幽门螺杆菌 H. pylori Open Access
Copyright ©The Author(s) 2004. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2004-03-15; 12(3): 630-634
在线出版日期: 2004-03-15. doi: 10.11569/wcjd.v12.i3.630
幽门螺杆菌对胃上皮细胞Cox-2表达与凋亡的影响
余琴, 刘南植, 龚建平
余琴, 刘南植, 华中科技大学同济医学院附属同济医院消化内科 湖北省武汉市 430030
龚建平, 华中科技大学同济医学院附属同济医院普外科 湖北省武汉市 430030
余琴, 女, 1977-04生, 湖北省宜昌市人, 汉族, 1998年湖北医科大学本科毕业, 2003年华中科技大学同济医学院硕士毕业, 主要从事胃癌的研究.
通讯作者: 刘南植, 430030, 湖北省武汉市汉口解放大道1095号, 华中科技大学同济医学院附属同济医院消化内科.
电话: 027-83663611
收稿日期: 2003-09-15
修回日期: 2003-09-25
接受日期: 2003-11-13
在线出版日期: 2004-03-15

目的: 研究Cox-2在胃癌细胞中及胃黏膜组织中表达的意义, 探讨其表达与胃上皮细胞凋亡的关系.

方法: 将粗制H. pylori总蛋白与胃上皮细胞株MKN28, AGS共同孵育, 用RT-PCR及免疫组化染色法测定孵育前后细胞Cox-2表达的情况, 同时检测了40例患者胃镜活检胃黏膜病变标本的Cox-2蛋白的表达及H. pylori感染情况. 用流式细胞术观察H. pylori、选择性Cox-2抑制剂NS-398及二者共同诱导细胞凋亡的情况.

结果: 与H. pylori孵育后的MKN28细胞株中Cox-2表达增加; Cox-2蛋白在与H. pylori共同孵育前后的MKN28细胞株中表达强度分别为0.26和0.40(P<0.05), AGS细胞中为0.29和0.31(P>0.05); Cox-2在胃癌中的表达明显高于浅表性胃炎(CSG)、萎缩性胃炎(CAG)组(P<0.05). 流式显示, 与H. pylori孵育24, 48 h MKN28细胞凋亡率分别为1.0%和5.7% (P<0.01); 与10, 100, 200 mol/L NS-398孵育24 h及48 h的MKN28细胞凋亡率分别为1.2%, 14.0%, 27.5%及1.5%, 31.2%, 51.8%, 具有浓度、时间依赖性(P<0.01). 与H. pylori, NS-398共同孵育24, 48h的MKN28细胞凋亡率分别为12.2%, 25.0%, 其凋亡率低于单用NS-398 (P<0.01).

结论: H. pylori感染上调MKN28细胞中Cox-2的表达; Cox-2基因可抑制细胞凋亡, 在胃癌发生发展过程中起重要作用, 可能为胃癌形成的机制之一.

关键词: N/A
引文著录: 余琴, 刘南植, 龚建平. 幽门螺杆菌对胃上皮细胞Cox-2表达与凋亡的影响. 世界华人消化杂志 2004; 12(3): 630-634
Cyclooxygenase-2 expression in gastric mucosal cells with H. pylori infection and its relationship with apoptosis
Qin Yu, Nan-Zhi Liu, Jian-Ping Gong
Qin Yu, Nan-Zhi Liu, Department of Gastroenterology, Tongji Hospital, Tongji Medical College, Hua Zhong University of Science and Technology, Wuhan 430030, Hubei Province, China
Jian-Ping Gong, Department of General Surgery, Tongji Hospital, Tongji Medical College, Hua Zhong University of Science and Technology, Wuhan 430030, Hubei Province, China
Correspondence to: Dr Nan-Zhi Liu, Department of Gastroenterology, Tongji Hospital, Tongji Medical College, Hua Zhong University of Science and Technology, 1095 Jie Fang Avenue, Wuhan 430030, Hubei Province, China
Received: September 15, 2003
Revised: September 25, 2003
Accepted: November 13, 2003
Published online: March 15, 2004

AIM: To investigate the expression of Cox-2 in MKN28 and AGS gastric cells and gastric mucosal lesions with H. pylori infection, to determine whether Cox-2 gene expression by H. pylori infection could influence gastric cell apoptosis and to identify the relationship between Cox-2 and gastric carcinoma.

METHODS: The total H. pylori proteins of various concentrations were incubated with MKN28 and AGS gastric cells in vitro. RT-PCR and S-P immunohistochemical staining were used to detect the expression of Cox-2 before and after the incubation. 40 patients who underwent endoscopy were detected with S-P method. Apoptosis induced by H. pylori or selective Cox-2 inhibitor NS-398 or both was characterized by cell cycle kinetics with flow cytometry.

RESULTS: Expression of Cox-2 in MKN28 gastric mucosal cells incubated with H. pylori was significantly higher than that in non-incubated cells. Expression of Cox-2 protein in MKN28 gastric cells before and after incubated with H. pylori was 0.26 and 0.40 respectively (P<0.05), but in AGS gastric cells, the expression of Cox-2 protein was 0.29 and 0.31 before and after the incubation (P>0.05). Expression of Cox-2 protein in gastric carcinoma (GC) was higher than that in chronic superficial gastritis (CSG) and chronic atrophic gastritis (CAG) (P<0.05). The apoptosis rate when cells were incubated with H. pylori for 24h and 48h was 1.0% and 5.7% (P<0.05). Apoptofic cells were also observed after treated with 10, 100, 200 mol/L NS-398 for 24 h and 48 h, and apoptosis rate was 1.2%, 14.0% and 27.5%, and 1.5%, 31.4% and 51.8% respectively. However, the apopotosis induced by H. pylori and NS-398 was lower than that induced by NS-398 alone (P<0.01).

CONCLUSION: H. pylori upregulates Cox-2 expression in MKN28 gastric mucosal cells in vitro. Cox-2 can inhibit the apoptosis, which may promote gastric carcingenesis.

Key Words: N/A
0 引言

幽门螺杆菌(Helicobacter pylori, H. pylori)作为I类致癌因子, 已得到公认[1-4], 但尚无证据阐明H. pylori感染如何引起胃癌的发生. H. pylori感染可诱导Cox-2的表达[5-8], 并被认为是H. pylori感染增加胃癌发生危险性的可能机制之一. Cox-2过表达可引起局部产生较多的前列腺素(PGs), 同时他可作为分化和生长因子, 发挥类似免疫抑制剂及血管合成药物样作用, 促进肿瘤的增生发展[9-12]. 研究表明, NSAID药物的使用可以降低胃肠道肿瘤的发生[13-16], NSAID的作用靶点是Cox, 即花生四烯酸转化成前列腺素代谢中的限速酶, 其至少有两种亚型, Cox-1是一种看家基因, 其产生的前列腺素与胃肠道黏膜的完整性相关; Cox-2则是一种早期诱导基因, 其与炎症及肿瘤发生相关. 为此, 研究了H. pylori感染胃上皮细胞及胃黏膜病变Cox-2表达情况及其细胞生物学行为, 观察H. pylori, 不同浓度的选择性Cox-2抑制剂NS-398及二者共同诱导细胞凋亡的情况, 并对其凋亡的动力学及机制进行探讨.

1 材料和方法
1.1 材料

人胃癌细胞MKN28(管状腺癌)由上海第二医科大学惠赠, AGS细胞(腺癌)购于中科院上海细胞生物研究所. 胃镜活检40例作胃黏膜组织学检查, 其中慢性浅表性胃炎(CSG)7例, 慢性萎缩性胃炎(CAG)7例, 肠化(IM)10例, 异型增生(Dys)6例, 胃癌(GC)10例, 其中低分化腺癌2例, 高分化腺癌2例, 黏液细胞癌4例, 印戒细胞癌2例.

1.2方法

人胃腺癌细胞系MKN28, AGS生长于RPMI1640培养液中, 按贴壁细胞传代培养法, 胰酶消化每2-3 d传代1次. 自胃溃疡患者活检胃黏膜组织中分离H. pylori, 37 ℃微需氧环境(50 mL/L O2, 100 mL/L CO2)培养. 将其超声粉碎(60 A×5 min ×3次, 间隔15 s), 20 000 r/min离心20 min, 取上清. 调整细胞浓度为1×109/L, 加入相当于1011CFu/L活菌量的H. pylori粗制总蛋白, 再加入RPMI1640 2 mL, 放入37 ℃孵育24 h, 以不加H. pylori的等体积的培养液为对照. RT-PCR: Cox-2上游引物序列: 5'-TCTGGTGCCTGGTCTGATGATGTA-3'; 下游引物序列为: 5'-CAGAAGGGGATGCCAGTGATAGAG-3'. 用Trizol法提取总RNA, 并进行逆转录. 待细胞与不同浓度的粗制H. pylori总蛋白 (相当于109-1011CFu/L的活菌量)孵育24 h后, 用SP法检测Cox-2蛋白的表达. 同法检测40例胃镜标本中Cox-2蛋白表达的情况. 调整细胞浓度为1×109/L, 加入相当于1011CFu/L活菌量的H. pylori, 不同浓度的NS-398(10, 100, 200 mol/L)及H. pylori +NS-398 (10, 100, 200 mol/L), 以不加H. pylori及NS-398的空白细胞为对照. 分别于培养24, 48 h后, PI染色(含Rnase 100 mg/L)上机测定凋亡细胞百分率及细胞同期.

统计学处理 所有实验数据以mean±SD表示. 采用t检验和精确概率法检验, 率的显著性差异检验选用方差分析, P<0.05认为差异有显著性.

2 结果
2.1 MKN28细胞Cox-2表达

MKN28细胞的RT-PCR产物经电泳分析, 可见1条约314 bp的扩增带, 特异性好, 阴性对照无相应条带出现. 在与H. pylori总蛋白孵育前后的平均Cox-2/-actin为0.2698±0.0124和 0.6720±0.0206, 提示Cox-2mRNA在孵育后的细胞中表达水平增强(P<0.05, 图1). MKN28细胞分别与相当于1011, 1010, 109CFu/L的H. pylori粗制总蛋白孵育24 h的平均吸光度为0.40±0.13, 0.40±0.08和0.30±0.14; 未与H. pylori孵育的MKN28Cox-2表达的平均吸光度为0.26±0.18, 与1011, 1010CFu/L H. pylori孵育后Cox-2的表达相差显著(P<0.05)(图2, 3).

图1
图1 MKN28细胞Cox-2mRNA表达. A, B: 未与H. pylori孵育; C: 空白对照; D: 与H. pylori孵育后.
图2
图2 未与幽门螺杆菌孵育×100.
图3
图3 与幽门螺杆菌孵育后×400.
2.2 AGS细胞Cox-2表达

AGS细胞未与H. pylori孵育及与相当于1011, 1010, 109CFu/L的H. pylori粗制总蛋白孵育24 h的平均吸光度分别为0.29±0.22, 0.30±0.19, 0.32±0.24, 0.31±0.15 (P>0.05)(图4, 5).

图4
图4 未与幽门螺杆菌孵育×400.
图5
图5 与幽门螺杆菌孵育×400.
2.3胃黏膜Cox-2蛋白表达

CSG, CAG, IM, Dys及GC中Cox-2表达呈平行递增趋势, GC与CSG, CAG中的表达差异有显著性(P<0.05, 表1). Cox-2主要表达在胃癌细胞中, 血管平滑肌细胞, 成纤维细胞, 炎性单核细胞及肠化上皮, 不典型增生腺上皮细胞亦表达, 胞质显色, 弥漫性分布; 胃炎, IM, Dys中H. pylori感染率与GC中H. pylori感染率差异有显著性(P<0.05)(图6-12).

表1 胃黏膜Cox-2蛋白的表达及H. pylori感染率.
病变nCox-2表达H. pylori感染n(%)
GC1010 (100)0 (0.0)
Dys65 (83.3)4 (66.7)
IM108 (80.0)6 (60.0)
CAG74 (57.1)6 (85.7)
CSG72 (28.6)6 (85.7)
图6
图6 CSG×100.
图7
图7 CAG×200.
图8
图8 IM×200.
图9
图9 DYS×200.
图10
图10 腺癌×100.
图11
图11 黏液细胞癌×100.
图12
图12 印戎细胞癌×100.
2.4 MKN28细胞凋亡

H. pylori可诱导MKN28细胞的凋亡, 且具有时间依赖性(表2), NS-398亦可诱导MKN28细胞的凋亡, 具有时间、剂量依赖性(表3), H. pylori+NS-398对MKN28细胞凋亡, 也具有时间、剂量依赖性(表4), 但其凋亡率低于单纯NS-398组.

表2 H. pylori诱导MKN28细胞的凋亡(%).
凋亡率G0/G1SG2/M
对照0.4±0.250.8±2.127.6±1.821.8±1.6
24 h1.0±0.358.7±2.519.3±1.621.5±0.9
48 h5.7±1.1b58.3±1.917.2±2.119.2±1.4
bP<0.01 vs 对照组.
表3 NS-398诱导MKN28细胞的凋亡(%).
NS-398凋亡率G0/G1SG2/M
24 h
对照0.5±0.351.1±2.629.5±1.619.9±2.8
10 mol/L1.2±0.848.1±3.028.1±2.123.0±1.9
100 mol/L14.0±2.1bd44.8±2.315.5±0.926.6±1.3
200 mol/L27.5±1.5bd34.9±0.85.8±2.232.4±2.3
48 h
对照1.0±0.555.3±3.023.0±1.721.2±2.2
10 mol/L1.5±1.153.5±2.622.9±1.822.7±2.7
100 mol/L31.2±2.5b33.1±2.815.3±0.920.9±2.0
200 mol/L51.8±2.2b25.2±1.48.2±1.915.4±3.0
bP<0.01 vs 对照组, 10 mol/L组;
dP<0.01 vs 48 h 100 mol/L及200 mol/L组.
表4 H. pylori+ NS-398诱导MKN28细胞的凋亡(%).
NS-398凋亡率G0/G1SG2/M
24 h
对照0.5±0.351.1±2.329.5±1.619.9±2.8
100 mol/L1.5±0.8d49.6±2.911.6±2.637.5±3.1
200 mol/L2.2±2.2bd40.4±1.78.4±1.239.5±3.2
48 h
对照1.0±0.555.3±3.023.0±1.721.2±2.2
100 mol/L4.5±1.661.1±3.19.4±0.925.2±2.3
200 mol/L25.0±2.5b30.7±2.417.6±0.527.3±2.2
bP<0.01 vs 对照组, H. pylori+ 100 mol/L组;
dP<0.01 vs 48 h H. pylori+ 100 mol/L及H. pylori+200 mol/L组.
3 讨论

H. pylori感染与GC发生关系密切[17-23], 而Cox-2是黏膜炎症和上皮细胞生长的重要调节物, 该基因表达是对H. pylori感染直接的反应[5-6,24-27]. 因此, Cox-2的表达可能参与了H. pylori相关胃炎向癌前病变和胃癌的演变过程. 我们发现, H. pylori感染可上调MKN28细胞中Cox-2的表达, 且具有浓度依赖性, 当H. pylori小于或等于109CFu/L时, 对MKN28 Cox-2的表达并无明显影响; H. pylori对AGS细胞中Cox-2表达无明显影响, 这可能与不同胃上皮细胞中Cox-2启动子甲基化水平相关. Akhtar et al[28]研究了H. pylori感染的胃上皮细胞中Cox-2启动子甲基化对Cox-2表达及其活性的影响. MKN28细胞中的Cox-2启动子是未甲基化的, 而AGS细胞中Cox-2启动子是甲基化的. 用H. pylori刺激Cox-2启动子未甲基化的MKN28细胞, 其Cox-2表达明显增加, 而用H. pylori刺激Cox-2启动子甲基化的AGS细胞, 其Cox-2表达无明显增加. 但当用H. pylori感染经去甲基化药物处理后的AGS, Cox-2表达呈5-10倍地增加, 表明Cox-2启动子甲基化的缺失可能促进了Cox-2表达和H. pylori感染诱导胃癌的发生. 胃上皮细胞Cox-2启动子甲基化的缺失可能H. pylori感染诱发胃癌的一个中心事件, 可促进Cox-2表达导致细胞凋亡和增生的失衡[29-31].

本结果表明, 从CSG→CAG→IM→Dys→GC, Cox-2表达率增加, 可能为胃癌形成的早期事件. CSG, CAG, IM, Dys中H. pylori感染率与GC中H. pylori感染率差异有显著性, 这与胃癌所致胃内H. pylori生存环境改变有关. 如果Cox-2为胃肿瘤形成的早期事件, 则胃内炎症及H. pylori感染为胃癌的更早期事件. H. pylori感染致炎症, 产生大量的炎性因子激活Cox-2, 这可能为诱发癌变的机制之一. Cox-2除了使细胞凋亡、增生失衡外, 还可促进肿瘤细胞相关血管的生成, 增加癌细胞的侵袭性, 激活基质金属蛋白酶2降解细胞外基质, 产生促血小板凝集的血栓烷等, 从而有助于肿瘤的侵袭和转移[32]. 在体外, H. pylori粗制总蛋白可诱导细胞凋亡, 并且孵育时间越长, 细胞凋亡越多. 选择性Cox-2抑制剂NS-398能有效地促进MKN28细胞的凋亡, 且具有时间、浓度依赖性. 作用24 h时, 其凋亡率不很明显, 但细胞周期明显受阻, 停滞于G2期; 作用48 h时, 其凋亡率明显升高. 不同浓度NS-398对细胞凋亡影响亦不相同. 当用小剂量(10 mol/L)作用时, 细胞凋亡不明显, 细胞周期受阻也不明显, 提示10 mol/L NS-398并不能有效抑制Cox-2, 诱导细胞凋亡; 而100, 200 mmol/LNS-398使MKN28细胞凋亡明显增加. 我们还发现, 尽管H. pylori或NS-398单因素均可促进MKN28细胞的凋亡, 但二者同时作用于细胞时, 并未出现凋亡增加的情况, 相反, 其凋亡率竟远低于单纯用药组, 但其G2阻滞明显增加. 可能是由于H. pylori感染上调了细胞Cox-2的表达, 而Cox-2促进了细胞的增生, 抑制了凋亡, 所以加同浓度的Cox-2抑制剂时, 其凋亡少于仅加同剂量的用药组.

总之, 我们认为H. pylori感染增加胃癌发生的危险性, 可能与其上调Cox-2表达及其相关事件有关. 选择性Cox-2抑制剂的上市可望为胃癌的NSAID化学预防提供有利优势[33-34].

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