【摘要】 目的探讨alpha bcrystallin 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rtpcr方法检测64例结肠癌组织及其癌旁正常组织中alpha bcrystallin 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 bcrystallin蛋白的表达变化。结果alpha bcrystallin 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 bcrystallin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 bcrystallin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 bcrystallin；结肠癌；rtpcr；western blot
alpha bcrystallin expression and its significance in colon carcinoma tissues
hang xiaosheng1，wang daoyuan2，wang weili3，li lihua4
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 bcrystallin in colon carcinoma, and its relation in clinical pathology of colon carcinoma.methodsthe expression of alpha bcrystallin mrna was detected using rtpcr in 64 colon carcinoma tissues and adjacent normal tissues. the expression level of alpha bcrystallin protein was detected by rtpcr and western blot. the detection rates of alpha bcrystallin mrna and protein expression in cancer tissues and adjacent normal tissues were analyzed.resultsalpha bcrystallin 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 bcrystallin 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 bcrystallin 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 bcrystallin was detected in cancer tissues but not in adjacent normal tissues (p<0.01) by western blotting.conclusionthe expression of alpha bcrystallin 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 bcrystallin；colon carcinoma；rtpcr；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.11665.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),rtpcr试剂盒(gibcobrl公司公司产品),alpha bcrystallin(nm_001885)上游引物：5′acatcgccatccaccacc3′，下游引物：5′attccacaaccctctacact3′，预计扩增片段长度为288bp；gapdh (ng_007073)上游引物：5′aacggatttggtcgtattg3′，下游引物：5′ggaggacgtggtggttgacgaatcg3′，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 bcrystallin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）。
1.3rtpcr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 bcrystallin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% sdspage，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% bsatbst 常温下封闭1h,分别加入alpha bcrystallin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 bcrystallin相对含量用alpha bcrystallin/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
rtpcr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 bcrystallin mrna在结肠癌组织中表达见表1。即alpha bcrystallin 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caspase3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caspase9(线粒体通路)和caspase8(死亡受体通路)对caspase3的激活，起到抗凋亡的作用[35]；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 bcrystallin以后，细胞对凋亡十分敏感[12]。最近研究资料显示alpha bcrystallin与多种肿瘤的发生相关。本研究发现alpha bcrystallin mrna在结肠癌组织中表达阳性率明显高于正常组织，alpha bcrystallin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 bcrystallin在其他肿瘤中的研究结果一致[710]。可能是由于alpha bcrystallin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 bcrystallin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 bcrystallin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 bcrystallin基因后，发现caspase3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 bcrystallin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 bcrystallin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 bcrystallin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 bcrystallin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 bcrystallin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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