Alpha Bcrystallin基因在结肠癌组织中的表达

【摘要】 目的探讨alpha bcrystallin 基因在结肠癌组织中的表达及其与结肠癌临床病理的相关性。方法采用rtpcr方法检测64例结肠癌组织及其癌旁正常组织中alpha bcrystallin 基因的表达，计算其阳性率；并用western blot方法检测结肠癌组织及其癌旁正常组织中alpha bcrystallin蛋白的表达变化。结果alpha bcrystallin mrna在结肠癌组织中表达的阳性率明显高于癌旁正常组织(p<0.01),在发生浆膜外转移者的表达阳性率明显高于浆膜内者(p<0.05)，在较大肿瘤者中的表达阳性率明显高于较小肿瘤者，在有淋巴结转移者中的表达阳性率明显高于无淋巴结转移者(p<0.05),但与患者的性别、年龄、肿瘤的分化程度无关(p>0.05)。alpha bcrystallin蛋白在结肠癌组织中表达明显高于癌旁正常组织(p<0.01)。结论alpha bcrystallin基因的高表达可能与结肠癌的发生及侵袭转移有关。

【关键词】 alpha bcrystallin；结肠癌；rtpcr；western blot

alpha bcrystallin expression and its significance in colon carcinoma tissues

hang xiaosheng1，wang daoyuan2，wang weili3，li lihua4

1. department of medical oncology，wuxi cancer hospital, 4th hospital affiliated to suzhou university, wuxi, 214062,china;2. ammed cancer center, shanghai ruijin hospital affiliated to medical school of shanghai jiaotong university;3. department of general surgery,wuxi cancer hospital, 4th hospital affiliated to suzhou university, 4.department of laboratoryabstract：objective to study the expression of alpha bcrystallin in colon carcinoma, and its relation in clinical pathology of colon carcinoma.methodsthe expression of alpha bcrystallin mrna was detected using rtpcr in 64 colon carcinoma tissues and adjacent normal tissues. the expression level of alpha bcrystallin protein was detected by rtpcr and western blot. the detection rates of alpha bcrystallin mrna and protein expression in cancer tissues and adjacent normal tissues were analyzed.resultsalpha bcrystallin mrna expression rate in cancer tissues was significantly higher than adjacent normal tissues (p<0.01). the patients with colon cancer involving the serosa obtained obviously higher alpha bcrystallin expression rate than the one not involving the serosa, the group which has bigger tumor or with lymph node metastasis has higher expression of alpha bcrystallin than those group with smaller size of tumor or without lymph node metastasis (p<0.05), and its expression has no correlation with age, sex and tumor differentiation (p>0.05). the protein of alpha bcrystallin was detected in cancer tissues but not in adjacent normal tissues (p<0.01) by western blotting.conclusionthe expression of alpha bcrystallin is positively correlated with tumorogenesis and progression of colon cancer. it may be used as a good marker for clinic therapeutic purpose and prognosis estimation of colon cancer.

key words：alpha bcrystallin；colon carcinoma；rtpcr；western blot

1材料与方法

1.1标本 收集苏州大学附属第四医院2003年3月~2006年10月结肠癌手术切除的新鲜标本64例，每例标本取材于癌组织及其远端正常组织，立即置液氮中保存备用。www.133229.COm其中男性38例，女性26例，年龄31～77岁，平均51岁，其中<60岁的有37例，≥60岁的有27例。术前未经任何治疗，病理学诊断64例结肠癌均为腺癌。分化程度：高分化17例,中分化26例，低分化21例；浸润深度：浸及黏膜下层或肌层（浆膜内）37例,浸润至浆膜及肠壁外脂肪组织者27例（浆膜外）；有淋巴结转移29例,无淋巴结转移35例；肿瘤原发灶<3cm的有33例，≥3cm的有31例。

1.2主要试剂trizol 试剂盒(invitrogen公司产品),rtpcr试剂盒(gibcobrl公司公司产品),alpha bcrystallin(nm_001885)上游引物：5′acatcgccatccaccacc3′，下游引物：5′attccacaaccctctacact3′，预计扩增片段长度为288bp；gapdh (ng_007073)上游引物：5′aacggatttggtcgtattg3′，下游引物：5′ggaggacgtggtggttgacgaatcg3′，预计扩增片段长度为449bp。所有引物序列均由上海英俊生物工程有限公司提供。alpha bcrystallin一抗为多克隆山羊抗人抗体(abcam公司),二抗为hrp 标记的兔抗山羊多克隆抗体(dako公司,美国),内参为单克隆鼠抗人gapdh 抗体(上海康成生物公司),二抗为hrp 标记的兔抗鼠多克隆抗体(dako公司,美国)，ecl 发光液(pierce 公司,美国),ripa裂解液、bca 蛋白浓度测定试剂盒（碧云天，中国）。

1.3rtpcr按照试剂盒的操作说明提取总rna，取50mg组织至2ml匀浆器中，加入1ml trizol试剂，匀浆，将匀浆后的液体转入1.5ml微量离心管中，加入0.2ml氯仿，振荡15sec，静置2min。4℃离心，12000r/min ×10min，将上层水相移至一新的1.5ml离心管中。加入0.5ml异丙醇，轻轻混匀，室温静置10min。4℃离心，15000r/min×10min，弃上清。加入1ml 75%乙醇，轻轻洗涤沉淀。4℃离心，10000g×5min，弃上清。晾干，加入20μl的depc水溶解（65℃促溶10～15min）。在20μl的反应体积中加入2.0μl总rna，置于37℃孵箱中反应1h，接着95℃灭活5min，再立即冰浴，-20℃备用。然后进行pcr扩增，在25μl pcr反应体系中加入cdna 5μl,扩增条件为：94℃，预变性3min；94℃，变性45sec；57℃，退火45sec；72℃，延伸45sec。循环30次。最后72℃延伸10min，经1.5％琼脂糖凝胶电泳鉴定pcr产物。分别取5μl pcr 产物,1.5%琼脂糖凝胶电泳检测，对凝胶进行灰度扫描及照相，出现288bp条带即为alpha bcrystallin阳性，内参gapdh（甘油醛3磷酸脱氢酶）片段大小为449bp。

1.4总蛋白提取及westernblot免疫印迹用ripa 裂解液裂解组织样品，提取总蛋白，用bca法测蛋白浓度,在每一个病例样品中取10μg混合作为结肠癌组织蛋白样品，癌旁正常组织蛋白样品也是由每一个癌旁蛋白样品分别取10μg混合而成。制备12% sdspage，每条泳道加总蛋白量50μg,进行电泳,经湿转印到pvdf膜上(amresco 公司,美国),5% bsatbst 常温下封闭1h,分别加入alpha bcrystallin山羊抗人多克隆抗体( 1∶1000稀释) 及gapdh 鼠抗人单克隆抗体(1∶300稀释),4℃孵育过夜,然后加入hrp 标记的兔抗山羊igg (1∶1000稀释)和hrp 标记的兔抗鼠多克隆抗体(1∶1000稀释),37℃孵育1h,tbst洗涤,ecl化学发光试剂自显影。alpha bcrystallin相对含量用alpha bcrystallin/gapdh 灰度比值表示(灰度用伯乐公司的quantity one软件分析)。每组重复3次。

1.5统计学方法 采用stata 7.0统计软件，进行χ2检验或t检验，p<0.05为有统计学意义。

2结果

　　rtpcr产物经1.5%琼脂糖凝胶电泳检测见一特异扩增条带，其分子量大小与预期结果(288bp)相符，结果见图1。alpha bcrystallin mrna在结肠癌组织中表达见表1。即alpha bcrystallin 基因在结肠癌组织中表达阳性率明显高于正常组织(p<0.01),在发生浆膜外转移者的表达阳性明显高于浆膜内转移者(p<0.05)，在较大肿瘤者中的表达阳性率明显高于较小肿瘤者，在有淋巴结转移者中的表达阳性率明显高于无淋巴结转移者(p<0.05),但与患者性别、年龄及肿瘤的分化程度无关(p>0.05)。

　　还可通过结合caspase3前体的活化中间产物p24，并阻止p24的进一步自体水解活化，从而抑制caspase9(线粒体通路)和caspase8(死亡受体通路)对caspase3的激活，起到抗凋亡的作用[35]；另外还可通过阻止ras的激活抑制erk1/2的活性，从而大大减轻ca2+诱导引起的凋亡作用[11]。通过rnai技术消除alpha bcrystallin以后，细胞对凋亡十分敏感[12]。最近研究资料显示alpha bcrystallin与多种肿瘤的发生相关。本研究发现alpha bcrystallin mrna在结肠癌组织中表达阳性率明显高于正常组织，alpha bcrystallin蛋白在结肠癌组织的中的表达明显高于癌旁正常组织，与alpha bcrystallin在其他肿瘤中的研究结果一致[710]。可能是由于alpha bcrystallin可以阻断细胞凋亡的信号传递，抑制了细胞的凋亡，从而有利于肿瘤的形成；本研究发现alpha bcrystallin在较大肿瘤者中的表达阳性率明显高于较小肿瘤者，在有淋巴结转移者中的表达阳性率明显高于无淋巴结转移者(p<0.05)，可能是由于alpha bcrystallin的表达抑制肿瘤细胞的凋亡，使肿瘤细胞进一步恶性增殖，从而使肿瘤不断增大；敲除alpha bcrystallin基因后，发现caspase3的活性明显增强，凋亡广泛，超微结构显示肿瘤血管明显少于野生型[12 13]。据此，我们可以推测alpha bcrystallin在结肠癌中也起到抑制肿瘤血管内皮细胞凋亡的作用，从而促进肿瘤血管的形成，使肿瘤的血供丰富，这样有利于肿瘤的进一步增大，同时可能也增加了肿瘤细胞侵润和转移的机会，这与本实验的结果相符。另外，我们发现alpha bcrystallin在发生浆膜外转移者的表达阳性明显高于浆膜内转移者。moyano等[8]体外研究发现alpha bcrystallin在乳腺癌中高表达可增加乳腺癌细胞的转移和浸润。由此我们可以推测alpha bcrystallin在结肠癌中高表达可能也增加了结肠癌细胞的转移和浸润。以上结果可以提示alpha bcrystallin的高表达在结肠癌的发生发展过程中可能起重要的作用。这或许可以为结肠癌的治疗提供一个潜在的靶标。

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