表达均增加, Fas的表达亦增加, 伴有肝细胞凋亡的发生, 进一步阻断TNF-α的作用后Fas的表达及肝细胞凋亡均被阻断, 提示在此种模型中TNF-α促进肝细胞凋亡的作用与Fas的表达增加有关.

备注

$[Footnotes]

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