大肠黏膜癌变过程中PGE2, Bcl-2及Bax表达相关性

摘要

目的: 探讨PGE₂, Bcl-2及Bax在大肠黏膜癌变过程中表达相关性.

方法: 采用免疫组织化学DAB法检测了正常结肠黏膜15例、慢性结肠炎20例、大肠癌旁组织50例、腺瘤样息肉30例及大肠癌组织50例中PGE₂,Bcl-2及Bax蛋白的表达.分析PGE₂、Bcl-2及Bax的表达与各种临床病理特征的关系及三者的相关性.

结果: PGE₂在正常结肠黏膜,慢性结肠炎,大肠癌旁组织,腺瘤样息肉及大肠癌组织中的表达阳性率分别是6.67%,10.0%,40.0%,56.7%,90.0%, 呈递增趋势.Bcl-2在5组中的表达阳性率分别为6.67%,10.0%,38.0%,46.7%,76.0%, 呈递增趋势.Bax在5组中的表达阳性率分别为86.67%,75.0%,78.0%,76.7%,82.0%, 五组之间阳性率比较均无显著性差异(P>0.05), 但其阳性表达程度在正常组织,腺瘤样息肉,大肠癌三组之间呈递减趋势(P<0.05).3者在大肠癌中的表达与患者性别、年龄、肿块大小无明显差异(P>0.05).PGE₂和Bcl-2、Bax和Bcl-2在大肠癌组织中阳性反应呈显著正相关(r = 0.532, P<0.05; r = 0.653, P<0.05).PGE₂、Bcl-2及Bax在大肠癌高、中分化组中的表达率分别为100%, 88.9%, 85.2%, 明显高于低分化组中的79.3%、60.9%、78.3%; 在大肠癌Duke's A, B期中Bcl-2表达却显著高于C, D期.

结论: PGE₂及Bcl-2在正常结肠黏膜、慢性结肠炎、大肠腺瘤样息肉、大肠癌组织中的表达均呈递增趋势, 而Bax在正常结肠黏膜、大肠腺瘤样息肉、大肠癌组织中的阳性表达程度呈递减趋势提示PGE₂及Bcl-2表达上调和Bax表达下调与大肠黏膜癌变过程有关.PGE₂在大肠癌中的表达与肿瘤分化程度有关, Bcl-2在大肠癌中的表达与肿瘤分化程度、Duke's分期有关.PGE₂与Bcl-2的表达呈正相关.

关键词: PGE₂; Bcl-2; Bax; 大肠癌

Expression of PGE2, Bcl-2 and Bax in carcinogenesis of colorectal mucosa

Miao Ouyang, Gui-Ying Zhang, Mei-Hua Xu

Miao Ouyang, Gui-Ying Zhang, Mei-Hua Xu, Department of Digestion, Xiangya Hospital, the Central South University, Changsha 410008, Hunan Province, China

Correspondence to: Miao Ouyang, Department of Digestion, Xiangya Hospital, the Central South University, Changsha 410008, Hunan Province, China.

Received: December 30, 2004
Revised: January 15, 2005
Accepted: February 26, 2005
Published online: June 15, 2005

Abstract

AIM: To investigate the expression of PGE₂, Bcl-2 and Bax in the carcinogenesis of colorectal mucosa and the relationship between them.

METHODS: The expression of PGE₂, Bcl-2 and Bax were detected in 15 normal colorectal mucous membrane (NCM), 20 chronic colonitis (CHC), 30 colorectal adenoma (CAA), 50 colorectal cancer (CC)and 50 cancer adjacent (CAT) tissues by DAB immunohistochemical staining. The correlations among PGE₂, Bcl-2 and Bax expression as well as the clinical and pathological characteristics were analyzed.

RESULTS: The rates of PGE₂ expression in NCM, CHC, CAT, CAA and CC tissues were 6.67%, 10%, 40%, 56.7% and 90% respectively and manifested an ascending trend. The rates of Bcl-2 expression in the five groups were 6.67%, 10%, 38%, 46.7%, and 76% respectively and also showed an ascending trend. The rates of Bax expression in the five groups was 86.67%, 75%, 78%, 76.7% and 82% respectively and there was no significant difference among the five groups (P>0.05). However, the positive degree in NCM, CA and CC group exhibited a descending trend (P<0.05). The expressions of PGE₂, Bcl-2 and Bax in human CC were not associated with sex, age and the size of tumor (P>0.05). A positive correlation was noted between expression of PGE₂ and Bcl-2 in CC tissue (r = 0.532, P<0.05). The same correlation also existed between Bax and Bcl-2 (r = 0.653, P<0.05). The rates of PGE₂, Bcl-2 and Bax expression in highly and moderately differentiated CC were significantly higher than those in lowly differentiated one (100%, 88.9%, 85.2% vs 79.3%, 60.9%, 78.3%; P<0.05). The expression of Bcl-2 was significantly higher in Duke's A, B stage than that in Duke's C, D (90%, 77.8% vs 60%, 57.2%; P<0.05).

CONCLUSION: The expression of PGE₂ and Bcl-2 increases while that of Bax decreases with the occurrence and development of CC, which indicates that PGE₂ and Bcl-2 play important roles in the carcinogenesis and development of CC. At the same time, there is a positive relationship between PGE₂ and Bcl-2.

Key Words: PGE₂; Bcl-2; Bax; Colorectal cancer

0 引言

原癌基因Bcl-2是最重要的抑制细胞凋亡的基因[1-3], 他在多种肿瘤组织中均有异常高表达, Bax是促进细胞凋亡的基因, 是细胞凋亡所必需的基因[4-6], 他和Bcl-2的比例与细胞凋亡的敏感性有关.大肠癌常见, 但发病机制不十分清楚, 是分泌PGE₂能力最高的肿瘤[7], 为其他肿瘤的1.7-5.2倍.关于PGE₂的表达高低与大肠癌的病理分型、临床分期及预后的关系, PGE₂在大肠黏膜癌变过程中与Bcl-2及Bax的关系尚未见报道.我们应用免疫组化的方法检测人正常结肠黏膜、慢性结肠炎、大肠癌旁组织、腺瘤样息肉及大肠癌组织中PGE₂, Bcl-2及Bax的表达, 分析其与大肠黏膜病理变化的关系, 以探讨PGE₂, Bcl-2及Bax在大肠癌的发生发展中的作用.

1 材料和方法

1.1 材料

2002-09/11大肠腺癌以及其配对的癌旁组织新鲜手术标本(距肿块边缘5 cm以上)50例.高分化21例, 中分化6例, 低分化23例; Duke's A期10例, B期18例, C期15例, D期7例; 男29例, 女21例, 年龄28岁-78岁.结肠腺瘤样息肉30例, 男16例, 女14例, 年龄24岁-78岁.慢性结肠炎20例及正常结肠黏膜15例, 男20例, 女15例, 年龄23岁-65岁.PGE2单克隆抗体购自华美公司, Bcl-2及Bax单克隆抗体为美国SantaCruze公司产品, S-P试剂盒及DAB显色剂均购自福州迈新公司.

1.2方法

标本均经40 g/L甲醛固定, 石蜡包埋, 5 μm厚连续切片先经多聚赖氨酸浸泡, 60℃烤片2 h.组织切片经二甲苯脱蜡、酒精梯度脱水后, 浸入30 mL/L H₂O₂室温下30 min灭活内源性过氧化物酶活性, 然后置入煮沸的0.01 moL/L枸橼酸盐缓冲液中修复抗原5 min, 冷却至室温后重复.滴加正常山羊血清封闭液覆盖组织, 置于37℃恒温箱中30 min.滴加PGE₂、Bcl-2及Bax一抗37℃孵育30 min, 4℃冰箱过夜.再先后滴加生物素化的二抗及链霉素亲和素-生物素-过氧化物酶复合物溶液, 各置湿盒内37℃温箱内孵育30 min.滴加DAB显色剂, 显微镜下控制显色时间8-10 min.蒸馏水冲洗终止染色, 苏木素液染色1 min, 盐酸酒精分化, 碳酸锂返蓝, 树胶封片.以胃癌PGE₂, Bcl-2及Bax阳性免疫组化染色片为阳性对照, 用PBS代替一抗作阴性对照.光镜下观察组织切片的显色反应.PGE₂, Bcl-2及Bax均以细胞质染色为主, 细胞膜可有少量染色.阳性判断标准为该位置出现了黄色或棕黄色颗粒状, 并用阳性片作为对照.每张切片随机选择5个视野, 40×10高倍显微镜下观察计数.参照文献[3]方法根据染色的浓度及阳性细胞的数量分别记分.不着色为0分, 浅黄色为1分, 黄色为2分, 棕黄色为3分.无阳性细胞染色为0分, 阳性细胞数少于30%为1分, 30-70%为2分, 70%以上为3分.根据染色深度与阳性细胞数得分之和分为4级, 即(-)为0分, 弱阳性(+)为1-2分, 阳性(++)为3-4分, 强阳性(+++)为5-6分.

统计学处理 采用统计学软件SPSS11.0进行χ²检验、相关分析及秩和检验, 取显著性检验水准α = 0.05.

2 结果

2.1 PGE₂、Bcl-2及Bax在大肠黏膜癌变过程中的表达

PGE₂在正常结肠黏膜、慢性结肠炎、大肠癌旁组织、腺瘤样息肉及大肠癌组中的表达阳性率为6.7%、10.0%、40.0%、56.7%、90.0%, 从正常结肠黏膜到癌前病变直至癌的发展过程中PGE₂的表达呈递增趋势(图1), 正常组与结肠炎组、癌旁组与腺瘤样息肉组比较无显著性差异(P>0.05), 而正常组与癌旁组、正常组与腺瘤样息肉组、正常组与大肠癌组、腺瘤样息肉组与大肠癌组、癌旁组与大肠癌组比较均有显著性差异(表1); Bcl-2在5组中的表达阳性率分别为6.7%、10.0%、38.0%、46.7%、76.0%, 从正常结肠黏膜到癌前病变直至癌的发展过程中PGE₂的表达呈递增趋势(图2), 正常组与结肠炎组、癌旁组与腺瘤样息肉组比较无显著性差异(P>0.05), 而正常组与癌旁组、正常组与腺瘤样息肉组、正常组与大肠癌组、腺瘤样息肉组与大肠癌组、癌旁组与大肠癌组比较均有显著性差异(P<0.05, 表1).大肠癌组中DUKES分期的A, B两期bcl-2阳性表达率显著高于腺瘤样息肉组(P<0.05), 而C, D期大肠癌与腺瘤样息肉组间无统计学差异; Bax在5组中的表达阳性率分别为86.7%、75.0%、78.0%、76.7%、82.0%, 五组之间阳性率比较均无显著性差异(P>0.05, 图3), 但其阳性表达程度在正常组织、腺瘤样息肉、大肠癌三组之间呈递减趋势(P<0.05).

表1 PGE₂, Bcl-2及Bax在5组中的表达阳性率及表达程度.

分组	n	PGE2				Bcl-2				Bax
		+	++	+++	阳性率(%)	+	++	+++	阳性率(%)	+	++	+++	阳性率(%)
正常黏膜	15	1	0	0	6.7	1	0	0	6.7	3	8	2	86.7
结肠炎	20	2	0	0	10	2	0	0	10	6	8	3	75
大肠癌旁	50	14	5	1	40	12	5	2	38	18	20	1	78
腺瘤样息肉	30	5	8	4	56.7	10	4	0	46.7	14	19	0	76.7
大肠癌	50	9	26	10	90	8	26	4	76	17	23	1	82

图1 PGE2在大肠组织中的表达程度. A: 正常; B: 癌旁; C: 腺瘤; D: 高分化癌; E: 低分化癌.

图2 Bcl-2在大肠组织中的表达程度. A: 正常; B: 癌旁; C: 腺瘤; D: 高分化癌; E: 低分化癌.

图3 Bax在大肠组织中的表达程度. A: 正常; B: 腺瘤; C: 高分化癌; D: 低分化癌.

2.2 PGE₂, Bcl-2及Bax在大肠癌中的表达

PGE₂在高、中分化组及低分化大肠癌组中的阳性表达率分别为100%、79.3%, Bcl-2在高、中分化组及低分化大肠癌组中的阳性表达率88.9%、60.9%, Bax在高、中及低分化大肠癌中的阳性表达率85.2%、66.7%.PGE₂, Bcl-2及Bax在高分化与低分化的大肠腺癌中的阳性表达率比较均有显著差异(P<0.05), 而且他们在高分化与低分化大肠癌中的阳性表达程度也有显著差异(P<0.05).大肠癌组织PGE₂的表达在Dukes分期的A, B, C, D的阳性率分别为100%、94.4%、86.7%、71.4%, 两两之间比较无显著差异(P>0.05).大肠癌组织Bcl-2的表达在A, B, C, D的阳性率分别为90%、77.8%、60.0%、57.2%, 在A, B期中Bcl-2表达显著高于C, D期(P<0.05).大肠癌组织Bax的表达在A, B, C, D的阳性率分别为100%、72.2%、86.7%、85.7%, 4期之间比较无显著差异(P>0.05, 表2).在大肠癌组中有85.4%的Bcl-2 阳性组织同时表达PGE2阳性, 经统计学计算, 二者呈显著正相关(P<0.05, r = 0.532); 在大肠癌组中有76.4%的Bax阳性组织同时表达Bcl-2阳性, 经统计学计算, 二者呈显著正相关(P<0.05, r = 0.653); 而PGE₂与Bax在大肠癌组中的表达无明显相关性(P>0.05, r = 0.365).

表2 PGE₂ Bcl-2及Bax的阳性表达率与Dukes分期关系(n, %).

Dukes	n	PGE2	Bcl-2	Bax
A	10	10(100)	9(90)	10(100)
B	18	17(94.4)	14(77.8)	13(72.2)
C	15	13(86.7)	9(60)	13(86.7)
D	7	5(71.4)	4(57.2)	6(85.7)

3 讨论

最近许多研究提示, Bcl-2蛋白家族在凋亡过程中尤为重要, 在正常及病理情况下, Bcl-2具有抑制细胞凋亡、延长细胞寿命的功能[8], 增加肿瘤的播散侵袭能力.国内报道大肠良、恶性肿瘤中均有Bcl-2蛋白的高表达[9-10].我们通常发现, Bcl-2蛋白在正常及各级病变大肠黏膜中均有表达, 其阳性表达强度和阳性表达率随着大肠黏膜的癌变过程呈从正常黏膜、癌旁组织、腺瘤样息肉至大肠癌递增趋势, 且癌旁、腺瘤和腺癌中的阳性率与正常及慢性结肠炎中的比较有显著性差异.结果表明, Bcl-2蛋白表达在大肠黏膜癌变过程中起重要的作用, 其作用可能是通过抑制细胞凋亡来实现的, 且在癌旁组织中的Bcl-2异常高表达提示Bcl-2表达异常发生在大肠黏膜癌变过程中出现细胞形态学改变之前, 这与Bronner et al[11]的研究一致.我们发现Bcl-2的阳性表达率与大肠癌的Duke's分期有关, 腺癌(A, B期)中Bcl-2表达显著高于中晚期腺癌(C, D期), 亦显著高于腺瘤, 腺瘤中的表达高于正常结肠黏膜, 因此我们认为Bcl-2不仅在腺瘤形成中有重要作用, 而且在腺瘤恶变过程中亦有明显的促进作用, Bcl-2 表达异常可能是大肠黏膜癌变过程中的早期事件, 这与Hague et al[12]的报道相似.Bax基因是Bcl-2基因家庭的成员之一[13-16], Bax基因受到重视是因为他与Bcl-2基因在细胞凋亡的调控中起着中心作用[17-21].本结果显示, 在正常结肠黏膜、慢性结肠炎、大肠癌旁、腺瘤、腺癌组中Bax的阳性表达率无明显规律, 各组间均无显著性差异, 但其阳性表达程度在正常组织、腺瘤样息肉、大肠癌3组之间呈递减趋势, 这说明Bax在大肠黏膜癌变过程中也起重要作用, 其作用是由于Bax表达减弱、细胞凋亡减少, 而促进大肠黏膜癌变过程.Bcl-2和Bax的作用是相辅相成、密不可分的关系, 细胞内的Bcl-2与Bax的比例调节了细胞凋亡的发生[22-25].本结果也证实了这一点, 在大肠黏膜癌变过程中Bcl-2蛋白的表达逐渐增强而Bax蛋白的表达逐渐减弱, Bcl-2与Bax的比例逐渐升高, 细胞凋亡的发生减少以至肿瘤的发生.我们还发现在大肠癌组中Bcl-2和Bax的阳性表达与肿瘤的分化程度相关.随着肿瘤细胞分化降低而表达逐渐减弱, 在高分化的大肠腺癌Bcl-2与Bax的阳性率明显高于中、低分化的大肠腺癌, 提示Bcl-2的抗凋亡作用可能主要体现在分化的中晚期, 这与Baretton et al[26]的结果一致.

PGE₂作为一重要的消化道激素, 可使局部血管通透性增加, 因其可扩张血管, 故可增加病变组织中的血液供应, 促进腺瘤和癌组织的生长[27-28].我们发现PGE₂在正常及各级病变结肠黏膜中均有表达, 其阳性表达强度和表达率随着大肠黏膜癌变过程呈递增趋势, 且腺瘤和腺癌中的阳性率与正常及慢性结肠炎中的比较有显著性差异, 这一实验结果表明, PGE₂表达在大肠黏膜癌变过程中起重要的作用, 可能是通过抑制肿瘤免疫来实现的[29], 但是PGE₂在大肠癌细胞的增殖与凋亡过程中起什么作用, 国内目前还未见有相关报道.国外有报道在培养结肠癌细胞时加入PGE₂可见Bcl-2的表达上调, 细胞凋亡减少, 而对Bax的表达无影响, 加入COX-2抑制剂后Bcl-2的表达下调[30].本结果显示: 有85.4%的大肠癌组织表达Bcl-2的同时PGE₂表达阳性, 呈显著正相关(P<0.05, r = 0.653), 提示PGE₂与Bcl-2在大肠癌的发生发展过程中相互作用, 共同促进肿瘤细胞生长, 抑制细胞凋亡.但是PGE₂通过何种途径上调Bcl-2的表达, 有待进一步研究.

备注

编辑: 潘伯荣 审读: 张海宁

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