CN108251529B - Ano5在制备治疗胰腺癌的药物中的应用 - Google Patents
Ano5在制备治疗胰腺癌的药物中的应用 Download PDFInfo
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Abstract
本发明涉及ANO5(Anoctamin5)在制备胰腺癌诊断标志物，以及制备治疗胰腺癌的药物中的应用。本发明利用CCK‑8实验与划痕实验检测了转染ANO5siRNA后对胰腺癌细胞增殖、迁移的影响，结果显示，转染ANO5siRNA后可显著降低胰腺癌细胞的增殖与迁移能力，抑制细胞中的ANO5后可抑制胰腺癌细胞的增殖与迁移能力，因此ANO5可成为胰腺癌治疗新的靶点，ANO5siRNA可用于制备治疗胰腺癌的药物。
Description
(一)技术领域
本发明涉及ANO5(Anoctamin5)在制备胰腺癌诊断标志物，以及制备治疗胰腺癌的药物中的应用。
(二)背景技术
胰腺癌是一种具有高发病率和高死亡率特点的恶性肿瘤，严重威胁人类的健康，其发生发展的机制一直是研究者们研究的热点。近年来，离子通道与肿瘤的关系受到了越来越多的关注，TMEM16跨膜蛋白家族(TMEM16A～K)也称为anoctamin家族(anoctamin1-10)为钙离子激活氯离子通道蛋白家族，研究显示，其多个家族成员在肿瘤的发生及肿瘤细胞的表型维持中发挥着重要的作用。
ANO5广泛表达于消化管各部上皮细胞中，但是其在正常胰腺组织及胰腺癌细胞的表达及对肿瘤细胞的影响还未见报道。
(三)发明内容
本发明目的是研究ANO5在胰腺癌细胞的表达及作用，提供ANO5在制备胰腺癌诊断标志物，以及制备治疗胰腺癌的药物中的应用。
本发明采用的技术方案是：
ANO5在制备胰腺癌诊断标志物中的应用。经发明人研究发现，ANO5在癌旁组织的表达不明显，与癌旁组织相比较，在胰腺癌组织中ANO5的表达显著升高，提示ANO5可作为胰腺癌诊断标志物。
ANO5在制备治疗胰腺癌的药物中的应用。
具体的，所述的应用是以ANO5siRNA制备抑制胰腺癌细胞的增殖与迁移的药物。
所述ANO5siRNA序列为：GCAGAGTTAAAGGCGGAAA。
发明人利用CCK-8实验与划痕实验检测了转染ANO5siRNA后对胰腺癌细胞增殖、迁移的影响，结果显示，转染ANO5siRNA后可显著降低胰腺癌细胞的增殖与迁移能力，抑制细胞中的ANO5后可抑制胰腺癌细胞的增殖与迁移能力，因此ANO5可成为胰腺癌治疗新的靶点，ANO5siRNA可用于制备治疗胰腺癌的药物。
本发明的有益效果主要体现在：提供了ANO5在制备胰腺癌诊断标志物，以及制备治疗胰腺癌的药物中的应用，为新药筛选提供了新的途径。
(四)附图说明
图1为胰腺癌及癌旁组织的组织切片染色结果；A为正常胰腺组织ANO5；B为胰腺癌ANO5；C为胰腺癌阴性对照；
图2为ANO5在人正常胰腺导管上皮细胞和胰腺癌细胞HPAC细胞的表达；
图3为ANO5mRNA在人正常胰腺导管上皮细胞和胰腺癌细胞HPAC细胞的表达；
图4为转染PANC-1细胞48h后，利用Wesrtern blot检测细胞中ANO5的表达情况；
图5为转染ANO5siRNA后PANC-1细胞的迁移情况；
图6为转染ANO5siRNA后PANC-1细胞的增殖情况。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
实施例1：
1.材料与方法
1.1材料：人胰腺癌组织购买于西安艾莉娜生物科技有限公司；人胰腺癌细胞HPAC细胞，PANC-1细胞，BxPC-3细胞，HEK293细胞，人正常胰腺导管上皮细胞(HPDE6-C7细胞)购自于中国细胞资源库武大细胞库。
1.2主要试剂：ANO5抗体美国Biorbyt公司，免疫组化染色试剂盒(SPN-9001)
1.3实验方法
1.3.1免疫组织化学
组织切片经二甲苯脱蜡，梯度乙醇水化，3％的H2O2室温封闭10min，以消除内源性过氧化物酶，枸橼酸盐抗原修复液微波热修复，正常山羊血清室温封闭1h，滴加ANO5抗体(1:100)4℃孵育过夜，复温后滴加免疫组化染色试剂盒中的B试剂，滴加免疫组化染色试剂盒中的C试剂,室温孵育30min，最后DAB、自来水充分冲洗，苏木素复染，封片，晾干后，显微镜下观察拍照。
1.3.2细胞培养
HPDE6-C7细胞、HEK293细胞、PANC-1细胞培养于DMEM高糖培养基(10％胎牛血清)，HPAC细胞、BxPC-3细胞培养于RPMI-1640培养基(10％胎牛血清)在37℃，5％CO2细胞培养箱中培养，取对数生长期的细胞用于实验。
1.3.3ANO5siRNA的转染
细胞分组：正常对照组；阴性对照组；ANO5siRNA转染组。
选择状态良好的PANC-1细胞接种于6孔板中，37℃，5％CO2细胞培养箱中培养24h后，ANO5siRNA组转染ANO5 siRNA(GCAGAGTTAAAGGCGGAAA)，阴性对照组转染siRNA control(UUCUCCGAACGUGUCACGUTT)，37℃，5％C O2细胞培养箱中培养4-6小时后，各组均更换含10％FBS的培养基继续培养48h，进行下一步实验。
1.3.4利用Western blotting检测各组细胞中ANO5蛋白的表达
正常培养的HPDE6-C7细胞、HEK293细胞、PANC-1细胞、HPAC细胞、BxPC-3细胞；转染ANO5siRNA 48h及各处理组的PANC-1细胞，弃去培养液，利用RIPA裂解液裂解细胞，提取总蛋白。常规SDS-PAGE电泳、转膜，10％脱脂奶粉室温封闭1h，ANO5抗体(1:1000)4℃孵育过夜，荧光二抗室温孵育1.5h后，Odyssey曝光，Image J软件分析灰度值。
1.3.5细胞划痕实验
取对数生长期，状态良好的PANC-1细胞，弃去培养液，用0.25％的胰酶室温消化后，加培养基重悬细胞，按照约2×105个细胞的密度种于6孔板中，培养24小时后用1ml的无菌蓝色吸头在孔的中间划痕，尽量保证每一个孔中划痕的宽度一致。弃去培养液，每孔加入1ml无血清培养液以洗去悬浮的细胞。
按照前面所述的方法分组转染细胞。分别在转染后24小时与48小时，镜下观察细胞愈合面积，并采集图像，利用Image J软件测量愈合面积。
1.3.6细胞增殖实验
取对数生长期，状态良好的PANC-1细胞，弃去培养液，用0.25％的胰酶室温消化后，加培养基重悬细胞，接种于24孔板中继续培养24h，细胞分组：正常对照组；阴性对照组；ANO5siRNA转染组。
ANO5siRNA组转染ANO5siRNA，阴性对照组转染siRNA control，37℃，5％CO2细胞培养箱中培养4-6小时后，各组均更换含10％FBS的培养基继续培养24和48h，弃去孔中的培养液，每孔新加入200μl培养液。每孔加入20μLCCK-8溶液，37℃，5％CO2孵育2小时。设置酶标仪，在450nm处测OD值。
1.4统计学分析采用SPSS19.0软件进行统计分析，数据以均数±标准差表示。两样本均数间比较采用t检验，P<0.05表示差异具有统计学意义。
2.结果：
2.1.ANO5在胰腺组织的表达情况
通过对12例胰腺癌及癌旁组织的组织切片染色结果发现，ANO5在癌旁的正常胰腺组织中的表达并不明显，而在胰腺癌组织中(8例)ANO5的表达显著升高(图1)，阳性率高达66.7％，这就提示ANO5可能参与肿瘤的发生发展。
2.2.ANO5在细胞中的表达
进一步检测了ANO5在人正常胰腺导管上皮细胞和胰腺癌细胞HPAC细胞的表达，Wesrtern blot结果显示，与人正常胰腺导管上皮细胞相比较，ANO5蛋白在胰腺癌细胞HPAC细胞的表达显著升高(图2)(HEK293细胞为阳性对照)；而在对三种胰腺癌细胞中ANO5蛋白的表达情况的检测结果中我们发现，在三种癌细胞中，ANO5蛋白的表达也不尽相同，ANO5蛋白在PANC-1细胞中的表达最高，且ANO5mRNA的表达与蛋白的表达相一致(图3)。
2.3.ANO5siRNA的干扰效果
ANO5siRNA转染PANC-1细胞48h后，利用Wesrtern blot检测细胞中ANO5的表达情况，结果显示，与阴性对照组相比较，转染ANO5siRNA组细胞中ANO5的表达显著降低(图4)，这就提示ANO5siRNA能有效沉默细胞中ANO5基因的表达。
2.4.ANO5对PANC-1细胞迁移的影响
利用划痕实验检测了转染ANO5siRNA后PANC-1细胞的迁移情况，结果显示转染ANO5siRNA后24h及48h，与阴性对照组相比较，细胞划痕的愈合面积显著减低(图5)，这就提示细胞的迁移能力下降。
2.5.ANO5对PANC-1细胞增殖的影响
进一步利用CCK-8实验检测了转染ANO5siRNA后PANC-1细胞的增殖情况，结果显示转染ANO5siRNA后24h及48h，与阴性对照组相比较，细胞的增殖能力显著下降(图6)。这些结果提示，ANO5对PANC-1细胞的迁移和增殖能力有一定的影响，抑制PANC-1细胞中ANO5的表达，可抑制其迁移和增殖的能力。
3.讨论
Anoctamin(TMEM16)家族的基因包括ANO1-ANO10，它们编码10个具有8次折叠的跨膜蛋白，而这些蛋白可作为钙激活的氯离子通道(Ca2+-activated Cl-channel，CaCC)，阳离子通道和脂质转运酶具有多种生理功能。在上皮细胞中，包括胰腺导管上皮细胞，CaCC为细胞分泌过程中重要的组成部分。研究显示，ANO1可高表达于多种肿瘤细胞中，参与肿瘤细胞的增殖，凋亡及迁移，而D.R.P.Sauter等人研究显示ANO1在胰腺癌三种细胞株中的表达均升高，抑制ANO1的表达后对肿瘤细胞的增殖并无明显的影响，但可显著抑制肿瘤细胞的迁移。Anoctamin家族的另一成员ANO5也为CaCC,前期的研究结果显示ANO5广泛表达于消化管各部上皮细胞中，但是其在正常胰腺组织及胰腺癌细胞的表达及对肿瘤细胞的影响还未见报道。
本发明中首先利用免疫组织化学的方法检测ANO5在胰腺癌及癌旁组织的表达情况，结果显示，ANO5在癌旁组织的表达不明显，与癌旁组织相比较，在胰腺癌组织中ANO5的表达显著升高。利用Western blot检测ANO5在胰腺癌细胞及人正常胰腺导管上皮细胞中的表达，结果显示，ANO5在胰腺癌HPAC细胞，PANC-1细胞中的表达显著高于其在人正常胰腺导管上皮细胞HPDE6-C7细胞中的表达，这就提示ANO5在胰腺癌的发生发展过程中发挥一定的作用。进一步利用ANO5siRNA转染至胰腺癌PANC-1细胞，检测ANO5对胰腺癌细胞生物学特性的影响，结果显示，转染ANO5siRNA后可显著降低PANC-1细胞的增殖活性，降低细胞的迁移能力。
本发明结果显示ANO5在胰腺癌细胞中的表达显著升高，其在癌细胞的增殖与迁移中发挥重要的作用，ANO5可作为临床胰腺癌诊断和治疗的潜在靶点。
序列表
<110> 新乡医学院
<120> ANO5在制备治疗胰腺癌的药物中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> 未知(Unknown)
<400> 1
gcagagttaa aggcggaaa 19
Claims (2)
1.ANO5 siRNA在制备抑制胰腺癌细胞的增殖与迁移的药物中的应用，其特征在于，所述胰腺癌细胞为PANC-1细胞。
2.如权利要求1所述的应用，其特征在于所述 A N O 5 s i R N A 序 列 为 ：GCAGAGUUAAAGGCGGAAA。
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