CN106645752B - Galectin‑10及其特异性抗体在制备检测鼻咽癌试剂盒中的应用 - Google Patents
Galectin‑10及其特异性抗体在制备检测鼻咽癌试剂盒中的应用 Download PDFInfo
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Abstract
本发明涉及诊断标志物，具体公开了一种鼻咽癌相关标志物Galectin‑10(genebank登录号ID1178)，并提供了该标志物及其特异性抗体在制备检测鼻咽癌试剂盒中的应用，所述试剂盒可为免疫组化试剂盒或酶联免疫检测试剂盒。本发明为有效判断人鼻咽疾病进程提供了新的科学依据，可实现鼻咽癌的早发现和早治疗。
Description
技术领域
本发明涉及诊断标志物，具体地说，涉及Galectin-10在检测鼻咽癌中的应用。
背景技术
鼻咽癌是一种频发于我国南方各省及东南亚地区的具有显著种族和地域分布特点的上皮源性恶性肿瘤。鼻咽癌的发病与遗传因素、EB病毒感染和其它环境因素多种因素相关。
随着医学科技的高速发展，越来越多的先进设备已用于鼻咽癌的检查。目前鼻咽癌的检测方法主要包括临床检查、鼻咽鼻镜检查、影像学检查、病理形态学检查和血清学检查。
影像学检查的灵敏度有限，且价格不菲；病理形态学检查有创；EB病毒抗体的血清学检查由于精密度和准确度问题也只能用于筛查，且对治疗后的观察无意义。
血清学检查方法简便，价格低廉，病人易于接受。因此寻找、鉴定鼻咽癌相关标志物对防治鼻咽癌非常重要。
Galectin-10是半乳糖凝集素家族的一员，人Galectin-10蛋白基因名为LGALS10(genebank登录号ID1178)，又名人晶体蛋白(Charcot Leyden Crystal Protein，CLC)，位于染色体19q13.2区段，全长6777bp，包含4个外显子。人Galectin-10蛋白分子量为16.5KDa，由142个氨基酸组成(蛋白质登录号Q05315，http://www.uniprot.org/)。有报道称Galectin-10参与调节调控细胞的生长、血管生成和炎症进展，但其在鼻咽癌中发挥的生物学功能尚未见报道。
发明内容
为了解决现有技术中存在的问题，本发明的目的是提供一种鼻咽癌相关标志物，并提供了该标志物及其特异性抗体在制备检测鼻咽癌试剂盒中的应用。
为了实现本发明目的，本发明的技术方案如下：
本发明首先提供了一种鼻咽癌相关标志物Galectin-10，基因名为LGALS10(genebank登录号ID1178)，又名人晶体蛋白(Charcot Leyden Crystal Protein，CLC)，位于染色体19q13.2区段，全长6777bp，包含4个外显子。人Galectin-10蛋白分子量为16.5KDa，由142个氨基酸组成(蛋白质登录号Q05315，http://www.uniprot.org/)。
进一步地，本发明提供了Galectin-10或其特异性抗体在制备检测鼻咽癌的试剂或试剂盒中的应用。
所述试剂盒可为免疫组化试剂盒或酶联免疫检测试剂盒。
本发明的有益效果在于：
本发明提供了鼻咽癌诊断与预后的生物标志物Galectin-10，根据Galectin-10的表达水平可实现鼻咽癌的早发现和早治疗。
本发明为有效判断人鼻咽疾病进程提供了新的科学依据。
附图说明
图1为检测Galectin-10在正常鼻咽上皮和鼻咽癌组织表达的免疫组化图。
图2为Galectin-10在永生化鼻咽上皮细胞NP69和鼻咽癌细胞CNE1，CNE2，HK1细胞中表达的western blot检测结果。
图3为ELISA法检测Galectin-10在鼻咽癌患者血清中的表达结果。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的，并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下，可以对本发明进行各种修改和替换。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
本实施例用于说明免疫组化分析Galectin-10在鼻咽癌组织中表达的临床意义：
以Galectin-10特异性抗体(sigma aldrich公司购买，C0998)联合迈新S-P免疫组化试剂盒(福建迈新公司)组成本实施例的诊断试剂盒。活检鼻咽部组织经10％多聚甲醛固定后，常规梯度酒精脱水透明，石蜡包埋，组织切成4μm厚的薄片，贴片烤片。按照免疫组织化学超敏试剂盒说明书步骤进行免疫组织化学染色操作：
1)经100％二甲苯对石蜡组织切片脱蜡二次，每次半小时，至完全脱蜡干净；
2)用新鲜配制的100％、95％、90％、75％梯度酒精水化后，PBS液(PH6.0)冲洗3次，每次3min；
3)抗原修复：处理好的切片置于切片架，置枸橼酸盐缓冲液的烧杯中电磁炉上加热至沸腾，维持15min，以达到抗原全面表达的最佳状态。自然冷却至室温，双蒸水冲洗二次，PBS冲洗二次；
4)加一滴过氧化物阻断溶液(试剂A)，37℃下孵育8min，PBS冲洗10min×3次；
5)用吸水纸从周边吸除多余的PBS液，加一滴正常非免疫动物血清(试剂B)，完全覆盖住样本，继续孵育10min；
6)同样吸除血清，分别加入稀释的兔抗人Galectin-10(1:200)。为避免假阳性结果，采用PBS代替一抗做阴性对照；
7)孵育完后，PBS冲洗10min×3次，同样吸除PBS液。滴加一滴生物标记的二抗(试剂C)，室温下孵育10min，PBS冲洗10min×3次。
8)吸除PBS液，加一滴链霉素抗生物素-过氧化物酶溶液(试剂D)，室温下孵育10min，PBS冲洗10min×3次。
9)吸除PBS液，加二滴新鲜配制的DAB溶液，显微镜下观察1-10min，至显色合适为止。
10)自来水冲洗，苏木素复染至合适程度，自来水冲洗泛蓝。
11)最后采用新鲜配制的梯度酒精脱水干燥，二甲苯透明，中性树胶封固玻片。
对组织化学染色图片进行分析。染色评分标准如下：随机选取至少10个高倍镜视野，计数1000个细胞，以积分法计算结果。即根据每张切片的染色强度和阳性细胞比例计分。染色强度计分原则为无色0分；浅黄色1分；棕黄色2分；棕褐色3分。着色细胞比例计分原则为无着色0分；＜30％为1分；30％-60％为2分；≥60％为3分，着色强度和着色细胞比例所得积分两者相加，0-2分为表达阴性，3分以上为表达阳性。应用SPSS15.0统计软件对蛋白质在组织间的表达差异进行分析，P＜0.05表示具有统计学意义。
按照上述方法对存档的石蜡包埋的组织标本包括24例正常鼻咽组织，53例鼻咽癌组织化学染色分析。
结果如下：
1)免疫组化分析Galectin-10在正常鼻咽上皮和鼻咽癌组织中的表达(图1)。图1A是Galectin-10在正常鼻咽上皮的典型图片，为阴性表达，图1B是Galectin-10在鼻咽癌组织中表达的典型图片，为阳性表达。
2)表1结果显示Galectin-10在正常鼻咽上皮和鼻咽癌组织中之间的表达存在差异且有统计学意义(P＜0.05)。Galectin-10在正常鼻咽上皮组织中大部分成阴性表达(阳性率为29.2％)，而Galectin-10在鼻咽癌组织中阳性表达明显升高(阳性率为77.4％)，阳性率存在显著性差异(P＜0.05)。galectin10的表达在正常和患者中存在统计学差异，但对个体而言，精密度和准确度不可兼得，所以在临床实践中得结合其他的诊断方法，辅助诊断。
表1 Galectin-10在鼻咽癌组织和正常鼻咽粘膜组织中的表达
实施例2
本实施例用于说明Western blotting检测Galectin-10蛋白的表达情况
蛋白准备：鼻咽癌细胞系CNE1、CNE2和HK1用含100U/ml抗生素(青霉素和链霉素)和10％小牛血清配制的RPMI 1640完全培养基进行常规培养(37℃、5％CO2、饱和湿度条件)。NP69细胞用含10％小牛血清和100U/ml抗生素的1×KSFM特殊培养基培养。待所有细胞生长至80％左右融合时，PBS冲洗细胞表层三次，离心收集约1×107个细胞，加入改良RIPA裂解液400μL(50mmol/L Tris HCl[pH 8.0]，150mmol/L NaCl，0.5％脱氧胆酸钠，1mmol/LEDTA1％，1％NP-40，0.1μg/ml PMSF，2μg/ml亮抑素)，充分混合后冰上裂解30min，4℃12000rpm离心30min，吸上清即为总蛋白。Bradford法测定蛋白质浓度：分别加入2mg/ml牛血清白蛋白(0、5、10、15和20μL)于5支微量离心管中设为标准孔，以0.9％NaCl补足至100μL稀释成蛋白浓度梯度。同时在每个样品管内加5μL待测样品，补95μL的0.9％NaCl。每管各加入lml考马斯亮蓝G-250溶液，振荡混匀，室温放置5min待充分反应。酶标仪上测定各孔A595的吸光度值，得到样品的蛋白浓度。调整至相同浓度后Western blotting检测目的蛋白表达。
Western blotting检测目的蛋白表达：每个泳道取50μg蛋白样品，加入等量2×SDS loading buffer(100mM Tris-Hcl(PH6.8)，200mM DTT，4％SDS，0.2％溴酚蓝，20％甘油)，沸水浴5分钟使蛋白变性，冰上冷却。加入样品于PAGE凝胶点样孔内，Tris-甘氨酸电泳缓冲液中8V/cm进行电泳。当溴酚蓝染料前沿进入分离胶后，把电压提高到15V/cm，继续电泳至溴酚蓝染料到达分离胶底部(约需1.5小时)，关闭电源。卸下分离胶，用电转膜缓冲液平衡洗涤1次10分钟。取下PVDF膜，标记好蛋白面，放入甲醇中平衡10分钟，再双蒸水中漂2分钟。PVDF膜、滤纸、海绵板放入电转膜缓冲液中平衡15分钟。从负极到正极方向依次安装转膜三明治：(黑，负极)海绵板-三层滤纸-胶-PVDF膜-三层滤纸-海绵板(红，正极)，按照凝胶面积以0.65mA/cm2恒流电转移1.5小时将蛋白自凝胶转印至PVDF膜，摇床上室温封闭液孵育1h，以封闭非特异性结合位点；加入1∶1000经封闭液稀释的Galectin-10一抗4℃孵育过夜，然后TBS-T缓冲液洗膜3×10分钟，加入1∶2000稀释的兔抗鼠二抗室温孵育1小时，同样TBS-T漂洗。加等量的发光剂A液和B液，Bio-Rad发光仪上获取条带图像。
结果显示：与永生化鼻咽上皮细胞NP69比较，Galectin-10在3株鼻咽癌细胞株中表达上调(图2)。
实施例3 ELISA检测鼻咽癌血清中Galectin-10的浓度
为验证Galectin-10在人鼻咽癌和癌旁血清中的差异，本实施例采用ELISA法检测了28例鼻咽癌患者及17例正常人血清中Galectin-10的表达水平。
操作流程如下：
1)收集约500μL的鼻咽癌或正常人的血清，-80℃保存备用。
2)购买Elabscience Biotechnology公司的人晶体蛋白(Charcot LeydenCrystal Protein，CLC，又名Galectin-10)酶联免疫吸附测定试剂盒(产品货号：E-EL-H1517c)，按照操作说明书检测血清中Galectin-10的浓度。
3)加样：分别设空白孔、标准孔、待测样品孔。空白孔加标准品&样品稀释液100μL，余孔分别加标准品或待测样品100μL，注意不要有气泡，加样时将样品加于酶标板底部，尽量不触及孔壁，轻轻晃动混匀。给酶标板覆膜，37℃孵育90分钟。
4)弃去液体，甩干，不用洗涤。每个孔中加入生物素化抗体工作液100μL(在使用前15分钟内配制)，酶标板加上覆膜，37℃温育1小时。
5)弃去孔内液体，甩干，洗板3次，每次浸泡1-2分钟，大约350μL/每孔，甩干并在吸水纸上轻拍将孔内液体拍干。
6)每孔加酶结合物工作液(临用前15分钟内配制)100μL，加上覆膜，37℃温育30分钟。
7)弃去孔内液体，甩干，洗板5次，方法同步骤3。
8)每孔加底物溶液(TMB)90μL，酶标板加上覆膜37℃避光孵育15分钟左右(根据实际显色情况酌情缩短或延长，但不可超过30分钟。当标准孔出现明显梯度时，即可终止)。
9)每孔加终止液50μL，终止反应，此时蓝色立转黄色。终止液的加入顺序应尽量与底物溶液的加入顺序相同。
10)立即用酶标仪在450nm波长测量各孔的光密度(OD值)。
结果显示，Galectin-10在鼻咽癌中的浓度明显高于正常人(图3)。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
Claims (1)
1.Galectin-10或其特异性抗体在制备检测鼻咽癌试剂盒中的应用，其特征在于，所述试剂盒为免疫组化试剂盒。
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