基础研究 Open Access
Copyright ©The Author(s) 2003. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2003-11-15; 11(11): 1711-1714
在线出版日期: 2003-11-15. doi: 10.11569/wcjd.v11.i11.1711
大鼠胃黏膜损伤修复时早期应答基因c-Jun及c-met的表达
姚永莉, 徐波, 宋于刚, 张万岱
姚永莉, 宋于刚, 张万岱, 中国人民解放军第一军医大学南方医院全军消化病研究所 广东省广州市 510515
姚永莉, 女, 1973-02-20生, 湖北省武汉市人, 汉族, 2001年第一军医大学博士, 讲师, 主要从事慢性胃病的防治研究.
徐波, 中国人民解放军第一军医大学南方医院侨科 广东省广州市 510515
基金项目: 军队"九五"卫生科研基金资助课题, No. 96M060.
通讯作者: 姚永莉, 510515, 广东省广州市同和, 中国人民解放军第一军医大学南方医院全军消化病研究所. xbyyl@fimmu.com
电话: 020-85141535 传真: 020-87280770
收稿日期: 2002-05-05
修回日期: 2003-03-20
接受日期: 2003-03-24
在线出版日期: 2003-11-15

目的

建立无水乙醇急性胃黏膜损伤大鼠模型并观察早期应答基因c-Jun和c-met对胃黏膜损伤修复的影响.

方法

采用无水乙醇1 mL胃饲诱发急性胃黏膜损伤大鼠模型, 并于伤后0, 4, 8 d分别处理一组大鼠, 观察损伤模型自然修复过程, 免疫组织化学技术检测早期应答基因c-Jun和c-met的表达.

结果

在损伤模型自然修复过程中, 损伤后4, 8 d组大鼠胃黏膜损伤指数(LI)为32±7, 18±3, 均显著低于损伤模型组75±11, (P<0.05); 免疫组织化学显示损伤后8 d组大鼠胃黏膜c-Jun的阳性表达率为87.5%, 显著高于正常对照组的12.5%, c-met的阳性表达率为62.5%, 均显著高于正常对照的0及损伤模型组的0(P<0.05).

结论

早期应答基因c-Jun和c-met的表达能促进急性胃黏膜损伤的修复, 对黏膜损伤的自愈具有重要作用.

关键词: N/A
引文著录: 姚永莉, 徐波, 宋于刚, 张万岱. 大鼠胃黏膜损伤修复时早期应答基因c-Jun及c-met的表达. 世界华人消化杂志 2003; 11(11): 1711-1714
c-met and c-Jun expression during acute gastric mucosal lesions in rats
Yong-Li Yao, Bo Xu, Yu-Gang Song, Wan-Dai Zhang
Yong-Li Yao, Yu-Gang Song, Wan-Dai Zhang, Institute of Digestive Disease of PLA, Nanfang Hospital, First Military Medical University, Guangzhou 510515, Guangdong Province, China
Bo Xu, Department of Overseas Chinese, Nanfang Hospital, First Military Medical University, Guangzhou 510515, Guangdong Province, China
Supported by: the Medical Science Foundation of Chinese PLA, No. 96M060.
Corresponding author: Yong-Li Yao, Institute of Digestive Disease of Chinese PLA, Nanfang Hospital, First Military Medical University, Tonghe Road, Guangzhou 510515, Guangdong Province, China. xbyyl@fimmu.com
Received: May 5, 2002
Revised: March 20, 2003
Accepted: March 24, 2003
Published online: November 15, 2003

AIM

To establish an ethanol-induced acute gastric mucosal lesions model in rats and to investigate the effect of c-met and c-Jun protein on healing of acute gastric mucosal lesions.

METHODS

Animal models of acute gastric mucosal lesions were established by intragastric instillation of 1 mL ethanol in the rats. On day 0, 4, 8, the rats were sacrificed respectively. Rats without any treatment served as control. The expressions of c-met and c-Jun were analyzed by immunohistochemical staining.

RESULTS

Gastric mucosal lesion indexes (LIs) were significantly lower after 4, and 8 days with acute gastric mucosal lesions (32±7, 18±3) than LI of acute gastric mucosal lesion model rats (75±11) (P<0.05). Immunohistochemical staining revealed higher positive staining of c-Jun after 8 days with acute gastric mucosal lesions (87.5%) than those of normal control rats (12.5%), and higher positive staining of c-met after 8 days with acute gastric mucosal lesions (62.5%) than those of normal control (0) and acute gastric mucosal lesions model rats (0) (P<0.05).

CONCLUSION

The expression of c-met and c-Jun could accelerate the healing of acute gastric mucosal lesions, which is important for the healing of acute gastric mucosal lesions.

Key Words: N/A
0 引言

即刻早期应答基因c-Jun及c-met参与细胞增生的调控, 与胃黏膜损伤修复的关系密切[1-12], 以大鼠无水乙醇急性胃黏膜损伤模型为基础, 检测早期应答基因c-Jun和c-met在胃黏膜损伤修复过程中的变化规律, 将有助于提高对胃黏膜损伤修复能力病理生理的认识.

1 材料和方法
1.1 材料

免疫组化染色试剂购自武汉博士德公司. 选用32只健康成年♂SD大鼠, 体质200 ± 10 g, 饮食不限, 适应性饲养7 d后用于实验. 实验用大鼠随机分成4组, 每组8只. 正常对照组: (1)禁食不禁水24 h后, 蒸馏水1 mL灌胃, 间隔1 h后断颈处死. 无水乙醇模型组; (2)禁食不禁水24 h, 无水乙醇1 mL灌胃, 间隔1 h后断颈处死. 自然恢复4 d组; (3)禁食不禁水24 h, 无水乙醇1 mL灌胃, 4 d后处死. 自然恢复8 d组; (4)禁食不禁水24 h, 无水乙醇1 mL灌胃, 8 d后处死. 全部实验大鼠均正中开腹取出全胃, 结扎食管及幽门, 向胃内注入40 mg/L多聚甲醛溶液2 mL, 并将全胃浸入40 mg/L多聚甲醛溶液中固定30 min后, 沿胃大弯切开, 平展在玻璃板上, 肉眼观察胃黏膜改变; 用微卡尺测定溃疡灶大小并按Guth法计算黏膜损伤指数[3]: 全胃各病灶长度之和为损伤指数(LI), 以mm表示, 损伤小于或等于1 mm (包括糜烂点)为1分, 大于1 mm而小于或等于2 mm为2分, 依此类推, 损伤宽度大于2 mm者指数加倍. 胃组织40 mg/L多聚甲醛溶液固定6 h, 石蜡包埋, 制成3-4 μm的连续切片, HE染色观察组织病理学改变.

1.2 方法

免疫组织化学染色采用武汉博士德公司的SABC免疫组化染色试剂盒, 按说明书操作. c-Jun阳性细胞的核或胞质可被染成棕黄色或深棕色, 而c-met阳性细胞的胞质可被染成棕黄色或深棕色. 染色结果分级评分方法为: 每张切片均观察胃窦部黏膜染色情况, 从中选择5个有代表性的区域, 每区计算200个细胞. 阳性细胞数计分: 阳性数小于或等于5%为0, 阳性数大于5%但小于或等于35%为1, 阳性数大于35%但小于或等于70%为2, 阳性数大于70%为3. 阳性强度记分: 不着色为0, 弱着色为1, 强着色为2. 阳性分级: 以上两项相加, 小于或等于2为(-), 3 = (+), 4 = (++), 5 = (+++). 以阳性分级(+)以上为阳性.

统计学处理 用SPSS统计软件进行数据处理, c-Jun和c-met表达阳性率的比较用Chi-square检验, 表达强度比较用非参数检验; 计量资料满足正态分布, 行方差分析.

2 结果
2.1 病理学观察

正常对照组大鼠胃黏膜红润、光泽, 未见糜烂、水肿及充血等病理改变, 光镜下未见病理表现. 无水乙醇模型大鼠胃黏膜表面见条状出血病灶, 少数呈斑片状及点状, 黏膜表面明显水肿, 光镜下见黏膜表层坏死组织, 并有中性粒细胞浸润(图1, 图2); 自然恢复4 d组大鼠胃黏膜的色泽浅淡, 光润度稍差, 仍可见糜烂、水肿及充血等改变, 但较无水乙醇模型大鼠程度明显减轻, 光镜下较少见黏膜表层坏死组织, 有中性粒细胞及少量单核细胞、淋巴细胞浸润; 自然恢复8 d组大鼠胃黏膜的色泽及光润度与正常相似, 偶见点状糜烂, 光镜下黏膜表层坏死组织基本消失, 炎性细胞已明显减少. 各组间胃黏膜表面损伤程度比较以LI为标准, A, B, C, D组分别为0, 75±11, 32±7, 18±3, 各组间皆有差别(P<0.05).

图1
图1 大鼠胃黏膜小片状烧伤. A: 大体标本; B: 侵入固有膜, 炎性细胞浸润 HE×200.
图2
图2 大鼠胃黏膜条状烧伤. A: 大体标本; B: 表层上皮缺损明显, 损伤侵入胃黏膜全层 HE×200.
2.2 c-Jun和c-met的表达

c-Jun阳性细胞的核或胞质可被染成棕黄色或深棕色, c-met阳性细胞的胞质可被染成棕黄色或深棕色. c-Jun核阳性细胞多位于黏膜固有层胃小凹区域的腺管上皮中, 呈带状分布, c-Jun胞质阳性细胞多位于胃黏膜, 散在分布或簇状分布(图3A). c-met阳性细胞多位于黏膜中上1/3, 散在分布(图3B). 正常胃黏膜及无水乙醇所致急性胃黏膜损伤时c-Jun的阳性信号表达较少, 而未见c-met的阳性信号表达; 自然恢复4 d组大鼠胃黏膜中出现c-met的阳性信号表达; 自然恢复8 d组大鼠胃黏膜中c-Jun的阳性信号表达明显增加, 与正常对照有显著差异, c-met的阳性信号表达明显增加, 与正常对照及损伤模型组大鼠均有显著差异见表1.

表1 c-Jun和c-met在无水乙醇所致急性胃黏膜损伤中的表达(n = 32).
组别c-Jun
c-met
++++++-阳性率++++++-阳性率
A1712.5%a80a
B4450.0%80a
C5362.5%2625.0%
D25187.5%14362.5%
aP<0.05 vs D.
图3
图3 大鼠胃黏膜损伤8 d. A: c-Jun基因的表达SABC×100; B: c-met基因的表达SABC×400.
3 讨论

胃黏膜在受到一定程度的损伤刺激后, 可启动修复机制使胃黏膜迅速恢复其完整性, 以避免损伤向深层发展, 这是胃黏膜抵抗疾病的一种重要屏障能力[13-18]. 胃黏膜的损伤修复是一个复杂的多因素、多途径的网络调控过程[19-28], 为进一步了解即刻早期应答基因c-Jun及c-met在胃黏膜损伤修复这一复杂调控过程中的作用, 我们检测了胃黏膜损伤修复过程中即刻早期应答基因c-Jun及c-met的变化规律.

原癌基因c-Jun及c-met属即刻早期应答基因, 其编码的蛋白为重要的转录因子, 可诱导其下游基因mRNA转录和蛋白表达, 参与细胞增生的调控, 与急性胃黏膜损伤修复的关系密切[29-41]. 我们的研究发现正常胃黏膜及无水乙醇所致急性胃黏膜损伤时c-Jun的阳性信号表达较少, 未见c-met的阳性信号表达, 自然恢复4 d组大鼠胃黏膜中出现c-met的阳性信号表达; 自然恢复8 d组大鼠胃黏膜中c-Jun的阳性信号表达明显增加, 与正常对照有显著差异, c-met的阳性信号表达明显增加, 与正常对照及损伤模型组大鼠均有显著差异. 提示早期应答基因c-Jun及c-met参与并促进损伤胃黏膜的修复过程, 这可能正是无水乙醇所致急性胃黏膜损伤修复过程得以进行的重要原因之一.

1.  Liu JD, Lin SY, Ho YS, Pan S, Hung LF, Tsai SH, Lin JK, Liang YC. Involvement of c-jun N-terminal kinase activation in 15-deoxy-delta12, 14-prostaglandin J2-and prostaglandin A1-induced apoptosis in AGS gastric epithelial cells. Mol Carcinog. 2003;37:16-24.  [PubMed]  [DOI]
2.  Zhuang X, Zheng J, Lin S, Sun G, Li Y. The prognostic significance of expression of c-met oncogene and its relation to gastric mucosal lesions. Zhonghua BingLiXue ZaZhi. 2000;29:409-411.  [PubMed]  [DOI]
3.  Wielenga VJ, van der Voort R, Taher TE, Smit L. Expression of c-Met and heparan-sulfate proteoglycan forms of CD44 in colorectal cancer. Am J Pathol. 2000;157:1563-1573.  [PubMed]  [DOI]
4.  Nishioka H, Baesso I, Semenzato G, Trentin L, Rappuoli R, Del Giudice G, Montecucco C. The neutrophil-activating protein of Helicobacter pylori (HP-NAP) activates the MAPK pathway in human neutrophils. Eur J Immunol. 2003;33:840-849.  [PubMed]  [DOI]
5.  Chu SH, Kim H, Seo JY, Lim JW, Mukaida N, Kim KH. Role of NF-kappaB and AP-1 on Helicobater pylori-induced IL-8 expression in AGS cells. Dig Dis Sci. 2003;48:257-265.  [PubMed]  [DOI]
6.  Lee HW, Lee SS, Lee SJ, Um HD. Bcl-w is expressed in a majority of infiltrative gastric adenocarcinomas and suppresses the cancer cell death by blocking stress-activated protein kinase/c-Jun NH2-terminal kinase activation. Cancer Res. 2003;63:1093-1100.  [PubMed]  [DOI]
7.  Rokutan K. Molecular chaperone inducers in medicine and diseases. Nippon Yakurigaku Zasshi. 2003;121:15-20.  [PubMed]  [DOI]
8.  Chen Y, Wu Q, Song SY, Su WJ. Activation of JNK by TPA promotes apoptosis via PKC pathway in gastric cancer cells. World J Gastroenterol. 2002;8:1014-1018.  [PubMed]  [DOI]
9.  Zhuang X, Lin S, Zheng J. The prognostic research of expression and relationship between Helicobacter pylori of c-met oncogene correlation with gastric mucosal proliferation. Zhonghua NeiKe ZaZhi. 2001;40:381-384.  [PubMed]  [DOI]
10.  Huang TJ, Wang JY, Lin SR, Lian ST, Hsieh JS. Overexpression of the c-met protooncogene in human gastric carcinoma-correlation to clinical features. Acta Oncol. 2001;40:638-643.  [PubMed]  [DOI]
11.  Chen JH, Liu TY, Wu CW, Chi CW. Nonsteroidal anti-inflammatory drugs for treatment of advanced gastric cancer: cyclooxygenase-2 is involved in hepatocyte growth factor mediated tumor development and progression. Med Hypotheses. 2001;57:503-505.  [PubMed]  [DOI]
12.  Heideman DA, Snijders PJ, Bloemena E, Meijer CJ, Offerhaus GJ, Meuwissen SG, Gerritsen WR, Craanen ME. Absence of tpr-met and expression of c-met in human gastric mucosa and carcinoma. J Pathol. 2001;194:428-435.  [PubMed]  [DOI]
13.  Ueyama T, Saika M, Senba E. Distinct gene expression in the stomach following stress and alcohol exposure. Kaibogaku Zasshi. 2001;76:435-441.  [PubMed]  [DOI]
14.  夏 敏, 陶 嘉泳. 乙醇对小鼠胃黏膜的损伤机制. 世界华人消化杂志. 1997;5; 211-212.  [PubMed]  [DOI]
15.  李 林, 王 竹立, 刘 芭, 吴 小林, 张 静. 一贯煎抗无水乙醇性胃黏膜损伤的作用. 世界华人消化杂志. 1998;6:519-520.  [PubMed]  [DOI]
16.  沈 鹰. 健脾方药对胃黏膜保护作用的研究及其意义. 世界华人消化杂志. 1999;7:357-358.  [PubMed]  [DOI]
17.  薛 会光, 易 粹琼. 胃黏膜对阿斯匹林适应性的实验研究. 世界华人消化杂志. 1999;7:703-704.  [PubMed]  [DOI]
18.  Xiao ZQ, Majumdar AP. Induction of transcriptional activity of AP-1 and NF-kappaB in the gastric mucosa during aging. Am J Physiol Gastrointest Liver Physiol. 2000;278:G855-865.  [PubMed]  [DOI]
19.  Meyer ter Vehn T, Covacci A, Kist M, Pahl HL. Helicobacter pylori activates mitogen-activated protein kinase cascades and induces expression of the proto-oncogenes c-fos and c-jun. J Biol Chem. 2000;275:16064-16072.  [PubMed]  [DOI]
20.  Tominaga K, Higuchi K, Tsuno M, Watanabe T, Fujiwara Y, Kim S, Arakawa T, Iwao H, Kuroki T. Induction of signal transduction pathways in rat gastric epithelial cells stimulated with interleukin-1beta. Aliment Pharmacol Ther. 2000;14:101-108.  [PubMed]  [DOI]
21.  Kim R, Ohi Y, Inoue H, Toge T. Enhancement of chemotherapeutic agents induced-apoptosis associated with activation of c-Jun N-terminal kinase 1 and caspase 3 (CPP32) in bax-transfected gastric cancer cells. Anticancer Res. 2000;20:439-444.  [PubMed]  [DOI]
22.  Tominaga K, Arakawa T, Tsuno M, Kim S, Iwao H, Kuroki T. Increased mitogen-activated protein kinase activities stimulated with interleukin-1-beta and mechanism(s) of the kinase signaling pathways in rat gastric epithelial cells. Digestion. 2000;61:30-38.  [PubMed]  [DOI]
23.  Haruma K, Ito M, Kohmoto K, Kamada T, Kitada Y, Yasui W, Tahara E, Kajiyama G. Expression of cell cycle regulators and growth factor/receptor systems in gastric carcinoma in young adults: association with Helicobacter pylori infection. Int J Mol Med. 2000;5:185-190.  [PubMed]  [DOI]
24.  Wu HM, Wen HC, Lin WW. Proteasome inhibitors stimulate interleukin-8 expression via Ras and apoptosis signal-regulating kinase-dependent extracellular signal-related kinase and c-Jun N-terminal kinase activation. Am J Respir Cell Mol Biol. 2002;27:234-243.  [PubMed]  [DOI]
25.  Xu YC, Wu RF, Gu Y, Yang YS, Yang MC, Nwariaku FE, Terada LS. Involvement of TRAF4 in oxidative activation of c-Jun N-terminal kinase. J Biol Chem. 2002;277:28051-28057.  [PubMed]  [DOI]
26.  Kang KW, Novak RF, Lee CH, Kim SG. Induction of microsomal epoxide hydrolase by sulfur amino acid deprivation via the pathway of C-Jun N-terminal kinase and its extracellular exposure during cell death. Free Radic Biol Med. 2002;32:1017-1032.  [PubMed]  [DOI]
27.  Heideman DA, Snijders PJ, Bloemena E, Meijer CJ, Offerhaus GJ, Meuwissen SG, Gerritsen WR, Craanen M ESO. Absence of tpr-met and expression of c-met in human gastric mucosa and carcinoma. J Pathol. 2001;194:428-435.  [PubMed]  [DOI]
28.  Zhao Y, Wu K, Xia W, Shan YJ, Wu LJ, Yu WP. The effects of vitamin E succinate on the expression of c-jun gene and protein in human gastric cancer SGC-7901 cells. World J Gastroenterol. 2002;8:782-786.  [PubMed]  [DOI]
29.  Guo YS, Cheng JZ, Jin GF, Gutkind JS, Hellmich MR, Townsend CM. Gastrin stimulates cyclooxygenase-2 expression in intestinal epithelial cells through multiple signaling pathways. Evidence for involvement of ERK5 kinase and transactivation of the epidermal growth factor receptor. J Biol Chem. 2002;277:48755-48763.  [PubMed]  [DOI]
30.  Sepulveda AR, Tao H, Carloni E, Sepulveda J, Graham DY, Peterson LE. Screening of gene expression profiles in gastric epithelial cells induced by Helicobacter pylori using microarray analysis. Aliment Pharmacol Ther. 2002;16:145-157.  [PubMed]  [DOI]
31.  Wu K, Liu BH, Zhao DY, Zhao Y. Effect of vitamin E succinate on expression of TGF-beta1, c-Jun and JNK1 in human gastric cancer SGC-7901 cells. World J Gastroenterol. 2001;7:83-87.  [PubMed]  [DOI]
32.  Ueyama T, Saika M, Senba E. Distinct gene expression in the stomach following stress and alcohol exposure. Kaibogaku Zasshi. 2001;76:435-441.  [PubMed]  [DOI]
33.  Saika M, Ueyama T, Senba E. Expression of immediate early genes, HSP70, and COX-2 mRNAs in rat stomach following ethanol ingestion. Dig Dis Sci. 2000;45:2455-2462.  [PubMed]  [DOI]
34.  Tominaga K, Arakawa T, Tsuno M, Kim S, Iwao H, Kuroki T. Increased mitogen-activated protein kinase activities stimulated with interleukin-1-beta and mechanism(s) of the kinase signaling pathways in rat gastric epithelial cells. Digestion. 2000;61:30-38.  [PubMed]  [DOI]
35.  Miyazaki Y, Hiraoka S, Tsutsui S, Kitamura S, Shinomura Y, Matsuzawa Y. Epidermal growth factor receptor mediates stress-induced expression of its ligands in rat gastric epithelial cells. Gastroenterology. 2001;120:108-116.  [PubMed]  [DOI]
36.  Ichikawa T, Endoh H, Hotta K, Ishihara K. Hepatocyte growth factor region specifically activates mucin synthesis in rat stomach. Eur J Pharmacol. 2000;392:87-91.  [PubMed]  [DOI]
37.  Hori K, Shiota G, Kawasaki H. Expression of hepatocyte growth factor and c-met receptor in gastric mucosa during gastric ulcer healing. Scand J Gastroenterol. 2000;35:23-31.  [PubMed]  [DOI]
38.  Doggett TA, Swarthout JT, Jefcoat SC, Wilhelm D, Dieckmann A, Angel P, Partridge NC. Parathyroid hormone inhibits c-Jun N-terminal kinase activity in rat osteoblastic cells by a protein kinase A-dependent pathway. Endocrinology. 2002;143:1880-1888.  [PubMed]  [DOI]
39.  Dunn C, Wiltshire C, MacLaren A, Gillespie DA. Molecular mechanism and biological functions of c-Jun N-terminal kinase signalling via the c-Jun transcription factor. Cell Signal. 2002;14:585-593.  [PubMed]  [DOI]
40.  Hurd C, Waldron RT, Rozengurt E. Protein kinase D complexes with C-Jun N-terminal kinase via activation loop phosphorylation and phosphorylates the C-Jun N-terminus. Oncogene. 2002;21:2154-2160.  [PubMed]  [DOI]
41.  She QB, Huang C, Zhang Y, Dong Z. Involvement of c-jun NH(2)-terminal kinases in resveratrol-induced activation of p53 and apoptosis. Mol Carcinog. 2002;33:244-250.  [PubMed]  [DOI]