幽门螺杆菌 Open Access
Copyright ©The Author(s) 2003. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2003-05-15; 11(5): 544-546
在线出版日期: 2003-05-15. doi: 10.11569/wcjd.v11.i5.544
胃癌细胞系幽门螺杆菌感染对金属蛋白酶表达的影响
李新华, 张桂英, 罗非君, 徐美华, 李乾
李新华, 张桂英, 徐美华, 李乾, 中南大学湘雅医院消化内科湖南省长沙市 410008
李新华, 女, 1969-10-19生, 湖南省邵东县人, 汉族, 1993年湖南医科大学本科毕业, 2001年中南大学湘雅医学院硕士研究生毕业, 主要从事消化系肿瘤的分子机制研究.
罗非君, 中南大学湘雅医学院肿瘤所湖南省长沙市 410078
基金项目: 湖南省科技厅资助项目, No. 01SSY2008-9; 湖南省卫生厅资助项目, No. 2001-Y23/1.0.
通讯作者: 张桂英, 410008, 湖南省长沙市湘雅路141号, 中南大学湘雅医院消化内科.
电话: 0731-4327249
收稿日期: 2002-07-23
修回日期: 2002-07-30
接受日期: 2002-08-02
在线出版日期: 2003-05-15

目的

建立胃癌细胞系H.pylori感染动物转移体外模型, 初步探讨H.pylori感染在胃癌转移过程中的可能作用及其机制.

方法

将H.pylori活菌与胃癌细胞系BGC-823低转移细胞株C8细胞共培养后, 行裸鼠接种, 观察H.pylori感染组与未感染组移植瘤生长情况及裸鼠肝、肺转移情况, 免疫组化检测两组移植瘤中MMP-2, MMP-3, TIMP-2及TIMP-3蛋白的表达.

结果

H.pylori感染组移植瘤重量与未感染组比较有差异(P<0.05), 病理检查示感染组2个肺转移, 而未感染组无转移. 免疫组化结果示感染组MMP-2, MMP-3, TIMP-2, TIMP-3蛋白的表达阳性率分别为: 77.89.6%, 64.56.9%, 57.612.2%, 40.09.2%; 而未感染组分别为61.29.7%, 53.15.8%, 54.310.9%, 53.06.6%, 二者比较除TIMP-3蛋白的表达无显著性差异外, MMP-2、MMP-3、TIMP-2蛋白的表达均有显著性差异(P<0.05).

结论

H.pylori感染能促使裸鼠移植瘤的生长及肺转移, 建立了H.pylori感染与胃癌的动物转移体外模型; H.pylori感染可能通过促使转移正相关因子MMP-2、MMP-3表达增高, 转移负相关因子TIMP-2蛋白表达降低, 而在胃癌转移过程中发挥重要作用.

关键词: N/A
引文著录: 李新华, 张桂英, 罗非君, 徐美华, 李乾. 胃癌细胞系幽门螺杆菌感染对金属蛋白酶表达的影响. 世界华人消化杂志 2003; 11(5): 544-546
Influence of expression of matrix metalloproteinase induced by H. pylori infection in gastric cancer cell line
Xin-Hua Li, Gui-Ying Zhang, Fei-Jun Luo, Mei-HuaXu, Qian Li
Xin-Hua Li, Gui-Ying Zhang, Mei-HuaXu, Qian Li, Department of Gastroenterology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
Fei-Jun Luo, Cancer Research Institute, Xiangya Medical College, Central South University, Changsha 410078, Hunan Province, China
Supported by: the Natural Scientific Foundation of Hunan Province, No. 01SSY2008-9; and the Foundation of Hunan Province, No. 2001-Y23/1.0.
Correspondence to: Prof. Gui-Ying Zhang, Department of Gastroenterology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province , China.
Received: July 23, 2002
Revised: July 30, 2002
Accepted: August 2, 2002
Published online: May 15, 2003

AIM

To establish an animal model to study the effect of H. pylori on metastasis, and to explore the possible mechanism and the relationship between H. pylori and gastric cancer metastasis.

METHODS

Using BGC-823 cell cocultured with H. pylori, the growth of tumors and metastasis were observed in 8-week nude mice after the first intraumoral injection of the group of H.pylori infection and the group of no infection. The expression of MMP-2, MMP-3, TIMP-2 and TIMP-3 were investigated by immunohistochemical staining.

RESULTS

There was a significant difference in the weight of tumors between the group of H.pylori infection and the group without H.pylori infection in 8-week nude mice after the first intratumoral injection (P<0.05). There were two lung metastases in group of H. pylori infection. The positive rates of MMP-2, MMP-3, TIMP-2 and TIMP-3 in the group of H. pylori infection by immunohischemical staining were 77.8±9.6%, 64.5±6.9%, 57.6±12.2% and 40.0±9.2%, and 61.2±9.7%, 53.1±5.8%, 54.3±10.9% and 53.0±6.6% in the group without H.pylori infection, respectively. The expression of MMP-2, MMP-3 and TIMP-2 were significantly higher in the group of H. pylori infection than those in the group without H.pylori infection (P<0.05), but the expression of TIMP-3 has no significantly change.

CONCLUSION

H.pylori infection can accelerate the tumor growth in nude mouse and lung metastasis. The expression of MMP-2 and MMP-3 increases and TIMP-2 decreases in H. pylori infection, suggesting H. pylori may enhance gastric cancer metastasis by regulating the expression of metastasis-correlated factors.

Key Words: N/A
0 引言

幽门螺杆菌(H. pylori)感染是胃癌发生的重要环境因素[1-11], 可能参与胃癌的转移过程[12]. 细胞外基质成分的降解对于肿瘤细胞侵犯周围组织和向远处转移是必不可少的, 而基底膜基质的降解与MMPs(matrix metalloproteinase, MMPs)和TIMP(tissue inhibitors of metalloproteinases, TIMP)的活性紧密相关[13]. 为此, 我们用H.pylori与细胞体外共培养技术, 建立动物转移实验体外模型, 观察H. pylori感染组与未感染组的裸鼠移植瘤生长情况及裸鼠肝、肺转移情况, 确定H.pylori感染能否促进人胃癌细胞的转移潜能. 利用裸鼠移植瘤标本, 采取免疫组织化学SABC法检测在H.pylori感染和无H.pylori感染时的裸鼠移植瘤中, 转移相关因子MMP-2, MMP-3, TIMP-2及TIMP-3蛋白表达的差异. 从而对H.pylori感染在胃癌转移过程中的作用及机制作一初步探讨.

1 材料和方法
1.1 材料

空肠弯曲菌选择性培养基(上海市疾病预防控制中心); 羊血(湖南临检中心及益阳生化试剂厂); RPMI 1 640(Gibco); 免疫组化主要试剂: 兔抗鼠MMP-2, MMP-3, TIMP-2, TIMP-3, 生物素化羊抗兔IgG; SABC试剂盒; DAB显色试剂盒 (武汉博士德公司). H.pylori标准株ATCC49503, 由中南大学湘雅医院范学工教授馈赠; 人胃癌细胞系BGC-823低转移单克隆株C8 (以下简称BGC-823), 购自北京肿瘤研究所遗传室. BALB/c裸鼠由北京军事医学科学院提供, 3-4周龄, 雌雄各半. 置中南大学湘雅医学院动物学部SPF环境中饲养.

1.2 方法

H.pylori与细胞的共同孵育 参照文献[14]方法.取对数生长期的BGC-823细胞用含0.2 g/LEDTA和1.5 g/L胰蛋白酶消化后计数, 接种于培养瓶或培养板中, 在37 ℃, 50 mL/L CO2培养箱培养24 h后, 弃去原有细胞培养液, 加入用含40 mL/L小牛血清的RPMI-1 640培养液稀释的不同浓度的H.pylori活菌菌液后继续置于37 ℃, 5% CO2培养箱培养. 收集处于对数生长期的BGC-823细胞, 以每只裸鼠2×106个/只接种于裸鼠右腋皮下, 每组5, 6只, 雌雄各半. 8 wk后处死裸鼠, 剥离肿瘤称重, 并对肿瘤组织学进行检查, 同时, 取小鼠的肝、肺进行病理切片分析, 寻找微转移灶. 免疫组化检测裸鼠移植瘤中MMP-2, MMP-3, TIMP-2及TIMP-3蛋白的表达采用链亲和素-生物素-过氧化氢酶法(SABC法)稍加修改, 用冰冷切片取代石蜡切片染色. 用PBS代替一抗作阴性对照, 结果判定: 光学显微镜下观察组织切片的显色反应. 阳性产物主要位于胞质, 阳性判断标准为细胞质内出现了明确的棕黄色颗粒. 免疫组化评分标准: 每张切片在光学显微镜下取5个高倍视野, 各计数200个细胞, 计算阳性细胞百分比, 得阳性表达率;

统计学处理 采用统计学软件SPSS10.0, 计量资料采用t检验或单因素方差分析(one-way ANOVA), 计数资料采用x2检验或Fisher精确分析. P<0.05有统计学意义.

2 结果

H. pylori活菌与BGC-823 C8细胞共同培养后, 接种裸鼠, 结果发现所有均能致瘤, 实验组与对照组的瘤体重量有显著性差异(6.91.7 vs 4.90.8 g , P<0.05), 实验组瘤体有大量坏死. 大体上可见与接种原发瘤包膜完全分开或转移生长于颈部、腹股沟等处的转移瘤, 实验组: 对照组为4/6: 2/5, 分别取两组肝、肺切片, 发现实验组有2个肺转移, 而对照组未发现. 免疫组化结果示, H. pylori感染组MMP-2, MMP-3, TIMP-2, TIMP-3蛋白的表达阳性率分别为: 77.8±9.6%, 64.5±6.9%, 57.6±12.2%, 40.0±9.2%, 而未感染组分别为61.2±9.7%, 53.1±5.8%, 54.3±10.9%, 53.0±6.6%, 二者比较除TIMP-3蛋白的表达无显著性差异外, MMP-2、MMP-3、TIMP-2蛋白的表达均有显著性差异(P<0.05).

3 讨论

近年来随着H.pylori感染与胃癌的关系的研究深入, 发现H.pylori感染使与转移相关的C-Ha-ras基因发生突变、IL-8、钙连接素及TNF等表达增加[15,16], 将H. pylori与AGS细胞一起培养后, 可引起AGS细胞分泌MMP-3、TIMP-3增加[17], 推测H. pylori感染可能通过引起MMPs、TIMP表达的改变而参与胃癌的转移过程. 因此推测H.pylori感染与胃癌转移过程有关. H. pylori感染不仅能诱导胃癌细胞凋亡, 而且能促进胃癌细胞增生[18-30]. H. pylori低浓度能促进细胞增生的作用, 为动物转移试验提供了基础. 本实验用低转移的胃癌细胞株与H. pylori共同培养后接种裸鼠, 结果显示, 低浓度H.pylori感染胃癌细胞后可能引起胃癌细胞生长转移能力的增强, 成瘤大小与对照组相比有差异, 且大体转移数及肺转移亦增多, 为进一步的致癌机制研究提供了很好的体外模型.

为进一步研究H.pylori促胃癌转移的机制, 本研究用免疫组织化学的方法检测了H.pylori感染组和未感染组裸鼠移植瘤中转移相关因子MMP-2、MMP-3、TIMP-2及TIMP-3蛋白的表达.结果显示: H.pylori感染组MMP-2、MMP-3蛋白表达增高、TIMP-2蛋白表达降低, 与未感染组相比有显著性差异(P<0.05). 因TIMP-2除了能选择性地与MMP-2形成复合物外, 还可抑制金属蛋白酶家族所有成员的水解活性, 故可出现MMP-2、MMP-3蛋白表达均增高, 而只有TIMP-2蛋白表达降低的趋势. 已有研究发现, MMP-2的激活与胃癌进展密切相关, 可作为胃癌的预后因素; H.pylori感染可引起胃癌细胞MMP-3的分泌增加及活性增强[17]. 而TIMP-2过度表达, 能抑制转移性ras转化小鼠胚胎成纤维细胞裸鼠静脉注射后肺转移灶的形成, 以及体内肿瘤的生长速度和癌细胞的浸润特性[31]用TIMP-2基因转染胃癌细胞系SGC-7901, 可减弱肿瘤细胞的侵袭[32]. 临床研究亦证实MMP-2、MMP-3表达增加、TIMP-2表达降低与胃癌的转移及预后密切相关, MMP-2、MMP-3和TIMP-2在胃癌中的负相关作用作为整体调节胃癌细胞的浸润转移, 可能具有重要的预后意义[33,34]. 本结果提示: H.pylori感染与胃癌的转移有关, H.pylori感染时通过调节MMPs及TIMP的表达, 可能是其参与胃癌转移过程的机制之一.

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