CN113249474A - Pcdh20在预测肝癌化疗敏感性中的应用 - Google Patents
Pcdh20在预测肝癌化疗敏感性中的应用 Download PDFInfo
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Abstract
本发明公开了一种PCDH20在预测肝癌化疗敏感性中的应用,属于生物医药技术领域,所述PCDH20作为标志物预测肝癌化疗敏感性,同时也可作为化疗敏感性的增效靶点。
Description
技术领域
本发明涉及生物医药技术领域,具体涉及一种PCDH20在预测肝癌化疗敏感性中的应用。
背景技术
肝细胞癌(Hepatocellular Carcinoma,HCC)是威胁人类健康的重大疾病全球范围内每年约有782,000例新发病例和746,000例死亡病例,其中高达55%患者集中在我国,给社会造成了严重负担。超过60%肝癌患者在确诊时已为中晚期,错失手术根治机会。基于铂类、抗代谢药物的化疗是中晚期HCC重要的治疗方案之一,方式包括全身化疗和肝动脉灌注化疗(Hepatic arterial infusion chemotherapy,HAIC)。全身化疗主要用于伴有肝外转移的晚期 HCC患者,而 HAIC多用于局部晚期HCC患者。但无论采取哪种化疗方式,化疗在HCC的局部及远期疗效上疗效均差强人意。即使是作为被亚太各大指南推荐的HAIC,通过导管技术找到肿瘤供血动脉并将抗癌药直接注入肿瘤组织或肿瘤床,可起到药物治疗的“首过效应”,能显著提高肿瘤局部药物浓度,并明显减轻全身不良反应。但据以往研究报道,晚期 HCC 患者接受HAIC 治疗也仅可获得 7.6 -17.6 个月的中位生存时间。
有文献报道,除化疗药物种类、肿瘤血液供应、肿瘤分期等因素外,原发性肝癌中存在的多药耐药(Multi-drug Resistance, MDR)机制可能是影响化疗疗效的关键因素,原因如下:1、肝癌细胞本身对化疗不敏感,即存在内源性耐药;2、肿瘤局部持续高浓度的药物接触,进一步诱导了获得性耐药的发生。因此,若能找到提高化疗敏感性的新策略,有望从根本上提高化疗疗效,联合基因治疗成为从根本上解决这一难题的潜在希望。
PCDH20是一种新型原钙黏蛋白,位于13q21.2,由6个细胞外钙黏蛋白结构域,1个跨膜区域以及1个胞质域,在相邻细胞间的黏附和连通中起着重要作用。其已被报道在非小细胞肺癌、大肠癌、鼻咽癌中发挥抑癌作用,但在HCC中的报道仅有两项。其中一项研究结果显示,PCDH20可通过拮抗Wnt / β-catenin通路抑制Akt及Erk的活性,增强GSK-3β的活性,在HCC中发挥抑癌作用。另一项研究发现,miR-122 / PCDH20 / Akt / mTOR信号轴在介导HCC化疗反应中具有重要作用,抑制miR-122或过表达PCDH20可降低HCC的耐药性。近有研究报道PCDH20可能是卵巢癌化疗的新靶点。上述信息提示我们,PCDH20可能在介导HCC的化疗效果中扮演了重要角色,其可能是HCC化疗增敏的重要潜在靶点。
发明内容
本发明提供一种PCDH20在预测肝癌化疗敏感性中的应用,所述PCDH20作为标志物预测肝癌化疗敏感性。
本发明解决其技术问题采用以下技术方案:
PCDH20作为标志物在制备用于预测肝癌化疗敏感性产品中的应用。
作为一种优选方案,所述产品包括芯片、试剂盒或试剂。
作为一种优选方案,所述产品通过PCDH20表达量进行预测。
作为一种优选方案,所述PCDH20过表达的受试者具有更高的化疗敏感性,所述PCDH20低表达的受试者具有更低的化疗敏感性。
本发明还提供了一种检测PCDH20表达量的试剂,用于预测肝癌化疗敏感性。
作为一种优选方案,所述试剂包括PCDH20抗体。
作为一种优选方案,所述试剂包括Real time-PCR的引物对。
作为一种优选方案,所述PCDH20过表达的受试者具有更高的化疗敏感性,所述PCDH20低表达的受试者具有更低的化疗敏感性。
本发明还提供了一种PCDH20在制备增强肝癌患者对化疗药物敏感性的产品中的应用。
本发明还提供一种用于增强肝癌患者对化疗药物敏感性的药物组合物,其特征在于,包括促进PCDH20表达的试剂。
发明人首先采用慢病毒感染SMMC7721肝癌细胞,构建PCDH20过表达稳转细胞株SMMC7721-PCDH20及对照空载细胞株SMMC7721-NC,并证实了PCDH20具有抑制肝癌细胞SMMC7721生长增殖、侵袭迁移、减慢细胞周期和促进细胞凋亡的功能,说明PCDH20可能在HCC的发生发展中起抑制作用。为进一步探讨PCDH20对HCC化疗敏感性的影响,发明人通过测定两组稳转细胞对奥沙利铂的IC50,发现SMMC7721-PCDH20细胞的IC50明显低于对照组。通过检测细胞活力发现,过表达PCDH20能增加奥沙利铂对肝癌细胞SMMC7721的杀伤作用,进一步说明了PCDH20可增加肝癌细胞SMMC7721的化疗敏感性。接下来发明人进一步深入探讨了PCDH20增加肝癌细胞的化疗敏感性的具体途径。发现经奥沙利铂处理后,PCDH20过表达细胞株的细胞调亡率明显高于空载细胞,并采用Western blot进行了验证,说明介导细胞凋亡是PCDH20发挥化疗增敏作用的机制之一。同时应用流式细胞仪对细胞的外排功能进行了检测,结果显示,SMMC7721-PCDH20细胞株内的化疗药物蓄积量明显高于对照细胞,提示PCDH20可能参与介导了ABC家族转运蛋白的调控,而MDR1是该家族的经典代表。发明人采用Western blot及Real-time PCR法对PCDH20过表达稳转株和空载对照细胞株中MDR1的mRNA及蛋白水平进行了检测,发现过表达PCDH20后MDR1的水平明显降低。并采用免疫组化法对30例肝癌组织中PCDH20及MDR1的表达水平同时进行了检测,结果提示,PCDH20与MDR1在蛋白水平呈明显负相关,且PCDH20低表达患者化疗效果差于PCDH20高表达者。接下来,发明人采用Real-time PCR法在五种不同肝癌细胞和正常肝细胞中也对两者的表达水平进行了检测,结果显示,两者在转录水平呈反向趋势。因此,抑制MDR1表达可能是PCDH20介导HCC化疗敏感性的重要机制。上述结果提示我们,PCDH20基因可作为肝细胞癌化疗疗效的预测因子及化疗增敏的潜在靶点。
本发明的有益效果:所述PCDH20作为标志物预测肝癌化疗敏感性,同时也可作为化疗敏感性的增效靶点。
附图说明
图1为目的质粒酶切鉴定图、部分测序图及荧光照片;
图2为Western blot及Real time-PCR鉴定结果图;
图3为细胞增殖实验图;
图4为细胞生长曲线图;
图5为细胞凋亡与周期图;
图6为划痕、细胞侵袭实验图;
图7为药物作用IC50实验图;
图8为细胞外排功能检测实验图;
图9为采取多种检测手段检测肝癌细胞中PCDH20及MDR1的表达水平图;
图中标记说明:图7中A:经奥沙利铂处理后细胞活力变化图;B:不同组别细胞对奥沙利铂的IC50检测结果。
图8中A:A中左上图为 SMMC7721-NC细胞经阿霉素处理前;A中右上图为SMMC7721-PCDH20细胞经阿霉素处理前;A中左下图为SMMC7721-NC细胞经阿霉素处理后;A中右下图为SMMC7721-PCDH20细胞经阿霉素处理后;
B:SMMC7721-PCDH20细胞内残留的阿霉素明显多于SMMC7721-NC细胞。
图9中A:Real time-PCR检测肝癌细胞中PCDH20及MDR1的mRNA表达水平图;
B:Western blot检测肝癌细胞中PCDH20及MDR1的蛋白表达水平图;
C:左上图为MDR1表达阴性组织(100 ×);右上为MDR1表达阳性组织(100 ×);左下图为MDR1表达阴性组织(200 ×);右下图为MDR1表达阳性组织( 200 ×);
D:免疫组化结果显示PCDH20与MDR1呈负相关图;
E:Western blot法检测肝癌细胞中PCDH20及MDR1的蛋白表达水平图;
F:荧光定量-PCR检测肝癌细胞中PCDH20及MDR1的mRNA表达水平图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
过表达PCDH20稳转细胞株及空载对照细胞株的构建及转染效能鉴定
①构建PCDH20过表达病毒载体
合成针对PCDH20全长表达序列的引物:
F:5’-GTACTCGAGATGCGCGGCCGAGGGAATGC-3’;
R:5’-GTAGGATCCTCAAATATTAGAAATATCCA-3’。
通过NEB公司高保真DNA聚合酶phusion对PCDH20模板进行扩增,扩增产物进行切胶回收后进行XhoI和BamHI双酶切3 h,同时慢病毒载体(pCDH-CMV-MCS-EF1-copGFP-T2A-Puro)进行XhoI和BamHI双酶切3 h,将双酶切后的载体进行切胶回收,将酶切回收后的PCR产物及酶切回收后的载体进行连接反应,连接体系采用NEB公司的快速T4连接酶及缓冲液,室温连接30 min后进行转化:从-80℃取出一支DH-5α感受态,快速溶解后冰浴10 min,然后将连接体系吸出缓慢与感受态混匀,继续冰浴30 min,将感受态与连接产物的混合物置于42℃水浴中进行热激,90s后将体系置于冰浴中5 min。加入1ml新鲜无抗性LB培养基,37℃摇床低速(170转 / min)复苏50 min。复苏结束后取出200 µl进行氨苄青霉素抗性板涂板,37℃细菌箱培养15 h,通过菌落PCR及测序对获得的阳性重组载体进行鉴定(图1)。接下来按照 QIAGEN Plasmid Maxi Kit试剂盒(Qiagen)说明抽提质粒。
1)293T细胞种于15cm培养皿生长,传代约3代后,待细胞汇合度至50~70%时,用于转染;
2)吸去培养基,用10 ml DPBS清洗两次,加3 ml胰酶消化,用含血清的培养基中和,收集细胞并计数,1000 rpm室温离心3 min,加入5 ml DPBS洗一次,1000 rpm室温离心3min,然后用不加抗生素的含血清5%的1640培养基重悬;
3)转染前,轻轻摇匀转染试剂Lipofectamine TM 2000,分别在2个2 mlEppendorf离心管中各加入750 µl Opti MEM。其中1个离心管另加35 µl LipofectamineTM 2000。另一个离心管加入8 µg目的质粒(PCDH20过表达)和包装质粒PMD2.G(8 µg)及PsPax2(16 µg),轻轻混和且在室温孵育5 min,孵育结束后,将上述两离心管液体混和,室温孵育20 min;
4)将上述准备好的293T细胞铺盘,每个15 cm 培养皿约1.2 × 107个细胞。将上述混合液加入至铺好细胞的培养皿中,轻轻混匀;
5)在37℃、湿度70%、5% CO2恒温培养箱中常规培养过夜,8 h更换新的无抗生素培养基。转染后60 h,收集细胞上清,2500 rpm离心,收集上清,用0.45 µm(MilliporeMillex-HV 0.45 µM PVDF)的过滤器过滤,收集滤液,分装,-80℃保存或立即使用。
加入病毒上清液500 µl及2 µl Polybrene(6 µg / mL),放置37°C培养箱中培养。细胞感染病毒后第二天,弃掉旧培养基,换不含Polybrene的新鲜完全培养液,继续放置培养箱中培养,感染后第三天,收集细胞检测目的蛋白的表达情况。挑选克隆,进行细胞扩增后,检测目的蛋白的表达情况(图2)。
发明人首先采用慢病毒感染SMMC7721肝癌细胞,构建PCDH20过表达稳转细胞株SMMC7721-PCDH20及对照空载细胞株SMMC7721-NC,并证实了PCDH20具有抑制肝癌细胞SMMC7721生长增殖、侵袭迁移、减慢细胞周期和促进细胞凋亡的功能,说明PCDH20可能在HCC的发生发展中起抑制作用。
实施例2
过表达PCDH20联合奥沙利铂对肝癌细胞形态及生物学功能的影响
将过表达PCDH20的稳转细胞株及对照细胞株分别分为四组:PCDH20高表达组,奥沙利铂组,PCDH20高表达联合奥沙利铂组及空白对照组,保证每个药物组奥沙利铂最终药物浓度20μg/ml。细胞处理24h后,采用光学显微镜观察上述细胞的形态变化,并采用CCK8法检测上述细胞的增殖能力,克隆形成、划痕及侵袭实验检测细胞的侵袭及迁移能力,流式细胞术检测细胞的凋亡率、细胞周期及外排功能。并设置不同的药物浓度,检测各组细胞对奥沙利铂的IC50。
(1)细胞种板:将处于对数生长期的细胞按5000个/孔接种到96孔板,置于5% CO2,37°C细胞培养箱培养24 h;
(2)结果检测与分析:24 h后,每孔加入含10% CCK-8试剂的培养基100 µl,于37°C温箱中孵育1 h,采用酶标仪双波长测定OD值(检测波长与观测波长分别为450 nm及630nm);
(3)上述实验重复3次,见图3,表明过表达PCDH20抑制细胞增殖;
(1)细胞种板:将处于对数生长期的细胞按500个/孔接种到96孔板,置于5% CO2,37°C细胞培养箱培养24 h。
(2)结果检测与分析:24 h后,每孔加入含10% CCK-8试剂的培养基100 µl, 37°C温箱中孵育1 h,酶标仪双波长测定OD值(检测波长与观测波长分别为450 nm及630 nm),测量结束后,弃掉含有CCK8的培养基,换用新鲜培养基,继续放回培养箱培养,以后每隔24 h,按上述步骤测量一次,连续计数5天;
(3)将数据输入GraphPad Prism 6.0绘图分析软件,以OD值为纵坐标,时间为横坐标,绘制生长曲线;
(4)上述实验重复3次,见图3,表明过表达PCDH20抑制细胞增殖;
细胞种板:将细胞种于6孔板,1×105个细胞/孔。
细胞凋亡检测:
(1)收集细胞:收集细胞废液,PBS洗涤细胞,用不含EDTA的胰酶消化3~5 min,加入含血清培养基终止消化,用移液枪将细胞混悬液转移至流式管中,1000 rpm离心5 min,弃上清;
(2)悬浮细胞:用500ul Binding Buffer混匀细胞;
(3)分别加入5 µl Annexin V-FITC和5 µl Propidiumlodide,然后混匀;
(4)室温条件下反应10 min,注意避光;
(5)上机检测,激发波长Ex = 488 nm;发射波长Em = 530 nm,见图5,表明过表达PCDH20促进细胞凋亡、减慢细胞周期;
细胞周期检测:
(1)用PBS洗涤细胞一次(1000 rpm,5 min),收集并调整细胞浓度为1× 106 /ml,取1ml单细胞悬液;
(2)单细胞悬液离心后,去除上清,在细胞中加入70%冷乙醇500 µl固定,4℃保存2h至过夜;
(3)倒掉固定液,用PBS清洗;
(4)然后加入RNase A 100 µl,放置于37℃水浴,约30 min;
(5)最后加入400 µl PI用于染色,放置于4℃,约30 min,注意避光;
(6)上机送检;
(7)以上实验均重复3次,见图5,表明过表达PCDH20促进细胞凋亡、减慢细胞周期;
(1)细胞种板:细胞计数,将处于对数生长期的细胞按1000个/孔种于6孔板,每孔加2 ml培养基,放置于5% CO2,37℃培养箱中培养;
(2)克隆计数:细胞培养3周后,显微镜下可观察到明显的单克隆( > 50个细胞)形成。吸走培养基后用PBS洗涤细胞两三次;甲醇固定15 min,0.1%结晶紫室温染色20 min,回收结晶紫,流水小心冲洗,待室温晾干后,于显微镜下随机选取5个视野,计数克隆个数,采用GraphPad Prism 6.0软件进行统计分析;
(3)以上实验重复3次。
(1)实验分组同上,将处于对数生长期的细胞分别以1.5 × 105/孔接种于6孔板上,放置于5% CO2,37℃培养箱中培养;
(2)在6孔板背面用黑色记号笔划三条横线做标记;
(3)待细胞融合度达80%时,用20 µl移液器枪头,垂直于已画的横线,划一条直线;
(4)用PBS清洗2遍,洗去漂浮的细胞,加入2 ml无血清培养基培养;
(5)用倒置显微镜分别于0、24和48 h拍照记录。24 / 48 h细胞迁移率=(0 h创口横径-24 / 48 h创口横径)/ 0 h创口横径 × 100%;
(6)以上实验重复3次,见图6,过表达PCDH20抑制细胞克隆形成、迁移及侵袭能力;
(1)细胞计数;
(2)分别取两组细胞配制成1 × 105 / ml细胞悬液,取100 µl细胞悬液分别加入两个小室内,做好标记。将含20%胎牛血清的1640培养基600 µl加入24孔板下室内;
(3)24 h后吸走培养基,用PBS清洗两三次,甲醇固定15 min,然后用0.1%结晶紫染色20 min,流水小心冲洗,用棉球轻轻擦掉小室内面未侵袭的细胞,空气中干燥,显微镜下随机选取5个视野观察,计数穿膜细胞数;
(4)以上实验重复3次,见图6,过表达PCDH20抑制细胞克隆形成、迁移及侵袭能力;
(1):奥沙利铂原液4℃保存。使用时按需要用1640培养液配制成预设的工作浓度。药物浓度设置:奥沙利铂药物浓度设置为1 µmol/L、10 µmol/L、25 µmol/L、50 µmol/L、100µmol/L、200 µmol/L;
(2)细胞种板:将SMMC7721-PCDH20及SMMC7721-NC细胞分别按1000个/孔种于96孔板,每孔加100 µl培养基;5% CO2,37℃培养;
(3)饥饿处理:种板18~24 h后,弃掉培养基,换成含1%血清的培养基,饥饿24 h;
(4)药物处理:用倍比稀释法配置含不同浓度梯度药物的培养基,加入到96孔板,100 µl / 孔。放回培养箱中继续培养;
(5)检测:药物处理24 h后,加入含10% CCK-8试剂的培养基,用酶标仪测定OD值;
(6)计算IC 50值:设定阳性对照:不加药物对照孔的OD平均值,空白对照:最高浓度抑制率为100%的OD平均值。抑制率计算公式:抑制率(%) = (阳性对照-实验组) / (阳性对照-空对照) × 100%。将获得的OD值录入Excel,四列数据分别为:药物浓度,与之对应的抑制率,对应的平均偏差,复孔数量。将数据复制进GraphPad prism 6.0作图软件,进行计算;
(7)以上实验重复3次,见图7。
(1)细胞种板:将SMMC7721-PCDH20及SMMC7721-NC细胞分别接种于6孔板中,5 ×105个细胞/孔;
(2)药物处理:将细胞在含阿霉素的培养基(10 µg/mL)中温育60 min,温育结束后吸走旧的培养基,用PBS洗涤两次,换成新鲜培养基(不含药物),继续放回培养箱中温育90min;温育结束后,胰酶消化收集细胞,l000 rpm离心5min;用冷PBS洗涤细胞两次,1000 rpm离心5 min;用1 ml PBS重悬细胞;
(3)送流式检测,结果分析;
(4)以上实验重复3次,见图8。
为进一步探讨PCDH20对HCC化疗敏感性的影响,发明人通过测定两组稳转细胞对奥沙利铂的IC50,发现SMMC7721-PCDH20细胞的IC50明显低于对照组。通过检测细胞活力发现,过表达PCDH20能增加奥沙利铂对肝癌细胞SMMC7721的杀伤作用,进一步说明了PCDH20可增加肝癌细胞SMMC7721的化疗敏感性。接下来发明人进一步深入探讨了PCDH20增加肝癌细胞的化疗敏感性的具体途径。发现经奥沙利铂处理后,PCDH20过表达细胞株的细胞调亡率明显高于空载细胞,并采用Western blot进行了验证,说明介导细胞凋亡是PCDH20发挥化疗增敏作用的机制之一。同时应用流式细胞仪对细胞的外排功能进行了检测,结果显示,SMMC7721-PCDH20细胞株内的化疗药物蓄积量明显高于对照细胞,提示PCDH20可能参与介导了ABC家族转运蛋白的调控,而MDR1是该家族的经典代表。
实施例3
采用荧光定量-PCR、Western blot及免疫组化法检测肝癌组织(30例)标本及多种肝癌细胞中PCDH20及MDR1的表达水平
(1)匀浆处理:
(a)肝癌组织:在高压好的研钵中加入少许液氮预冷,将100 mg组织置于研钵中,边研磨边加入液氮至充分研磨成粉末状,加入1 ml Triozol,冰上放置5 min;待Triozol融化后将研钵内混悬液移至1.5 ml EP管中,用匀浆仪进行匀浆处理;
(b)肝癌细胞:去除培养瓶中的旧培养基,用PBS冲洗3遍,然后在培养瓶中加入Triozol 1ml裂解细胞,用移液器反复吸打,直至细胞完全溶解至无絮状物,将裂解液移置无RNA酶的EP管中;
(2)向EP管中加入氯仿200 μl,剧烈振荡30 s混匀,室温放置10 min,至自动分层;
(3)1,5000 rpm,4℃离心15 min,使样品分为3层:上层水相,中间层以及下层有机相;
(4)将上层水相小心转移至新的EP管中;
(5)向新的EP管中加入等体积的异丙醇,充分混匀,室温放置10 min;
(6)1,5000 rpm,4℃离心10 min,管底可见RNA;
(7)弃掉上清,加入1ml冰冷的75%乙醇漂洗RNA沉淀;
(8)8000 rpm,4℃离心5 min;
(9)弃掉上清,加入1ml冰冷的无水乙醇继续漂洗RNA沉淀;
(10)弃掉上清,室温干燥10 min;
(11)加入20 μl RNase-free水,充分溶解RNA。进行后续操作或保存于-80℃。
RNA的纯度和浓度采用Nanodrop 2000超微量核酸蛋白分析仪测定。若RNA的A260/ A280的值在1.8~2.0之间,则表示纯度较高。
RNA测定纯度和浓度后,逆转录为cDNA。采用ABI 9700 PCR扩增仪。
逆转录反应体系见表1:
表1 逆转录反应体系
PCR仪(顶部加热型)65℃加热10 min后,立即将EP管放入冰浴中;
添加剩余组分(表2):
表2 剩余组分
加样完毕后,轻轻混匀,稍离心,放入PCR 仪。按以下条件设置逆转录反应程序:25°C 10 min,50°C 60 min,85°C加热5 min,最后至于冰浴上。
表3 引物列表
(1)取出cDNA产物于冰上;将100 µM PCR引物用无酶水稀释成10 µM;
(2)按照SYBR Green Mix 试剂说明,加入以下成分,表4:
表4 加入成分
表5Real-time PCR 扩增反应条件
⑤Western blot 实验步骤
(1)提取组织总蛋白和蛋白浓度的测定:
在高压好的研钵中加入少许液氮预冷,将组织置于研钵中,边研磨边加入液氮至充分研磨成粉末状,加入细胞裂解液,冰上孵育15 min,4℃ 1,4000 rpm 离心15 min,收集上清。按照BCA法测定蛋白浓度,用3 × loading buffer及蛋白裂解液配平,将配平好的蛋白样品于95℃中煮10 min,使蛋白变性,最后进行分装,取部分进行后续操作或保存于- 80℃;
(2)SDS-PAGE凝胶的制备及电泳:
(a)洗净玻璃板,晾干或用吹风机吹干,安装电泳装置;
(b)灌分离胶:根据所研究的蛋白质分子量选择相应的凝胶浓度,本实验采用8%分离胶,5%浓缩胶。将制好的分离胶混匀后均匀灌入玻璃板间,避免产生气泡,加1ml异丙醇封闭分离胶上层,放置约30 min,直到凝胶与异丙醇之间有一条明显的分隔线。
表6 8%分离胶30mL配胶体系
(c)灌浓缩胶:分离胶凝聚后,倒掉分离胶上的异丙醇,将混匀的浓缩胶加到分离胶上面,立即插上梳子,放置约20 min。待浓缩胶完全聚合后,洗净玻璃板上残留的胶,放置好胶板,固定卡槽,向电泳槽中加入电泳液,然后小心垂直向上拔掉梳子。
表7 5%浓缩胶 20mL配胶体系
(d)蛋白上样:取40 µg蛋白上样;
(e)电泳:先以80V等压电泳30 min,后转为120V继续电泳1~2h至溴酚兰到达分离胶的底部,即可终止电泳;
(3)转膜;
(a)提前备好PVDF膜,将纤维垫片、滤纸、PVDF膜等浸泡在转膜液中待用;
(b)按照黑面对胶、白面对膜的顺序安装“三明治”,避免夹层中残留气泡,将“三明治”固定入槽,加入电转缓冲液和冰袋;
(c)以250 mA恒流转膜100 min。
(4)封闭、孵育抗体;
(a)转膜结束后,取出PVDF膜,用TBST缓冲液洗膜5 min;
(b)将PVDF膜用含5%脱脂奶粉的TBST缓冲液室温封闭2 h;
(c)封闭结束后用TBST洗膜10 min × 3次;
(d)加入PCDH20抗体(1: 500),4°C孵育过夜;
(e)第二天回收一抗,TBST洗膜10 min × 3次;
(f)加入二抗(1: 3000缓冲封闭液稀释),室温孵育1.5 h;
(g)TBST洗膜10 min × 3次。
(5)化学发光显影;
(a)将发光试剂盒中A和B两种试剂等量混和均匀;
(b)取出膜甩去多余液体,将发光剂滴至PVDF膜上,将膜放入化学成像分析系统曝光;
(c)结果分析:采用 Image J图像分析软件定量分析靶蛋白及和内参GAPDH条带,求其比值进行统计分析;
阳性对照:正常肝脏组织;阴性对照:PBS替代一抗。
(1)烤片:将石蜡切片置于65℃保温培养箱中烤1h;
(2)脱蜡:将切片依次放入二甲苯I、II、III中各20 min;
(3)水化:将染色架放入100%、95%、85%、75%乙醇中各5 min;
(4)自来水冲洗2min。然后用双蒸水浸泡2次,每次2 min;
(5)抗原修复:抗原修复液置于玻璃缸中,微波炉高火煮沸,将切片架放入其中并全部浸没,继续煮沸5 min,温火20 min,关闭电源,待水温慢慢冷却至室温 (约1h),取出切片;
(6)双蒸水浸泡2次,每次2 min;
(7)PBS浸泡2次,每次2 min;
(8)用0.1%吐温+3%H2O2 PBS液浸泡30 min (1%吐温20 ml,30% H2O2 20 ml,PBS160 ml,现配现用);
(9)PBS浸泡3次,每次3 min;
(10)甩干片子,用棉球拭去周边水迹,用免疫组化笔标记组织;
(11)封闭:用3% BSA室温封闭1h;
(12)甩去液体,加入一抗,抗体用PBST稀释,放入湿盒,4℃过夜;
(13)取出切片,PBS浸泡3次,每次5 min;
(14)加入A液 (二抗),室温下封闭1h;
(15)PBS浸泡3次,每次5 min;
(16)染色:加入染色液(B液、C液以 1:50稀释,现配现用),染色时间根据显微镜下观察的着色情况而定。自来水终止染色;拭干玻片,苏木素复染2 min;自来水返蓝20 min;
(17)梯度酒精(50%,75%,95%,100%)脱水各5 min;
(16)在二甲苯I 中放置10 min,在二甲苯II中放置1h;
(19)中性树脂封片,显微镜下观察并拍片;
(20)结果评分,评分标准如下:
高倍镜下随机选取5个视野;
由2位病理医生按照染色强度和染色百分率进行评分。
阳性细胞染色:胞浆中出现棕黄色物且染色强度高于背景中非特异染色。
着色强度评分:无 = 0分;弱 = 1分;中 = 2分;强 = 3分。
阳性细胞率评分:0% = 0分; 0~10% = 1分;10~50% = 2分;50~100% = 3分。
将着色强度评分和阳性细胞率相乘得到最终的免疫组化评分(最小0分,最大9分,0~5分为阴性,6~9分为阳性)。
发明人采用Western blot及Real-time PCR法对PCDH20过表达稳转株和空载对照细胞株中MDR1的mRNA及蛋白水平进行了检测,发现过表达PCDH20后MDR1的水平明显降低。并采用免疫组化法对30例肝癌组织中PCDH20及MDR1的表达水平同时进行了检测,结果提示,PCDH20与MDR1在蛋白水平呈明显负相关,且PCDH20低表达患者化疗效果差于PCDH20高表达者。接下来,发明人采用Real-time PCR法在五种不同肝癌细胞和正常肝细胞中也对两者的表达水平进行了检测,结果显示,两者在转录水平呈反向趋势。因此,抑制MDR1表达可能是PCDH20介导HCC化疗敏感性的重要机制。上述结果提示我们,PCDH20基因为肝细胞癌化疗疗效的预测因子及化疗增敏的潜在靶点。
以上述依据本发明的理想实施例为启示,通过上述的说明内容,相关工作人员完全可以在不偏离本发明技术思想的范围内,进行多样的变更以及修改。本发明的技术性范围并不局限于说明书上的内容,必须要根据权利要求范围来确定其技术性范围。
Claims (10)
1.PCDH20作为标志物在制备用于预测肝癌化疗敏感性产品中的应用。
2.根据权利要求1所述的应用,其特征在于,所述产品包括芯片、试剂盒或试剂。
3.根据权利要求1所述的应用,其特征在于,所述产品通过PCDH20表达量进行预测。
4.根据权利要求1所述的应用,其特征在于,所述PCDH20过表达的受试者具有更高的化疗敏感性,所述PCDH20低表达的受试者具有更低的化疗敏感性。
5.检测PCDH20表达量的试剂,其特征在于,用于预测肝癌化疗敏感性。
6.根据权利要求5所述的试剂,其特征在于,所述试剂包括PCDH20抗体。
7.根据权利要求5所述的试剂,其特征在于,所述试剂包括Real time-PCR的引物对。
8.根据权利要求5所述的试剂,其特征在于,所述PCDH20过表达的受试者具有更高的化疗敏感性,所述PCDH20低表达的受试者具有更低的化疗敏感性。
9.PCDH20在制备增强肝癌患者对化疗药物敏感性的产品中的应用。
10.一种用于增强肝癌患者对化疗药物敏感性的药物组合物,其特征在于,包括促进PCDH20表达的试剂。
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