GSTM1, GSTT1基因多态与胃腺癌及幽门螺杆菌感染的关联

摘要

目的

探讨GSTM1, GSTT1基因多态与胃腺癌(GAC)临床特征及其与幽门螺杆菌(Hp)感染的相关性.

方法

应用多重聚合酶链反应(PCR)技术, 检测GSTM1, GSTT1基因多态性; ¹⁴C尿素呼气试验和外周血ELISA法检测Hp感染情况.

结果

GAC患者GSTM1空白基因型频率明显高于健康人, 其差异有统计学意义(x² =5.40, P <0.05), GSTT1空白基因型频率在GAC患者与对照组间的差异无统计学意义; GSTM1空白基因型频率在早期GAC患者与对照组间的差异有统计学意义(x² =4.74, P <0.05), 在进展期、肠型及弥漫型GAC与对照组间的差异均无统计学意义; GSTT1空白基因型频率在肠型GAC患者与对照组间的差异有统计学意义(x² =4.09, P <0.05), 在早期, 进展期及弥漫型GAC与对照组间的差异均无统计学意义. GSTM1, GSTT1均为空白基因型的个体患GAC的危险性是GSTM1, GSTT1均为非空白基因型个体的3.38倍(校正P=3.38, 95 % CI=1.58- 7.51). GSTM1空白基因型频率, 在Hp感染的GAC患者与Hp感染的对照组间的差异有统计学意义(x² =6.68, P <0.01).

结论

GSTM1空白基因型与GAC易感性有关, 肿瘤多处于早期, GSTT1空白基因型与肠型GAC易感性有关, 与肿瘤的临床分期无关; GSTM1, GSTT1均为空白基因型的个体患GAC的危险性增加; GSTM1基因多态和Hp感染有协同促GAC发生的作用.

关键词: N/A

Association of glutathione S-transferase M1 and T1 genetic polymorphisms with Helicobacter pylori infection and gastric adenocarcinoma

You-Cai Zhang, Chang-Sheng Deng, Yan Zhou, You-Qing Zhu

You-Cai Zhang, Chang-Sheng Deng, Yan Zhou, You-Qing Zhu, Department of Gastroenterology, Zhongnan Hospital, Wuhan University, Wuhan 430071, Hubei Province, China

Correspondence to: You-Cai Zhang, Department of Gastroenterology, Zhongnan Hospital, Wuhan University, Wuhan 430071, China. zhayc0701@163.com

Received: November 12, 2002
Revised: November 20, 2002
Accepted: December 3, 2002
Published online: September 15, 2003

Abstract

AIM

To study the association of genetic polymPphisms of glutathione S-transferase M1 and T1 (GSTM1, GSTT1) with clinicopathological features of gastric adenocarcinoma(GAC) and Helicobacter pylPi (Hp) infection.

METHODS

All subjects were unrelated Han people in Hubei Province of China. Using multiplex PCR, we studied the genetic polymPphisms of the GSTM1, GSTT1 genes. Hp infection was determined by IgG antibodies to Hp in stPed serum samples using enzyme-linked immunosobant assay and 14C urea breath test.

RESULTS

The null genotype fP GSTM1 was mPe significantly common in GACs when compared with controls (x²=5.40, P <0.05), and mPe common in early stage of GACs when compared with controls too (x²=4.74, P <0.05). All the differences of the frequency of GSTM1 null genotype between advanced stage GACs P intestinal type carcinomas P diffuse type carcinomas and controls did not reach statistical significance. The null genotype fP GSTT1 was significantly mPe common among intestinal type GACs when compared with controls (x²=4.09, P <0.05), but all the differences of the frequency of GSTT1 null genotype between early stage GACs P advanced stage GACs P diffuse type carcinomas and controls did not reach statistical significance. The subjects carrying both of the null genotypes fP GSTM1 and GSTT1 had mPe than 3.38-fold risk fP developing GAC compared with the subjects carrying both of the nonull genotypes fP GSTM1 and GSTT1 (adjusted odds ratio, P=3.38,95 % confidence interval, CI=1.58-7.51). The null genotype fP GSTM1 was mPe significantly common among those patients with Hp positive GAC compared with Hp positive controls (x²=6.68, P <0.01).

CONCLUSION

The null genotype fP GSTM1 has an increased risk of GAC, and most tumPs are in early stage GACs. The null genotype fP GSTT1 is significantly related to the intestinal type GAC,but not significantly related to the tumP stage. Subjects carrying both of the null genotypes fP GSTM1 and GSTT1 have increased risks fP GAC, GSTM1 gene polymPphisms and Hp infection may interact with each other in the initialization of GAC.

Key Words: N/A

0 引言

幽门螺杆菌(Helicobacter pylPi, Hp)感染是胃癌发生的高危因素[1-12], 然其确切的致癌机制并未明了. 谷胱甘肽S-转移酶(glutathione S-transferase, GST) M1, T1是II相毒物代谢酶的重要成员, 广泛存在多态性, 具GSTM1, GSTT1基因多态的个体, 其酶活性下降, 对进入体内的前致癌物降解能力下降, 机体患肿瘤的危险性增加[13-21]. 我们探讨GSTM1, GSTT1基因多态与胃腺癌(gastric adenocarcinoma, GAC)及其与Hp感染的相关性.

1 材料和方法

1.1 材料

武汉大学中南医院肿瘤科经病理组织学确诊的GAC患者127例, 其中早期GAC39例, 进展期88例; 肠型GAC76例, 弥漫型51例. 排除近期有抗生素及非甾体抗炎药服用史者; 合并有食管或肠道恶性病变者; 有胃手术史及胃癌家族史者. 对照组114例, 为同期在本院体检的无血缘关系的健康人. 研究对象均为湖北地区汉族人, 所有研究对象均行¹⁴C尿素呼气试验和血清抗Hp-IgG抗体检测, 二者均为阳性者, 确定为Hp感染阳性.

1.2 方法

GSTM1, GSTT1基因型检测(多重PCR法)以研究对象外周血提取的基因组DNA为模板, 行多重PCR检测. 扩增GSTM1的引物序列为5'-GAACT CCCTGAAAAGCTAAAGC-3'和5'-GTTGGGCTCAAATATACGGTGG-3', 扩增GSTT1的引物序列为5'-TTCCTTACTGGTCCTCACATCTC-3'和5'-TCACCGGATCATGGCCAGCA-3', 同时扩增b-globulin为内参照, 所用引物为5'-CAACTTCATCCACGTTCACC-3'和5'-GAAGAGCCAAGGACAGGTAC-3'. 反应条件为: 97 °C预变性5 min, 进入热循环后, 95 °C变性45 s, 57 °C退火45 s, 72 °C延伸60 s, 循环35次后, 72 °C再延伸5 min. 反应终止后经80 g/L聚丙烯酰胺凝胶电泳(PAGE)(扩增GSTT1时退火温度为55 °C, 用50 g/L PAGE), 1.8 g/L硝酸银染色分析电泳结果(图1, 2: GSTM1非空白基因型的PCR产物长230 bp, GSTT1非空白基因型的PCR产物长480 bp, b-globulin的PCR产物长268 bp).

图1 1: pBR322/HaeIII markers DNA分子质量标志物; 3, 4, 7: GSTM1空白基因型GSTM1基因和b-globulin基因PCR扩增产物PAGE图.

图2 1: pBR322/HaeIII markers DNA分子质量标志物; 2, 3: GSTT1非空白基因型. GSTT1基因和b-globulin基因PCR扩增产物PAGE图.

统计学处理 以x²检验分析GSTM1, GSTT1空白基因型频率差异, 以非条件Logistic回归法计算表示相对危险度的比值比(odds ratio, P)及95 %可信区间(confidence interval, Cl). P值均以潜在的混淆因素如年龄、性别等校正.

2 结果

GSTM1空白基因型在GAC和对照组中的频率分别为61.4 %和46.5 %, 其差异有统计学意义(校正P=1.80, 95 % CI=1.08-3.10, x²=5.40, P <0.05, 表1), GSTT1空白基因型在二者间的频率差异无统计学意义; GSTM1 空白基因型在早期、进展期、肠型及弥漫型GAC患者中的频率分别为66.7 %, 59.1 %, 60.5 %和62.7 %, GSTT1的频率分别为61.5 %, 59.1 %, 63.2 %和54.9%, 与对照组比较, 早期GAC患者GSTM1频率的差异有统计学意义(校正P=2.26, 95 % CI=1.05-4.90, x² =4.74, P <0.05), 肠型GAC患者GSTT1空白基因型的频率差异也有统计学意义(校正P=1.80, 95 % CI=1.78-3.38, x² =4.09, P <0.05). 提示, GSTM1空白基因型与GAC易感性有关, 肿瘤多处于早期, GSTT1空白基因型与肠型GAC易感性有关, 与肿瘤有临床分期无关.

表1 GSTM1, GSTT1空白基因型与GAC及其临床病理特征关联.

分组	n	M/F	年龄(mean±SD)	GSTM1空白基因型n(%)	校正P (95 % CI)	GSTT1空白基因型n (%)	校正P (95 % CI)
对照组	114	78/36	54?/FONT>10	53 (46.5 %)		55 (48.2 %)
GAC	127	89/38	55?/FONT>9	78 (61.4 %)a	1.80 (1.08-3.10)	76 (59.8 %)	1.58 (0.92-2.86)
早期	39	32/7	54?/FONT>11	26 (66.7 %)b	2.26 (1.05-4.90)	24 (61.5 %)	1.70 (0.80-3.62)
进展期	88	57/31	57?/FONT>9	52 (59.1 %)	1.62 (0.90-2.91)	52 (59.1 %)	1.52 (0.82-2.75)
弥漫型	51	38/13	56?/FONT>12	32 (62.7 %)	1.92 (0.93-3.84)	28 (54.9 %)	1.27 (0.65-2.56)
肠型	76	51/25	54?/FONT>9	46 (60.5 %)	1.75 (0.95-3.20)	48 (63.2 %)c	1.80 (1.78-3.38)

^aP<0.05, ^bP<0.05, ^cP<0.05 vs对照组

在GAC组, a联合型(GST M1/GSTT1非空白基因型)频率为15.7 %, b 联合型(GSTM1非空白基因型/GST T1空白基因型)频率为18.9 %, c 联合型(GSTM1空白基因型/GSTT1非空白基因型)频率为27.6 %, d 联合型(GSTM1, T1均为空白基因型)频率为37.8 %; 对照组分别为28.9 %, 27.2 %, 23.7 %和20.2 %(表2). 将a联合型视为暴露因素, 则b, c, d联合型的个体患GAC的危险性分别上升1.24倍(校正P=1.24, 95 % CI=0.55-2.84), 2.10倍(校正P=2.10, 95 % CI=1.00-4.80)和3.38倍(校正P=3.38, 95 % CI=1.58-7.51).可认为:GSTM1, GSTT1均为空白基因型的个体患GAC的危险性显著增加, 该联合基因型为GAC的易感基因型.

表2 胃腺癌GSTM1, GSTT1基因联合多态分析n (%).

分型	对照组(114)	GAC(127)	校正P(95 % CI)
a型: GSTM1, GSTT1均
为非空白基因型	33 (28.9 %)	20 (15.7 %)
b型: GSTM1非空白基因型
/ GSTT1空白基因型	31 (27.2 %)	24 (18.9 %)	1.24 (0.55-2.84)
c型: GSTM1空白基因型
/GST T1非空白基因型	27(23.7 %)	35 (27.6 %)e	2.10 (1.00-4.80)
d型: GSTM1, GSTT1均
为空白基因型	23 (20.2 %)	48 (37.8 %)f	3.38 (1.58-7.51)

^eP <0.05, x²=4.01,

^fP <0.01, x²=10.05 vs a组.

GAC患者Hp检出率为70.1 % (89), 对照组为61.4 % (70), 其检出率差异无统计学意义; GSTM1空白基因型的频率在Hp阳性GAC患者、Hp阳性对照组间的差异有统计学意义(66.3 % vs 45.7 %, 校正P=2.30, 95 % CI=1.20-4.78, x²=6.68, P<0.01), GSTT1空白基因型的频率在二者间的差异无统计学意义(68.5 % vs 52.9 %, 校正P=1.70, 95 % CI=0.85-3.52, x² =2.76, P >0.05), 具GSTM1空白基因型的个体感染Hp后, 患GAC的危险性上升了2.3倍. 提示, GSTM1基因多态、Hp感染间有协同促进GAC发生的作用.

3 讨论

GST和谷胱甘肽(GSH)在人消化道上皮细胞中广泛存在, 是多种潜在致癌物如亚硝胺类混合物、苯并芘等的重要解毒系统[22]. GSTM1, GSTT1互为同功酶, 均存在空白基因型, 其空白基因型的个体在体内不能表达GSTM1, GSTT 1蛋白, 对上述致癌物的降解能力下降, 肿瘤易感性增加. 近年, GSTM1, GSTT1基因多态与GAC易感性关联的结论尚有分歧[13,19,23]. Saadat et al [13]报道, GSTM1空白基因型的个体GAC的危险性增加(P =2.3, 95 % CI =1.2-5.0), GSTT1空白基因型与GAC的易感性无关, 具GSTM1, GSTT1均为空白基因型的个体患GAC的危险性上升1.3倍. 本实验结果显示, GSTM1空白基因型与GAC易感性有关, 肿瘤多处于早期, 而GSTM1, GSTT1均为空白基因型的个体患GAC的危险性是GSTM1, GSTT1均为非空白基因型个体的3.38倍, GSTT1空白基因型与肠型GAC易感性有关, 与肿瘤的临床分期无关.此与Saadat et al [13]的结论相似.

大量的临床、病理学和血清学的调查结果在一定程度上肯定了Hp与胃癌发生的相关性. 然胃癌的发生是多因素的结果, Hp感染本身可能以一种促癌剂的形式存在[24,25]. Hp阳性患者胃黏膜GSTs活性及GSH含量均低于Hp阴性的患者(P <0.001), 认为慢性Hp感染减少胃黏膜GSH的含量, 降低GST 活性, GST解毒能力下降, 导致胃癌的危险性增加[26]. Hp阳性GAC患者GSTM1空白基因型频率显著高于Hp阳性的对照组(P =0.031), 认为Hp感染可增加GSTM1空白基因型个体患GAC的危险性[27]. 本结果也证实GSTM1基因多态、Hp感染有协同促GAC发生的作用. 而Hp感染率在GAC及对照组之间的差异无显著性, 可能与样本量较小, 或有低毒力菌株混杂有关.

Hp所含的空泡毒素、尿素酶、磷脂酶等物质, 损伤胃黏膜上皮细胞, 破坏胃黏膜屏障, 进入胃黏膜上皮内的致癌物含量增加[6,28,29] ; Hp感染后, 黏膜内炎性细胞浸润并释放炎性递质、氧自由基等, 引起细胞炎症反应、增生加快, 易受到基因毒致癌物的损伤; 而GST空白基因型的个体, 对致癌物降解能力下降, 致癌物与细胞内生物大分子形成加成物, 导致肿瘤相关基因活化或失活, 终致肿瘤形成[3,9,30,31]. 这可能是GSTM1空白基因型的个体, Hp感染后胃腺癌危险性增加的原因.

备注

$[Footnotes]

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