CN117420309B - 一种用于评价结直肠癌患者免疫治疗反应性和评估直肠癌预后的三联标志物及其应用 - Google Patents
一种用于评价结直肠癌患者免疫治疗反应性和评估直肠癌预后的三联标志物及其应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域,具体涉及一种用于评价结直肠癌患者免疫治疗反应性和评估直肠癌预后的三联标志物及其应用,其中,所述标志物组合包括OCT1、CRK和PD‑L1,所述肿瘤为结直肠癌。可以通过检测外周血白细胞中OCT1、CRK和PD‑L1的含量,基于OCT1、CRK和PD‑L1这3个指标建立了评估结直肠癌免疫治疗疗效的数学模型,此数学模型预测免疫治疗可能无效的AUC为0.957,在治疗前可以很好地评估肿瘤患者免疫治疗的疗效,为精准免疫治疗方案的确定提供新方法和分子靶点。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种用于评价结直肠癌患者免疫治疗反应性和评估直肠癌预后的三联标志物及其应用。
背景技术
肿瘤的发生与宿主免疫调控密切相关:机体免疫***中具有细胞毒性的细胞(巨噬细胞、树突状细胞、调节性T细胞、髓系抑制性细胞、NK细胞等)可通过免疫监视及时清除癌变细胞;部分肿瘤细胞通过突变从而失去自身的免疫原性,或改变表观遗传等方式而躲过免疫***清除进入“平衡期”;在此期间免疫***和癌变细胞互相编辑,当这一平衡状态被肿瘤细胞打破后,肿瘤细胞将实现“免疫逃逸”。目前研究显示,肿瘤免疫逃逸的机制错综复杂,相关机制可能有:⑴肿瘤细胞自身参与免疫逃逸:肿瘤细胞通过改变自身的免疫原性和生物学特性,使得免疫***无法对其进行识别,逃避免疫杀伤;⑵肿瘤细胞改变微环境参与免疫逃逸:如通过释放炎症因子改变肿瘤信号转导通路、肿瘤代谢重编程、肿瘤细胞表面表达抑制性配体等方式抑制免疫细胞发挥作用。近年来,结直肠癌的免疫治疗受到人们的广泛关注,但目前尚没有评估结直肠癌免疫治疗反应和预后的有效方法。
肿瘤抗原特异性T细胞的诱导凋亡和无反应性是肿瘤细胞免疫逃逸中的主要机制,而PD-1/PD-L1信号通路正是存在于肿瘤微环境中介导免疫逃逸的关键分子。PD-1是一种跨膜蛋白受体,可在T细胞、B细胞、NK细胞以及单核细胞表面表达,PD-L1表达于肿瘤细胞表面,机制是通过T细胞表面的PD-1蛋白与肿瘤细胞表面产生的PD-L1结合,活化PD-1/PD-L1下游通路,传递负性调节信号,导致肿瘤微环境中PD-1通路持续激活,T细胞功能被抑制而不能识别肿瘤以至于不能攻击和杀伤肿瘤细胞。
PD-1/PD-L1信号通路介导的机体免疫抑制主要依赖于表达PD-1与其配体细胞的相互作用,PD-1/PD-L1在体内分布的差异及其在细胞膜上非恒定表达的特点决定了其生物学效应的不同,其在机体的表达与调控受多种因素的影响。为了增强结直肠癌免疫治疗的有效策略,结直肠癌中PD-L1调节的潜在机制需要进一步研究。
PD-L1的表达也是个动态变化的过程。在多种类型肿瘤中,一些信号通路的异常激活以及染色体畸变等与其表达上调密切相关。OCT1是POU结构域转录因子家族成员之一,又名POU2F1。作为转录因子,OCT1可以激活或是抑制B细胞中的免疫球蛋白基因以及白介素相关基因等多种转录因子。对TCGA结直肠癌测序数据分析发现,OCT1表达与CRK表达呈正相关,生物信息学显示OCT1能够同CRK启动子相结合。既有的研究显示CRK是促进PD-1/PD-L1的分子调控机制之一。越来越多的研究表明OCT1和CRK在细胞恶性转变过程扮演着至关重要的角色,但其在结直肠癌免疫中的作用以及在免疫反应中的具体作用机制仍不清楚。
发明内容
本发明的目的在于提供一种用于评价结直肠癌患者免疫治疗反应性和评估直肠癌预后的三联标志物及其应用,为精准免疫治疗方案的确定提供新的筛选指标,提高患者的治疗效果。
本发明的目的是通过如下技术方案实现的:
本发明提供了一种用于评价肿瘤患者免疫治疗反应性和评估肿瘤预后的标志物组合,所述标志物组合包括OCT1、CRK和PD-L1。
进一步的,所述肿瘤为结直肠癌。
进一步的,所述免疫治疗为免疫检查点抑制剂-抗PD-1/PD-L1治疗。
本发明还提供了一种用于评价结直肠癌患者免疫治疗反应性和评估结直肠癌预后的产品,所述产品中含有定量检测权利要求1所述的标志物组合的试剂。
进一步的,所述产品包括检测试剂盒、多聚酶链反应试剂、芯片检测试剂或测序试剂。
进一步的,所述定量检测权利要求1所述的标志物的试剂为能够定量检测OCT1、CRK和PD-L1的引物或探针。
本发明还提供了一种所述的标志物组合或所述的产品在评价肿瘤患者免疫治疗反应性和评估肿瘤预后中的应用,所述肿瘤为结直肠癌。
本发明还提供了一种利用所述的标志物组合评估结直肠癌免疫治疗疗效的***,所述***包括蛋白浓度检测装置、免疫治疗疗效评估装置及结果输出装置。
进一步的,所述蛋白浓度检测装置用于检测所述的标志物组合的含量;所述免疫治疗疗效评估装置通过蛋白浓度检测装置的检测结果,评估免疫治疗疗效;所述结果输出装置用于输出免疫治疗疗效评估结果。
进一步的,评估免疫治疗疗效的公式通过回归分析得到;所述评估免疫治疗疗效的公式为:风险值=3.0933×OCT1+2.0124×CRK+3.1292×PD-L1-11.1842;
式中,OCT1、CRK和PD-L1表达量的单位为ng/ml。
申请人基于大量的验证试验,确认了由OCT1、CRK和PD-L1所组成的三联标志物可用OCT1调控CRK/PD-L1轴促进结直肠癌免疫逃逸的机制预测结直肠癌免疫治疗反应性及预后。基于此,申请人可以在采用上述三联标志物的基础上制备一种用于评估肿瘤免疫治疗疗效的***,包括:
蛋白浓度检测装置:用于检测患者样本中的OCT1、CRK和PD-L1的含量;
免疫治疗疗效评估装置:根据OCT1、CRK和PD-L1的含量,评估免疫治疗疗效;
结果输出装置:输出免疫治疗疗效评估结果。
在一些实例中,评估免疫治疗疗效的公式通过回归分析得到。
在一些实例中,评估免疫治疗疗效的公式为:
风险值=3.0933×OCT1+2.0124×CRK+3.1292×PD-L1-11.1842
式中,OCT1、CRK和PD-L1的表达量的单位为ng/ml。
本发明和已有技术相比,其技术进步是显著的。
有益效果:
本发明的一些实例,可以通过检测外周血白细胞中OCT1、CRK和PD-L1的含量,基于OCT1、CRK和PD-L1这3个指标建立了评估结直肠癌免疫治疗疗效的数学模型,此数学模型预测免疫治疗可能无效的AUC为0.957,在治疗前可以很好地评估肿瘤患者免疫治疗的疗效,为精准免疫治疗方案的确定提供新方法和分子靶点。
本发明一些评估肿瘤免疫治疗疗效***的实例,可用于治疗前预评估肿瘤患者免疫治疗的疗效。本发明聚焦“OCT1调控CRK/PD-L1轴促进结直肠癌免疫逃逸的机制预测结直肠癌免疫治疗反应性的三联标志物”,为精准免疫治疗方案的确定提供新的筛选指标,提高患者的治疗效果。
附图说明
图1为OCT1在结直肠癌肿瘤样本中的蛋白表达结果图,其中,图1A为OCT1在结直肠癌组织与正常组的表达差异,图1B为qPCR结果,图1C为相关性;
图2为OCT1调控PD-L1蛋白的表达结果图,其中图A、图C为PCR实验结果图,图B、图D为Western Blot实验结果图及相应的条带光密度统计图,图E、图F为流式细胞分析图;
图3为OCT1作为启动子调节蛋白调控CRK结果图,其中图3A为验证OCT1作为RNA结合蛋白,图3B为检测RNA水平CRK的变化,图3C为使用放线菌素D(1μM)按时间梯度处理细胞,图3D为OCT1在CRK的启动子区的转录结合位点示意图,图3E为野生型和突变型两种荧光素酶报告质粒对比图;
图4为OCT1通过CRK调控PD-L1轴促进结肠癌细胞增殖结果图,其中图4A、图4B、图4C分别表示qPCR和western blot检测PD-L1的mRNA和蛋白表达情况以及生存几率;
图5为OCT1通过CRK增强PD-L1转录并且促进结肠癌细胞免疫抑制结果图,其中图5A、图5B为流式细胞术验证,图5C、图5D为采用免疫荧光实验验证,图5E为体外T细胞杀伤实验验证;
图6为38例免疫治疗无效患者和24例免疫治疗有效患者外周血白细胞中OCT1、CRK和PD-L1表达水平比较情况,其中图6A为检测OCT1,图6B为检测CRK,图6C为检测PD-L1;
图7为OCT1、CRK和PD-L1在结直肠癌中作为预测肿瘤免疫治疗反应性的三联标志物结果图。
具体实施方式
本发明聚焦“OCT1调控CRK/PD-L1轴促进结直肠癌免疫逃逸的机制预测结直肠癌免疫治疗反应性的三联标志物”,为证实这一机制通路,拟从表观遗传调控角度,进行以下七个实施例的试验进行验证:
实施例1OCT1在结直肠癌肿瘤样本中的蛋白表达
1、从美国癌症基因组图谱(TCGA)数据库选取539例结直肠癌病人mRNA芯片;预测OCT1的表达变化。
2、数据分析:
①通过TCGA数据库预测OCT1在正常和肿瘤组织中的表达差异;
②通过qPCR分析62例临床样本确定OCT1在正常和肿瘤组织中的表达差异;
③利用相关性分析确定OCT1与PD-L1和PDCD1的相关性。
具体分析结果见图1,由图1A可以看出:OCT1在结直肠癌组织中的表达高于正常组织;qPCR结果如图1B所示,OCT1在结直肠癌组织中的表达高于正常组织。细胞正相关基因与结肠癌免疫反应和T细胞活性密切相关,说明OCT1参与调节结肠癌免疫反应和免疫细胞,同时OCT1与结肠癌免疫抑制微环境相关。由图1C可以看出:OCT1与CD271(PD-L1)在结肠癌病人数据库中的相关性为0.56。
实施例2OCT1与PD-L蛋白的相关性研究
结直肠癌细胞株SW620和HCT116的培养建立步骤如下:取临床切除的新鲜结肠癌组织,使用无菌器械将组织剪成1×1cm大小,放入培养皿中,加入适量DMEM/F12含10%FPS的培养基,置于37℃恒温培养箱中,期间定时换液,细胞长满后进行传代收集,进行后续实验。在细胞中借助慢病毒载体外源性过表达PTRF,进行如下操作和分析:
(1)分别提取细胞的RNA进行RT-PCR分析检测mRNA水平PD-L1的表达变化
RNA提取操作步骤如下:
1)移除细胞培养基,使用无菌PBS清洗三次,加入Trizol提取试剂,室温静置5min,使用移液枪反复抽吸Trizol吹打细胞,移至无酶EP管中。
2)向EP管中加入五分之一Trizol体积的氯仿,上下颠倒,手动振荡30s,4℃离心机,12000rpm离心15min。
3)离心后将EP管从离心机中小心取出,管中液面分为三层,上层水相为RNA,下层有机相为蛋白质和DNA,小心吸取上层水相的RNA,至另一个无酶的EP管中。
4)向EP管中加入与水相RNA等体积的异丙醇,上下翻转混匀,室温静置10min。
5)将EP管放于4℃离心机中,12000rpm离心15min,取出EP管,可在离心管底部看到片状RNA沉淀,小心将上清倒掉。
6)向EP管中加入1mL 75%的乙醇,上下颠倒,涡旋振荡RNA沉淀30s。EP管于4℃离心机中12000rpm离心15分钟。
7)倒掉EP管中的上清,向EP管中加入1mL无水乙醇,上下颠倒,涡旋振荡RNA沉淀30s,4℃离心机,12000rpm离心15分钟。
8)弃去EP管中的上清,将EP管倒置于超净工作台中,通风干燥10-20分钟。
9)使用移液枪吸取20μL DEPC水溶解RNA沉淀。
荧光定量PCR实验步骤如下:
1)在NCBI使用Primer-BLAST设计引物并在数据库进行比对验证,之后找公司合成引物。
2)在qPCR八联管中按照10μL的2×SYBR Green qPCR混合物、1μL的基因上游引物(10μM)、1μL的基因下游引物(10μM)、2μL的模板DNA/cDNA、6μL的无核酸酶DEPC水总体积为20μL的配制混合液体系,然后使用实时荧光定量PCR***进行荧光定量PCR实验。
3)每个样本分别设置三个重复,根据PCR实验的熔解曲线和CT值,通过ΔΔCT计算公式得出实验结果,以GAPDH为内参比较各组之间目的基因的相对表达水平。
(2)分别提取细胞的蛋白进行Western Blot分析检测蛋白水平PD-L1的表达变化
其中,细胞总蛋白提取具体步骤如下:
1)在提取蛋白前,先配制细胞蛋白裂解液,一般使用高效RIPA裂解液,添加PMSF使其终浓度为10mM,颠倒混匀,置于冰上待用。
2)去除培养基,使用无菌PBS清洗细胞三遍,向培养皿中添加含PMSF的RIPA裂解液。
3)将培养皿晃动使裂解液浸润细胞,然后置于冰上放置30分钟进一步裂解细胞。
4)使用移液枪将细胞裂解液混合物吸取到干净1.5mL的EP管,在4℃离心机中,12000rpm离心15分钟。
5)离心后,使用移液枪将细胞裂解上清转移到新的EP管中,进行BCA蛋白浓度检测,对细胞总蛋白进行定量。
6)Western blot蛋白免疫印迹实验具体步骤如下:
配制SDS-PAGE凝胶,按装置夹好胶板放入电泳槽中。加入电泳缓冲液,将样品加入相应的孔中,盖好电泳槽的盖子,连接电泳仪,设置为恒压80kv-100kv。大约90-120分钟,当上样缓冲液中的溴酚蓝跑出胶板底部时,即可终止电泳,进行转膜。将PVDF膜裁剪为大小为5×8cm的长方形膜,并用无水甲醇浸泡激活PVDF。配制转膜缓冲液,将转膜夹放入盘中,将胶板上的胶小心取下,置于转膜夹的海绵垫子和滤纸上,盖上已经激活的PVDF膜,再用另一层滤纸和海绵垫子将胶和PVDF膜覆盖,最后将夹子夹紧合上。将转膜槽放于冰上并倒入转膜缓冲液,将转膜夹按照正负极放入转膜槽相应的位置,盖上转膜槽的盖子,连接电泳仪并通电,将电压设置为80kv,时间为90分钟。配制牛血清白蛋白封闭缓冲液(BSA封闭液),打开夹子将PVDF膜取出,置于含有5%BSA封闭液的封闭小盒中放在脱水摇床上缓慢摇动,室温孵育1-2小时进行封闭。按照抗体说明书的抗体使用浓度配制一抗工作液,将封闭液从封闭小盒中取出并回收,直接加入配好的一抗工作液,置于4℃摇床上,缓慢摇动过夜。转天,将一抗工作液回收,向PVDF膜加入PBST缓冲液,室温置于脱色摇床上快速摇动,清洗PVDF膜,时间8-10分钟,使用PBST清洗三次。配置相应种属的二抗工作液,待清洗PVDF膜最后一次后,将PBST缓冲液倒掉,重新加入二抗工作液,室温置于摇床上缓慢摇动,孵育1-2小时。将配制化学发光液进行Westernblot PVDF膜曝光。
(3)将细胞消化成单细胞悬液进行流式染色检测细胞中PD-L1的表达水平变化,以确定OCT1对PD-L1的调控作用。具体步骤如下:
1)将孔板从培养箱中拿出,使用PBS迅速清洗三遍,弃去PBS,向孔板中加入胰蛋白酶,待细胞消化下来后使用培养基终止消化。
2)将细胞悬液转移到流式管中,使用水平转子离心机,室温1000rpm离心5-8分钟。
3)PBS清洗,室温1000rpm离心5-8分钟,重复三遍,加入10%血清重悬细胞,室温30分钟进行封闭。
4)PBS清洗,室温离心5-8分钟,重复三遍,将抗体使用100-200μL细胞染色缓冲液稀释,重悬细胞沉淀,室温孵育30-40分钟。
5)PBS清洗,室温离心5-8分钟,重复三遍,使用100-200μL适宜浓度的荧光染色二抗将细胞沉淀重悬,室温避光孵育30分钟。
6)PBS清洗,室温离心5-8分钟,重复三遍,最后以100-200μLPBS将细胞沉淀重悬,使用BD流式细胞仪上机检测细胞染色蛋白的表达水平。
结果见图2,由图2A和图2C可以看出:在SW620细胞中,PCR实验证明过表达OCT1升高PD-L1mRNA表达水平;由图2B和图2D可以看出:在SW620和HCT116细胞中,Western Blot实验证明过表达OCT1升高PD-L1蛋白表达水平;由图2E和图2F可以看出:在SW620和HCT116细胞中,流式细胞术分析证明过表达OCT1促进PD-L1表达水平的升高。
实施例3OCT1作为启动子调节蛋白调控CRK表达
(1)在SW620细胞中进行RIP实验,利用RT-PCR进行验证OCT1作为RNA结合蛋白是否与CRK结合。结果如图3A所示,结果显示OCT1作为RNA结合蛋白可以与CRK结合。
(2)OCT1调控CRK的mRNA水平。
在细胞中过表达OCT1和敲低OCT1,通过RT-PCR实验检测RNA水平CRK的变化。
具体操作步骤如下:使用GV348载体构建的OCT1过表达慢病毒载体转染SW620细胞。使用敲低OCT1慢病毒载体转染SW620细胞。RT-PCR实验步骤同实施例2。
结果见图3B可以看出:在SW620中过表达OCT1能够促进CRK的表达;
(3)利用放线菌素D(1μM)按时间梯度处理过表达OCT1的细胞,运用RT-PCR实验与对照组细胞的CRK水平对比,分析OCT1对CRK稳定性的影响。
具体操作步骤为:将放线菌素D配置成1mM浓度,将细胞铺在6孔板中,每毫升培养基中加入1微升放线菌素D,将一定培养基加入培养板中,分别培养2,4,6和8小时,然后收集细胞进行实验。RT-PCR实验步骤同实施例2。
结果见图3C:使用放线菌素D(1μM)按时间梯度处理细胞发现,过表达OCT1抑制了CRKRNA的降解,提高了其稳定性。
(4)确定OCT1转录激活CRK:在CRK的启动子区寻找OCT1的转录结合位点,通过构建野生型和突变型两种CRK荧光素酶报告质粒(操作方法同实施例2)并结合CHIP和荧光素酶报告实验确定OCT1转录激活CRK及结合位点。
结果见图3,图3D为OCT1在CRK的启动子区的转录结合位点示意图。由图3E可以看出:通过构建野生型和突变型两种荧光素酶报告质粒并结合荧光素酶报告实验进一步确定了OCT1可以转录激活CRK及转录结合位点。
实施例4OCT1通过CRK调控PD-L1轴促进结肠癌细胞增殖
将肿瘤组织使用无菌器械取下,然后将胰蛋白酶和无菌PBS按1:1稀释并加入DNA酶配成消化液。使用无菌眼科剪,将肿瘤组织块剪成小米粒大小,然后加入消化液放入大皿置于37℃培养箱中,消化1-2小时,每隔15分钟镜下观察消化情况并使用移液枪吹打。待消化后,将消化混合物加入到40μm细胞过滤网中过滤单个肿瘤细胞,将细胞悬液加入到15mL离心管中,室温1000rpm离心10分钟。将上清弃除,使用PBS将细胞沉淀重悬,向肿瘤细胞悬液中加入过表达OCT1慢病毒(慢病毒购于上海吉凯基因化学技术有限公司)以及荧光素酶慢病毒,37℃培养箱反应4小时左右。将转染过表达OCT1慢病毒载体或者敲低CRK慢病毒载体和荧光素酶的细胞收集至15mL离心管中,1000rpm离心十分钟,去除上清,使用无菌PBS重悬洗涤细胞并离心。计数细胞,将细胞数量调成至合适密度,移至无菌1.5EP管中1000rpm离心10分钟。
将细胞铺板,分为四组:ex-NC、ex-OCT1、ex-NC+shCRK、OCT1+shCRK。采用CCK8法检测细胞的增殖情况。采用qPCR和western blot检测PD-L1的mRNA和蛋白表达情况。
结果见图4,由图4A可以看出:过表达OCT1促进肿瘤的生长,其作用可以被敲低CRK所阻断。由图4B和4C可以看出:过表达OCT1的荷瘤小鼠中生存时间最短,敲低CRK可以抑制肿瘤的生长,延长荷瘤小鼠生存时间。
实施例5OCT1促进CRK转录,增强PD-L1表达并且促进结肠癌细胞免疫抑制
检测OCT1/CRK对结直肠癌细胞免疫功能的影响:
1)通过流式细胞术和免疫荧光检测肿瘤细胞PD-1结合能力。
2)利用体外T细胞杀伤实验分析OCT1/CRK对结直肠癌细胞的免疫反应的影响。
具体操作步骤如下:SW620细胞中过表达OCT1以及敲低CRK。使用GV348载体构建的OCT1过表达慢病毒载体转染SW620细胞。使用CRK siRNA转染SW620细胞敲低CRK。当细胞密度至70-90%汇合度时进行转染,在生物安全柜中进行操作。在1.5mL的无菌EP中加入无血清培养基,加入Lipofectamine3000试剂进行充分混匀。在另外一个1.5mL的无菌EP中加入无血清培养基,加入siRNA制成预混液,然后加入相应的P3000试剂充分混匀。然后在稀释Lipofectamine 3000试剂的1.5mL EP管中以1:1比例加入稀释的siRNA,室温孵育5-10分钟。向相应的细胞培养皿中加入siRNA脂质体复合物,细胞在37℃二氧化碳培养箱中孵育2-4天,然后对转染细胞进行相应的分析实验。
结果见图5。由图5A和5B可以看出:流式细胞术验证过表达OCT1可以促进肿瘤细胞PD-1结合能力。由图5C和5D可以看出:免疫荧光实验验证过表达OCT1可以促进肿瘤细胞PD-1结合能力。由图5E可以看出:体外T细胞杀伤实验验证过表达OCT1抑制结直肠癌细胞免疫反应,敲低CRK逆转OCT1引起的免疫抑制微环境。
实施例638例免疫治疗无效患者和24例免疫治疗有效患者外周血白细胞中OCT1、CRK和PD-L1表达水平比较情况
收集本院CRC患者ICI治疗前血清标本:评估患者ICI的疗效;收集临床资料(姓名,性别,年龄,诊断,PD-1开始时间,PD-1治疗方案,TNM分期,转移数量,转移部位,病理类型,吸烟,饮酒,BMI等);ELISA检测其中CRC患者血浆中相关指标水平:OCT1、CRK、PD-L1;收集患者相关炎症指标。在ICI治疗有效组和进展组的血清中采用两独立样本比较的秩和检验比较,最终筛选出两组差异具有统计学意义(P<0.05)的指标:OCT1、CRK、PD-L1(图6)。
实施例7OCT1、CRK和PD-L1在结直肠癌中作为预测肿瘤免疫治疗反应性的三联标志物
收集临床结直肠癌病人组织样本,以免疫组织化学技术联合多种生物标志物检测为手段,研究OCT1、CRK、PDL1和CD8+T细胞的表达空间特征。
具体操作步骤为:
1)石蜡切片脱蜡水化
2)配制EDTA缓冲液,加入抗原修复盒中置于微波炉里加热至沸腾,将组织切片***抗原修复盒中,持续时间为15min,置于室温冷却。
3)向组织滴加适量的内源性过氧化物酶阻断剂,室温孵育反应10min。
4)将阻断剂去除,加入PBS溶液,室温摇动5min冲洗三次。滴加适量的封闭用正常山羊血清工作液,室温孵育反应40分钟至1小时。
5)反应完成后,弃去血清,向组织滴加适量一抗,置于湿盒中,4℃过夜孵育反应。
6)将一抗稀释液回收,加入PBS溶液,室温摇动5min冲洗三次,滴加适量的生物素标记山羊抗兔IgG聚合物,室温孵育反应40分钟至1小时。
7)将生物素标记山羊抗兔IgG聚合物弃去,加入PBS溶液,室温摇动5min清洗三次,滴加辣根酶标记链霉素卵白素工作液,室温孵育反应40分钟至1小时。
8)加入适量新鲜配制的DAB显色液,室温孵育,计时直至镜下观察出现阳性染色。
9)使用苏木素染色液室温孵育反应5分钟,PBS溶液冲洗,盐酸酒精分化,氨水反蓝。
10)将组织石蜡切片脱水透明,使用中性树胶封片。
结果见图7,由图7可以看出:免疫组织化学染色在病人组织样本中验证OCT1高表达病人CRK和PDL1表达水平增加。由图7可以看出:在公共数据库中通过OCT1-CRK-PD-L1三者表达水平可以区分患者的预后和预测免疫反应。
基于上述实施例,申请人确认了由OCT1、CRK和PD-L1所组成的三联标志物可用OCT1调控CRK/PD-L1轴促进结直肠癌免疫逃逸的机制预测结直肠癌免疫治疗反应性及预后。基于此,申请人可以在采用上述三联标志物的基础上制备一种用于评估肿瘤免疫治疗疗效的***,包括:
蛋白浓度检测装置:用于检测患者样本中的OCT1、CRK和PD-L1的含量;
免疫治疗疗效评估装置:根据OCT1、CRK和PD-L1的含量,评估免疫治疗疗效;
结果输出装置:输出免疫治疗疗效评估结果。
在一些实例中,评估免疫治疗疗效的公式通过回归分析得到。
在一些实例中,评估免疫治疗疗效的公式为:
风险值=3.0933×OCT1+2.0124×CRK+3.1292×PD-L1-11.1842
式中,OCT1、CRK和PD-L1的表达量的单位为ng/ml。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (1)
1.一种利用标志物组合评估结直肠癌免疫治疗疗效的***,其特征在于,所述***包括蛋白浓度检测装置、免疫治疗疗效评估装置及结果输出装置;所述标志物组合包括OCT1、CRK和PD-L1;所述免疫治疗为免疫检查点抑制剂-抗PD-1/PD-L1治疗;
所述蛋白浓度检测装置用于检测所述的标志物组合的含量;所述免疫治疗疗效评估装置通过蛋白浓度检测装置的检测结果,评估免疫治疗疗效;所述结果输出装置用于输出免疫治疗疗效评估结果;
评估免疫治疗疗效的公式通过回归分析得到;所述评估免疫治疗疗效的公式为:
风险值=3.0933×OCT1+2.0124×CRK+3.1292×PD-L1–11.1842 ;
式中,OCT1、CRK和PD-L1表达量的单位为ng/ml。
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