CN113138273A - 一种应用于肺癌快速筛查及免疫靶向治疗检测的试剂盒及其用途 - Google Patents
一种应用于肺癌快速筛查及免疫靶向治疗检测的试剂盒及其用途 Download PDFInfo
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Abstract
本发明公开了血浆和肺组织中的Siglec‑14蛋白及其肺癌快速筛查和免疫靶向治疗检测的试剂盒，具体提供了选择性地检测肺癌蛋白Siglec‑14的试剂及在制备肺癌快速筛查及免疫靶向治疗检测的试剂盒中的用途。同时，还提供了能够快速筛查受试者中是否患有肺癌及免疫靶向治疗检测的试剂盒，所述试剂盒包括血浆和肺组织样品中蛋白Siglec‑14的特异性抗体、免疫组化相关试剂以及定量ELISA、WB相关试剂。本发明首次采用Siglec‑14作为肺癌检测的生物标记物，用于肺癌的快速筛查以及免疫靶向治疗检测，为肺癌的临床诊断和靶向治疗提供了新的方向。
Description
技术领域
本发明涉及生物技术和医学领域，具体涉及一种来源于肺部组织高表达的肿瘤蛋白标志物及其用途。本发明首次发现肺癌患者血浆和肺组织中的Siglec-14含量均显著高于健康和良性肺部疾病患者。通过针对Siglec-14定量检测和分析，实现对肺癌进行临床早期诊断和靶向治疗。
背景技术
肺癌是全球发病率和死亡率增加最快的肿瘤，在男性常见肿瘤中肺癌居首位，在女性常见肿瘤中肺癌居于二、三位。每年大概有120万患者被诊断为肺癌，110万人死于肺癌。肺癌依据其组织病理学可分为两大类：小细胞肺癌和非小细胞肺癌。非小细胞肺癌又包含鳞状细胞癌、腺癌、大细胞癌等。迄今为止，在肺癌快速筛查和诊断的方案尚不成熟，靶向治疗也缺乏系统的手段，因此，开发早期筛查和靶向治疗技术己经成为目前肺癌防治中亟待解决的重大科学问题。肿瘤标志物的发现和合理应用是肿瘤早期发现及个体化治疗的前提。
目前有关肺癌的发病机制尚不清楚，研究显示直接从蛋白质整体含量入手有可能寻找出更合适的肺癌特异性分子标志，为肺癌的诊断预防和治疗提供依据。本发明首次发现肺癌患者血浆和肺组织中的唾液酸结合免疫球蛋白样凝集素-14(sialic acid-bindingimmunoglobulin-likelectin-14，Siglec-14)含量均显著高于健康对照，其中含量可以为单位体积的血浆所含的蛋白的质量。免疫球蛋白超家族(IgSF)是一类介导细胞之间或细胞与细胞基质之间相互结合和作用的粘附分子，参与细胞的识别、活化和信号转导，包括免疫应答、炎症反应、肿瘤转移等一系列重要生理和病理过程。免疫球蛋白样凝集素(Ⅰ型凝集素)(immunoglobulin-type lectin，I-type lectin)家族是免疫球蛋白超家族的重要一支，可与聚糖结构的化合物相结合，从而影响免疫应答。不同于其他免疫球蛋白超家族，唾液酸结合免疫球蛋白样凝集素Siglecs的特性是其具有高特异性的糖链结构配体——唾液酸化的碳水化合物。其表达于免疫细胞表面，介导抑制性信号。近年来研究表明Siglec广泛参与调节天然免疫和获得性免疫细胞功能，以及参与病原体识别和吞噬。同时，Siglec家族成员也参与免疫耐受的调控，并在自身免疫病、炎症反应和肿瘤发生中发挥重要的免疫调控作用。因此，越来越多的靶向Siglecs抗体或糖基化配体的治疗药物被相继研发并用于淋巴瘤、白血病和自身免疫疾病等多种Siglecs相关疾病的治疗。
目前已知Siglec-14抑制由病原体诱导的宿主免疫抑制效应，一些研究显示纯合Siglec-14空表达的中性粒细胞更易于免疫监视研究B型链球菌(GBS)，Siglec-14缺失也可能会阻止慢性阻塞性肺疾病(chronic obstructive pulmonary disease，COPD)进一步加重。
总之，深入揭示Siglec-14与肺癌发生、发展和转移的相关性，研究其潜在的临床诊治价值意义重大。本发明开发出基于肺癌高表达的Siglec-14的快速检测及靶向治疗的方法，对癌症患者提供更好的精准诊疗方法。
发明内容
本发明的目的是提供一种用对肺癌患者的支气管组织和肺泡灌洗液(BALF)进行处理，以免疫组化方法进行肺癌诊断为个体化靶向治疗提供依据。
为实现上述目的设计一种有关肺癌诊断试剂盒用于肺癌的检测方法，包括试剂盒、Siglec-14蛋白，其特征在于该方法包括以下步骤：
(1)肺癌诊断试剂盒设有若干个容器或液态芯片和标签，所述的容器或液态芯片中分别设有：
a、Siglec-14蛋白的特异性抗体；
b、磷酸缓冲盐溶液(PBS缓冲液)pH7.2-7.4，或0.01mol柠檬酸钠缓冲液pH6.0，或3％H2O2溶液，或辣根酶检测试剂；
(2)重组蛋白制备，扩增Siglec-14的核苷酸序列，或用含有该核苷酸的重组表达载体转化或转导至合适的宿主细胞；
(3)在合适的培养基中培养宿主细胞；
(4)从培养基或细胞中分离、纯化蛋白质；
(5)纯化的Siglec-14基因产物或者其具有抗原性的片段，注射于动物体内以诱导多克隆抗体制备的产生，同时特异性结合Siglec-14蛋白的抗体，也可以是用于检测比较的抗体；
(6)免疫组化：脱蜡和水化：取5μm的石蜡切片置于60℃烘箱，20min；待凉至室温。石蜡切片浸泡于二甲苯Ⅰ、Ⅱ各15min脱蜡；通过无水乙醇Ⅰ浸泡5min，无水乙醇Ⅱ浸泡5min，95％酒精浸泡5min，70％酒精浸泡5min等一系列不同浓度的乙醇浸泡至水化；石蜡切片置于摇床PBS洗2次，每次5min。
(7)封闭内源性过氧化物酶：每张切片滴加3％H2O2 l00ul，室温避光10min。石蜡切片置于摇床PBS洗3次，每次5min。
(8)抗原修复：配制300ml 0.01M PH6.0柠檬酸钠缓冲液，置于微波炉中火加热20min，等缓冲液自然冷却至室温后，置于摇床PBS洗3次，每次5min。
(9)封闭：取出切片，擦净切片背面水分及切片正面组织周围的水分(保持组织呈湿润状态)，先用免疫组化笔画个圈圈，后每张切片滴加5％正常山羊血清100ul,室温孵育1h。
(10)一抗孵育：用纸巾吸走封闭液，保持湿润，直接每张切片滴加siglec-14一抗100ul(1:100，用5％正常山羊血清稀释)。室温孵育1h后置于摇床PBS洗3次，每次5min。
(11)二抗孵育：每张切片滴加二抗100ul(1:1000，用5％正常山羊血清稀释)。室温孵育1h后置于摇床PBS洗3次，每次5min。
(12)DAB显色：按照DAB试剂盒说明：滴加1滴(大约30ul)发色剂浓缩液至1ml稀释液中混匀，每片加入100ul，然后避光室温孵育3min，然后自来水浸泡、冲洗。肉眼观察或镜下观察，呈棕黄色。
(13)苏木素复染：复染30s，然后用自来水浸泡2次，每次约10s，上下涮动，然后自来水冲洗，洗去苏木素颜色，纸巾擦去周围水分，保持湿润，准备拿去通风橱。
(14)梯度酒精脱水：70％酒精浸泡10s，95％酒精浸泡10s，无水乙醇Ⅰ浸泡10s，无水乙醇Ⅱ浸泡10s，二甲苯Ⅱ透明5min。
(15)封片：用1ml大枪头吸滴加一滴中性树胶于盖玻片上，然后封片，用镊子按压，保持无气泡，显微镜下观察组织，拍照。
重组蛋白制备：Siglec-14的编码序列通过逆转录-聚合酶链式反应(Real-timePCR，RT-PCR)获得，模板用的是肺癌组织互补DNA(cDNA)。获得的DNA片段经限制性内切酶BamHI/XohI双酶切后插入经BamHI/XohI双酶切的pGEX-4T-1中，构建重组质粒pGEX-4T-1-Siglec-14。转化重组质粒到BL21(DE3)中诱导表达，诱导表达的Siglec-14重组蛋白经过纯化，并通过凝胶电泳(SDS-PAGE)检测。
多克隆抗体制备：用生理盐水稀释Siglec-14重组蛋白，然后与弗氏不完全佐剂进行等比混合，吹打至抗原和佐剂完全混合形成稳定的乳剂，将该乳剂在兔子双肩周围的皮肤下进行皮下注射和后大腿进行肌肉注射。每次免疫前采血2ml(获得0.5-1ml免疫前血清)，并进行酶联免疫吸附试验(ELISA)和免疫印迹试验(Western Blotting，WB)检测。如果检测为阴性或抗体效价较低，则需要再次免疫。共免疫4次，末次放血20-30ml，纯化混合血样，平均每次纯化可以获得100-150mg抗体，最后进行ELISA和WB等质量检测。
本发明开发出可供临床应用的检测试剂，以深入地研究和大规模验证Siglec-14与肺癌的相关性，对癌症进行诊断及为个体患者提供更好的辅助治疗方法。
附图说明
图1(a)为：Siglec-14蛋白检测的肺癌组织；
图1(b)为：Siglec-14蛋白检测的癌旁组织。
具体实施方式
结合具体实施例对本发明做进一步说明，这种装置的制造技术对本专业的人来说是非常清楚的。
采用比较蛋白质组学方法对肺癌细胞系/组织和正常对照细胞/组织的蛋白质表达谱，或者肺癌BALF等体液进行蛋白谱比较分析，质谱鉴定其中部分差异蛋白质点。Siglec-14蛋白为其中一个差异点，即其在癌细胞中比正常细胞高表达，且可明显区分。
采用RT-PCR和免疫组化的技术，分别检测了肺癌患者及其配对的正常组织中的Siglec-14的信使RNA(mRNA)和蛋白表达含量，发现Siglec-14在肺癌组织中表达明显高于其配对的正常组织。该发现与比较蛋白质组的实验结果一致。表明Siglec-14能够作为一种肺癌的诊断工具。
因此，设计了一种试剂盒，通过Siglec-14抗体来检测Siglec-14的存在。
目前，已经可以通过重组DNA技术，来表达或生产重组Siglec-14蛋白。一般来说有以下步骤：
(1)扩增Siglec-14的核苷酸序列，或用含有该核苷酸的重组表达载体转化或转导至合适的宿主细胞；
(2)在合适的培养基中培养宿主细胞；
(3)从培养基或细胞中分离、纯化蛋白质。
纯化的Siglec-14基因产物或者其具有抗原性的片段，可注射于动物体内以诱导多克隆抗体的产生，同时特异性结合Siglec-14蛋白的抗体，也可以是商业化的抗体。
免疫组化具体包括：免疫组化具体包括：(1)脱蜡和水化：取5μm的石蜡切片于二甲苯中30min，脱蜡；然后通过一系列不同浓度的乙醇浸泡至水化。(2)封闭内源性过氧化物酶：每张切片滴加3％H2O2 l00ul，室温避光10min。石蜡切片置于摇床PBS洗。(3)抗原修复：配制300ml 0.01M PH6.0柠檬酸钠缓冲液，置于微波炉中火加热20min，等缓冲液自然冷却至室温后，置于摇床PBS洗。(4)封闭：取出切片，用免疫组化笔画个圈圈，后每张切片滴加5％正常山羊血清100ul,室温孵育1h。(5)一抗孵育：用纸巾吸走封闭液，直接在每张切片滴加siglec-14一抗100ul(1:100，用5％正常山羊血清稀释)。室温孵育1h后置于摇床PBS洗。(6)二抗孵育：每张切片滴加二抗100ul。室温孵育1h后置于摇床PBS洗。(7)DAB(二氨基联苯胺)底物染色。(8)切片用苏木精复染30s，自来水冲洗。(10)梯度酒精脱水：通过一系列不同浓度的乙醇浸泡至二甲苯。(11)处理完的切片，封片、镜检。实验中以水或缓冲液代替多克隆抗体做阴性对照，以Siglec-14过表达的标本为阳性对照。
实施例1
重组蛋白制备：Siglec-14的编码序列通过RT-PCR获得，模板用的是肺癌组织cDNA。获得的DNA片段经限制性内切酶BamHI/XohI双酶切后插入经BamHI/XohI双酶切的pGEX-4T-1中，构建重组质粒pGEX-4T-1-Siglec-14。转化重组质粒到BL21(DE3)中诱导表达，诱导表达的Siglec-14重组蛋白经过纯化，并通过SDS-PAGE检测。
多克隆抗体的制备免疫：用生理盐水稀释Siglec-14重组蛋白，然后与弗氏不完全佐剂进行等比混合，吹打至抗原和佐剂完全混合形成稳定的乳剂，将该乳剂在兔子双肩周围的皮肤下进行皮下注射和后大腿进行肌肉注射。每次免疫前采血2ml(获得0.5-1ml免疫前血清)，并进行ELISA和Western Blotting检测。如果检测为阴性或抗体效价较低，则需要再次免疫。共免疫4次，末次放血20-30ml，纯化混合血样，平均每次纯化可以获得100-150mg抗体，最后进行ELISA和WB等质量检测。
免疫组化：收集肺癌患者及其配对的正常组织切片各20例。(1)脱蜡和水化：取5μm的石蜡切片于二甲苯中30min，脱蜡；然后通过一系列不同浓度的乙醇浸泡至水化。(2)封闭内源性过氧化物酶：每张切片滴加3％H2O2 l00ul，室温避光10min。石蜡切片置于摇床PBS洗。(3)抗原修复：配制300ml 0.01M PH6.0柠檬酸钠缓冲液，置于微波炉中火加热20min，等缓冲液自然冷却至室温后，置于摇床PBS洗。(4)封闭：取出切片，用免疫组化笔画个圈圈，后每张切片滴加5％正常山羊血清100ul,室温孵育1h。(5)一抗孵育：用纸巾吸走封闭液，直接在每张切片滴加siglec-14一抗100ul(1:100，用5％正常山羊血清稀释)。室温孵育1h后置于摇床PBS洗。(6)二抗孵育：每张切片滴加二抗100ul。室温孵育1h后置于摇床PBS洗。(7)DAB(二氨基联苯胺)底物染色。(8)切片用苏木精复染30s，自来水冲洗。(10)梯度酒精脱水：通过一系列不同浓度的乙醇浸泡至二甲苯。(11)处理完的切片，封片、镜检。实验中以水或缓冲液代替多克隆抗体做阴性对照，以Siglec-14过表达的标本为阳性对照。结果，肺癌患者中12例有7例均有不同程度高表达，而正常组织中未见高表达。因此，Siglec-14高表达与肺癌有紧密的相关性，可供临床对癌症进行诊断及为个体患者提供更好的辅助治疗的有效方法。
实施例2
取受检者组织，经免疫组化检测，观察抗体染色得到诊断标准。染色阳性的判断为肺癌，参见图1(a)；阴性为癌细胞周围的正常组织，即癌旁组织，参见图1(b)。结果显示，应用组织进行免疫组化检测，肺癌检测敏感性为66.7％，特异性为100％(表1)
表1肺癌组织Siglec-14免疫组化检测结果
以上是对本发明的描述而非限定，基于本发明思想的其它实施方式，包括基于本发明标志进行的ELISA，免疫组化，Western blotting检测，及其药物靶点均在本发明的保护范围之中。
Claims (7)
1.选择性地检测一种蛋白Siglec-14的试剂在制备肺癌快速筛查及免疫靶向治疗检测的试剂盒中的用途，其特征在于，所述试剂用于测定得自肺癌患者血浆和肺组织中的蛋白Siglec-14含量，其中所述蛋白Siglec-14的含量均显著高于健康和良性肺部疾病患者。
2.根据权利要求1所述的用途，其特征在于，其中使用通过抗体和重组DNA技术来检测所述蛋白Siglec-14的存在。
3.根据权利要求2所述的用途，其特征在于，使用ELISA和免疫组化来检测所述蛋白Siglec-14的含量。
4.根据权利要求3所述的用途，其特征在于，所述试剂包括所述蛋白Siglec-14的反转录引物、定量聚合酶链式反应上游引物及下游引物。
5.根据权利要求1所述的用途，其特征在于，所述试剂盒用于：
a.测定得自所述肺癌患者血浆和肺组织中的蛋白Siglec-14的含量；
b.将血浆和肺组织中的蛋白Siglec-14的含量与一组数据对比，所述数据为正常受试者中的蛋白Siglec-14的含量；
c.基于步骤b做出的对比结果，确定受试者是否患有肺癌。
6.一种应用于肺癌快速筛查及免疫靶向治疗检测的试剂盒，其特征在于，具有如权利要求1中所述的用途，所述试剂盒包括若干个容器或液态芯片和标签，所述容器或液态芯片中分别设有：
a、蛋白Siglec-14的特异性抗体；
b、PBS缓冲液pH7.2-7.4，或0.01mol柠檬酸钠缓冲液pH6.0，或质量分数为3％H2O2溶液，或辣根酶检测试剂。
7.根据权利要求6所述的试剂盒，其特征在于，所述试剂盒还包括血浆样品中总RNA提取相关试剂、Siglec-14基因产物、其具有抗原性的片段的RT-PCR相关试剂的任一种或多种。
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