CN106383230B - 一种肺癌诊断试剂盒 - Google Patents
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Abstract
本发明公开了一种肺癌诊断试剂盒，属于生物科学技术领域。所述肺癌诊断试剂盒包括用于检测肺组织中非磷酸化JAK1表达水平的试剂。本发明该公开了检测肺癌组织中非磷酸化JAK1表达水平的试剂在制备肺癌诊断用试剂中的用途。通过检测肺组织中非磷酸化JAK1表达水平，可以判断患肺癌的风险，为患者采取相关的治疗措施或者决策提供有效的依据，对肺癌的诊断和治疗具有重大意义。
Description
技术领域
本发明涉及一种肺癌诊断试剂盒，属于生物科学技术领域。
背景技术
肺癌是发病率和死亡率增长最快，对人群健康和生命威胁最大的恶性肿瘤之一。近50年来许多国家都报道肺癌的发病率和死亡率均明显增高，男性肺癌发病率和死亡率均占所有恶性肿瘤的第一位，女性发病率占第二位，死亡率占第二位。肺癌的病因至今尚不完全明确。
患肺癌的原因有多种，包括吸烟、职业和环境接触、电离辐射、既往肺部慢性感染、遗传、大气污染等等；目前认为吸烟是肺癌的最重要的高危因素，烟草中有超过3000种化学物质，其中多链芳香烃类化合物（如：苯并芘）和亚硝胺均有很强的致癌活性。多链芳香烃类化合物和亚硝胺可通过多种机制导致支气管上皮细胞DNA损伤，使得癌基因（如Ras基因）激活和抑癌基因（如p53，FHIT基因等）失活，进而引起细胞的转化，最终癌变。
为了对肺癌进行诊断，一般都综合运用多种方法。到目前为止，通过对 肺部原发肿瘤以及转移性肿瘤（包括淋巴结及其它组织器官）进行病理活检并通过免疫组织化学方法进行分析是当前国际公认的疾病诊断的金标准。此外，还可以结合胸部X-线摄影、胸部计算机断层扫描、纤维支气管镜等进行辅助诊断。
与病理诊断相比，其它辅助诊断方式均有不同的缺陷与不足；如采用胸部计算机断层扫描，肺癌的大小必须达到约0.1cm以上才能够进行测定，在这一时期，癌症很可能已经转移到了其它的组织；再如纤维支气管镜虽然可以将内镜插入支气管直接对肺内部进行直接观察，但对可疑肿瘤部位也仅仅能根据临床经验进行怀疑，最终准确诊断还需活检可疑组织进行病理诊断。
国家知识产权局于2015年6月10日公告的公告号为CN102959398B的发明专利，该专利公开了一种含有以HpαR为有效成份的肺癌诊断用标记，其使用血液作为检体，不会给患者带来任何负担，其存在以下技术问题：（1）外周血与局部肿瘤组织相比，无法做到及时准确地反映肿瘤的真实情况；（2）外周血无法反映出肿瘤局部微环境（肿瘤局部微环境在肿瘤发生、发展、侵袭及转移中扮演着极为重要的角色）；（3）外周血是机体绝大多数蛋白、因子等的终末汇集处、机体任何一种损伤或应激都可能导致许多蛋白、因子表达不稳定，因此单一检测外周血中的某种因子其敏感性和特异性均差强人意。
又如公告号为CN104694623A的发明专利，公开了一种同于肺癌诊断的血浆miRNA标志物及应用。所述标志物包括miRNA-486、miRNA-150、miRNA-205和miRNA-210，其存在以下技术问题：miRNA在血浆中极其容易降解，对血浆的保存要求较高。
目前，暂未见有包括有用于检测肺组织中非磷酸化JAK1表达水平的试剂的试剂盒以及检测肺组织中非磷酸化JAK1表达水平的试剂在制备肺癌诊断用试剂中的用途的相关报道。
发明内容
本发明目的之一在于提供一种新的肺癌诊断试剂盒，以解决现有技术中肺癌诊断方式不够高灵敏性、高特异性等问题。
为了实现上述发明目的，本发明的技术方案如下：
一种肺癌诊断试剂盒，其特征在于：包括用于检测肺组织中非磷酸化JAK1表达水平的试剂，非磷酸化JAK1为非磷酸化Janus蛋白激酶1。
进一步地，所述检测肺组织中非磷酸化JAK1表达水平为免疫组化检测方法用试剂。
所述检测肺组织中非磷酸化JAK1表达水平为Western Blot或ELISA检测方法用试剂。
所述试剂盒内的试剂包括：JAK1抗体、抗体稀释液、抗原修复液、洗涤液、二抗、DAB显色剂和免疫组化笔。
本发明的另一个目的是提供检测肺组织中非磷酸化JAK1表达水平的试剂在制备肺癌诊断用试剂中的用途。
所述检测肺组织中非磷酸化JAK1表达水平的试剂为免疫组化检测方法用试剂。
所述检测肺组织中非磷酸化JAK1表达水平的试剂为Western Blot或ELISA检测方法用试剂。
本发明的有益效果：
本发明通过检测非磷酸化JAK1表达水平，根据肺癌组织与正常肺组织中非磷酸化JAK1表达水平的差异，可以判断出待检人患肺癌的风险：若非磷酸化JAK1高，则患肺癌的风险高，若非磷酸化JAK1低，则患肺癌的风险低，非磷酸化JAK1可以作为肺癌早期诊断的标志物，可用于肺癌的辅助诊断，临床应用前景良好，对于肺癌的诊断、治疗具有重大意义。
具体实施方式
下面结合实施例对本发明作进一步地详细说明，但本发明的实施方式不限于此。
实施例1
以免疫组化方法为例253例肺癌患者石蜡组织进行非磷酸化JAK1表达水平的检测，并以70例肺癌远端石蜡组织作为正常对照。
（一）试剂来源
JAK1抗体：购自美国affinity公司，型号为AF5012；
Dako HRP标记的二抗工作液、抗体稀释液、显色剂（DAB）、抗原修复液、WashBuffer、免疫组化笔，均购自丹麦Dako公司。
（二）临床资料
选取肺癌患者石蜡切片253例，远端对照70例，其临床资料如下：
上述表格中，其他包括大细胞癌、腺鳞癌、小细胞癌。
（三）检测方法
（1）封闭过氧化物酶：取石蜡切片进行脱蜡，脱至水化后，用3% H2O2溶液于暗处封闭内源性过氧化物酶，室温孵育15 min；
脱蜡的操作过程如下：二甲苯Ⅰ10min→二甲苯Ⅱ 10min→无水乙醇 5min→95%乙醇5min→85%乙醇 5min→75%乙醇 5min。
（2）蒸馏水漂洗：切片置于蒸馏水中，摇床上洗5 min/次，共三次；
（3）抗原修复：加入pH 8.0的Tris-EDTA或pH 6.0的柠檬酸钠溶液，在95 ℃水浴锅中水浴50 min，进行抗原修复；
（4）蒸馏水漂洗：取出切片，自然冷却至室温后，用蒸馏水洗三遍，再用WashBuffer润洗一次；
（5）孵育一抗：用免疫组化笔将组织圈住，防止抗体流出，然后在圈内滴加JAK1抗体稀释液（抗体稀释液是抗体与稀释液按1:200的比例配置而成），4 ℃孵育过夜；
（6）蒸馏水漂洗：用蒸馏水洗三遍，Wash Buffer润洗一次；
（7）滴加二抗：滴加Dako HRP标记的二抗工作液（二抗与稀释液的比例为1:50），室温孵育60 min；
（8）蒸馏水漂洗：蒸馏水洗，5 min/次，共三次；
（9）DAB显色：滴加显色底物DAB，在显微镜下观察显色情况，于蒸馏水中终止反应；
（10）苏木素复染：将切片放入苏木素染液中染色2 min，1%盐酸酒精分化后，流水冲洗10 min；
（11）脱水透明：经80%、95%、100%梯度酒精脱水、二甲苯透明，于通风厨中干燥；
（12）封片：用中性快干胶封片，在光学显微镜下观察，DP Controller图像采集系统采图。
（四）免疫组化评分标准
肿瘤细胞染色强度*肿瘤细胞阳性面积为0～9分；
（五）检测结果
由两名经验丰富的病理医生采用SPSS17软件对实验组（肺癌组织）和对照组（远端对照组织）进行统计分析，得到以下结果，结果中，阴性为0分，低度阳性为1～3分，高度阳性＞3分。
阳性表达率：
（六）结果分析
由以上结果可以看出，与癌旁正常肺组织相比，肺癌组织的非磷酸化JAK1显著升高，说明肺癌与非磷酸化JAK1表达水平呈正相关，非磷酸化JAK1的高表达会显著提高患肺癌的可能性。由于癌旁正常肺组织的FRK水平可反映正常人肺组织的非磷酸化JAK1水平，因此，可以通过检测待检人群的非磷酸化JAK1的表达水平，将肺癌的易感人群筛查出来。
实施例2
本实施例给出一种肺癌诊断试剂盒，具体如下：
1、试剂盒的组成
检测试剂盒（50人份）：
备注：本表格按每张切片滴加各类实验试剂为100μL/张，具体用量可根据组织大小适当增减。
2、试剂盒的使用方法同实施例1。
以上所述，仅是本发明的较佳实施例，并非对本发明做任何形式上的限制，凡是依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化，均落入本发明的保护范围之内。
Claims (7)
1.检测肺癌组织中非磷酸化JAK1表达水平的试剂在制备肺癌诊断用试剂中的用途。
2.如权利要求1所述的用途，其特征在于：所述检测肺组织中非磷酸化JAK1表达水平的试剂为免疫组化检测方法用试剂。
3.如权利要求1所述的用途，其特征在于：所述检测肺组织中非磷酸化JAK1表达水平的试剂为Western Blot或ELISA检测方法用试剂。
4.如权利要求1所述的用途，其特征在于：所述试剂包括用于检测肺组织中非磷酸化JAK1表达水平的试剂。
5.如权利要求4所述的用途，其特征在于：所述检测肺组织中非磷酸化JAK1表达水平为免疫组化检测方法用试剂。
6.如权利要求4所述的用途，其特征在于：所述检测肺组织中非磷酸化JAK1表达水平为Western Blot或ELISA检测方法用试剂。
7.如权利要求5所述的用途，其特征在于：试剂还包括：JAK1抗体、抗体稀释液、抗原修复液、洗涤液、二抗、DAB显色剂和免疫组化笔。
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