CN107632160A - Celsr3蛋白在制备肝癌术后预后评估试剂盒中的应用、肝癌预后评估试剂盒及方法 - Google Patents

Celsr3蛋白在制备肝癌术后预后评估试剂盒中的应用、肝癌预后评估试剂盒及方法 Download PDF

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CN107632160A
CN107632160A CN201710759221.0A CN201710759221A CN107632160A CN 107632160 A CN107632160 A CN 107632160A CN 201710759221 A CN201710759221 A CN 201710759221A CN 107632160 A CN107632160 A CN 107632160A
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林尧
王清水
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Xiamen Baiheng Biotechnology Co ltd
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Fujian Normal University
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Abstract

本发明提供了CELSR3蛋白在制备肝癌术后预后评估试剂盒中的应用、肝癌预后评估试剂盒及方法。本发明采用免疫组化方法检测CELSR3蛋白在肝癌组织中的相对表达量,以此判断肝癌患者出现肝癌复发转移的风险。本发明的有益效果主要体现在:本发明提供了CELSR3蛋白在制备肝癌术后预后评估试剂盒中的应用,提示该蛋白能用于制备判断肝癌患者预后的蛋白质分子标记,对于肝癌病人术后监控和序贯治疗也具有重要的指导意义。

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CELSR3蛋白在制备肝癌术后预后评估试剂盒中的应用、肝癌 预后评估试剂盒及方法
技术领域
本发明属于生物技术领域,具体涉及CELSR3蛋白在制备肝癌术后预后评估试剂盒中的应用、肝癌预后评估试剂盒及方法。
背景技术
肝癌即肝脏恶性肿瘤,可分为原发性和继发性两大类。原发性肝脏恶性肿瘤起源于肝脏的上皮或间叶组织,前者称为原发性肝癌,是我国高发的,危害极大的恶性肿瘤;后者称为肉瘤,与原发性肝癌相比较为少见。继发性或称转移性肝癌系指全身多个器官起源的恶性肿瘤侵犯至肝脏,一般多见于胃、胆道、胰腺、结直肠、卵巢、子宫、肺、乳腺等器官恶性肿瘤的肝转移。
肝癌结节外观多数呈球状,边界不甚规则,肿瘤周围可出现“卫星结节”。肝脏周边部靠近包膜的癌结节一般凸出表面但无中心凹陷。癌结节切面多呈灰白色,部分可因脂肪变性或坏死而呈黄色,亦可因含较多胆汁而显绿色,或因出血而呈红褐色。出血坏死多见于大结节的中央部。癌结节质地与组织学类型有关,实体型癌切面呈均质、光滑且柔软;梁状型癌切面则干燥呈颗粒状;胆管细胞癌因富含胶原纤维质地致密。肝癌体积明显增大,重量可达2000~3000g,不伴肝硬化的巨块型肝癌体积更大,重量可达7000g以上。多数肝癌伴大结节性或混合性肝硬化,部分门静脉、肝静脉腔内可见癌栓形成。
肝癌的发生发展及预后与癌基因的激活和抑癌基因功能失活密切关联,是多因素诱导,多基因参与的复杂病理过程。近年来,肝癌早期复发转移的分子机制是该领域的热门课题,深入阐明该分子机制,并在其特异性环节中导入靶向性治疗措施,有望很大幅度提高肝癌术后总体治疗效果。因此,探寻切实有效的肝癌肝切除术后早期复发相关预警分子标志,阐明其与肝癌早期复发转移的关系,这对提高肝癌术后治疗效果、评估肝癌早期复发风险、判断预后和个体化治疗有着至关重要的意义。
CELSR3(Cadherin EGF Laninin Seven Pass Receptor 1-3)编码属于钙粘蛋白家族的非典型钙粘蛋白。CELSR3蛋白在皮层中间神经元迁移,神经元轴突、树突的发育上发挥了重要作用。然而,相对于其广泛的表达,CELSR3及其家族在其他组织中的功能却未明。
肝癌做为对人类健康威胁最大的肿瘤之一,至今其发生的分子机制仍不清楚,对肝癌的治疗也缺少特异性的分子靶点,而CELSR3做为十分重要的肿瘤癌基因,目前尚无文献报道CELSR3蛋白与判断肝癌预后或肝癌转移相关。本发明研究发现在肝癌中的CELSR3表达与病人术后预后存在显著的关系,暗示CELSR3可作为肝癌的术后预后的有效预警蛋白。
发明内容
本发明目的在于提供CELSR3蛋白在制备肝癌术后预后评估试剂盒中的应用,并提供用于肝癌术后预后评估的试剂盒及方法。
本发明经过广泛而深入的研究,首次发现,采用免疫组化方法检测CELSR3蛋白在肝癌组织中的相对表达量,能够判断肝癌患者出现肝癌复发转移的风险。基于CELSR3蛋白表达量与肝癌复发转移的相关性,以CELSR3蛋白作为预后标记物对其表达量进行检测可以用于指导肝癌的预后判断。
因此,本发明提供了CELSR3蛋白在制备肝癌术后预后评估试剂盒中的应用,该应用是以CELSR3蛋白作为分子标记,利用CELSR3单克隆抗体或CELSR3多克隆抗体,结合免疫组化实验试剂,检测CELSR3蛋白在肝癌组织中的相对表达量。
本发明还提供了所述应用中用于肝癌术后预后评估的试剂盒,该试剂盒包括:人源CELSR3单克隆抗体或CELSR3多克隆抗体、免疫组化实验试剂。所述免疫组化实验试剂为本领域免疫组化实验中的常用试剂。
发明人经过研究发现,CELSR3在复发转移肝癌组织中表达高于未复发转移肝癌组织,可以推测CELSR3在肝癌术后复发转移中发挥重要作用。查阅国内外文献,CELSR3与肝癌的发生以及复发转移的相关研究少,在本研究中,CELSR3在肝癌组织中的表达上调,而在癌旁和正常肝组织中则表达下调,且与未复发转移组相比,CELSR3在复发转移组中表达也上调,表明CELSR3可能作为促进因子参与肝癌发生发展过程。通过Kaplan-Meier生存曲线分析,CELSR3表达程度与肝癌患者的预后有关,CELSR3高表达的患者预后不良(P<0.05)。
综上所述,CELSR3在肝癌组织中高表达,CELSR3的高表达与肝癌患者术后复发转移有关。CELSR3可以作为肝癌预后的一个重要候选分子标记物。
优选的,所述人源CELSR3多克隆抗体由序列为SEQ ID NO.1所示的CELSR3蛋白免疫兔子获得,可自行制备,也可采用市购商品。
本发明所述免疫组化实验试剂包括:二甲苯、乙醇、3%H2O2(水溶液)、3%BSA封闭液(以PBS配制)、DAB显色试剂、苏木素、辣根过氧化物酶(用于标记二抗)、PBS(pH7.4)、0.01M EDTA修复液。
利用本发明所述试剂盒进行肝癌术后预后评估的方法如下:
(1)取肝癌患者病理标本,利用人源CELSR3单克隆抗体或人源CELSR3多克隆抗体,以及免疫组化实验试剂,进行免疫组化染色;
其中,病理标本来自于肝癌患者活检组织或术中、术后的病理取材。免疫组化方法利用SP染色法,具体步骤如下:
(a)制备肝癌组织石蜡切片,60℃烤箱过夜。
(b)切片脱腊。依次浸泡:二甲苯I:10min;二甲苯II:10min;二甲苯III:10min。
(c)切片水化。依次浸泡:无水乙醇:3min;90%(v/v)乙醇:3min;80%乙醇:3min;75%乙醇:3min。
(d)PBS清洗3次,每次5min。
(e)EDTA抗原高压修复:切片放入0.01M EDTA修复液浸泡,沸水浴5min,冷却至室温。PBS清洗3次,每次5min。
(f)加入300μL的3%(w/w)过氧化氢水溶液,37℃10min。PBS清洗3次,每次5min。
(g)加入300μL的3%(w/w)BSA封闭液(PBS配制),37℃1h。PBS清洗3次,每次5min。
(h)加入一抗:CELSR3抗体浓度:1:500,4℃冰箱放置16h后取出,室温复温15min,然后PBS洗4次,每次5min。
(i)滴加二抗,所述的二抗为辣根过氧化物酶标记羊抗兔IgG(购自福州迈新试剂公司,即用型,无需稀释),37℃45min。PBS洗4次,每次5min。
(j)PBS洗3次,每次5min。DAB(DAB显色试剂盒,购自上海生工)显色2-10min,镜下观察;双蒸水洗止显色,苏木素复染10s,用自来水冲洗浸泡。
(k)脱水。依次浸泡:75%乙醇:2min;80%乙醇:2min;90%乙醇:2min;无水乙醇:2min。
(L)用电吹风吹干,加入中性树胶,盖玻片覆盖。
(2)利用显微镜和成像装置随机选取肝癌组织和癌旁组织3个视野拍摄,利用Aperio Image Scope软件对组织样本的相片进行扫描,扫描后采用该软件的Algorithms(Positive Pixel Count V9)程序对每个样本进行阳性强度计算,计算数据如下:
(3)每个组织样本的免疫组织化学评分计算为Positivity×Log10[255/Iavg],其中Positivity=NPositive/NTotal,即阳性率,计算方法为阳性象素数量/显色总数量;Iavg=(Iwp+Ip+Isp)/(Nwp+Np+Nsp),即阳性平均强度,计算方法为阳性平均强度=(弱阳性象素总强度+阳性象素总强度+强阳性象素总强度)/(弱阳性象素数量+阳性象素数量+强阳性象素数量),即为该组织的免疫组化评分,用于后续分析。
(4)采用SPSS18.0进行统计分析,检验指标与临床资料之间的计数资料采用Pearson卡方检验,计量资料采用t检验。检测指标与临床预后的分析采用KaPlan-Meier生存分析,对数秩和检验(log-ranktest)比较生存曲线的差别。本发明显示CELSR3蛋白与肝癌的预后具有显著的相关性,为预测肝癌的复发转移及术后的生存率提供一条全新途径,对肝癌患者的预后有重要作用。当癌组织CELSR3免疫组化评分高于0.462时,肝癌易出现复发转移,肝癌患者术后易死亡。
本发明的有益效果主要体现在:本发明研究发现在肝癌中的CELSR3表达与病人术后预后存在显著的关系,暗示CELSR3可作为肝癌的术后预后的有效预警蛋白。因此,本发明提供了CELSR3蛋白在制备肝癌术后预后评估试剂盒中的应用,提示该蛋白能用于制备判断肝癌患者预后的蛋白质分子标记,对于肝癌病人术后监控和序贯治疗也具有重要的指导意义。
附图说明
图1为CELSR3在肝癌患者术后1年内无复发转移的组织样本中弱表达;
图2为CELSR3在肝癌患者术后1年内复发转移的组织样本中高表达;
图3为CELSR3在肝癌旁组织中弱表达;
图4为肝癌组织中CELSR3低表达组与高表达组生存曲线。
具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不限于此:
实施例1
用于肝癌术后预后评估的试剂盒,该试剂盒包括:用于对肝癌组织中的CELSR3蛋白的相对表达量进行检测的人源CELSR3单克隆抗体或CELSR3多克隆抗体、免疫组化实验试剂。其中,免疫组化实验试剂包括:二甲苯、乙醇、3%H2O2、3%BSA封闭液、DAB显色试剂、苏木素、辣根过氧化物酶、PBS以及0.01M EDTA修复液。
本发明所述人源CELSR3多克隆抗体由序列为SEQ ID NO.1所示的CELSR3蛋白免疫兔子获得。可自行制备,也可采用市购商品。
本发明所述免疫组化实验试剂包括:二甲苯、乙醇、3%H2O2(水溶液)、3%BSA封闭液(以PBS配制)、DAB显色试剂、苏木素、辣根过氧化物酶(用于标记二抗)、PBS(pH7.4)、0.01M EDTA修复液。
实施例2
CELSR3蛋白在制备肝癌术后预后评估试剂盒中的应用,该应用是利用本发明试剂盒进行肝癌术后预后评估,方法如下:
(1)取肝癌患者病理标本,利用人源CELSR3单克隆抗体或人源CELSR3多克隆抗体,以及免疫组化实验试剂,进行免疫组化染色;
其中,病理标本来自于肝癌患者活检组织或术中、术后的病理取材。免疫组化方法利用SP染色法,具体步骤如下:
(a)制备肝癌组织石蜡切片,60℃烤箱过夜。
(b)切片脱腊。依次浸泡:二甲苯I:10min;二甲苯II:10min;二甲苯III:10min。
(c)切片水化。依次浸泡:无水乙醇:3min;90%(v/v)乙醇:3min;80%乙醇:3min;75%乙醇:3min。
(d)PBS清洗3次,每次5min。
(e)EDTA抗原高压修复:切片放入0.01M EDTA修复液浸泡,沸水浴5min,冷却至室温。PBS清洗3次,每次5min。
(f)加入300μL的3%(w/w)过氧化氢水溶液,37℃10min。PBS清洗3次,每次5min。
(g)加入300μL的3%(w/w)BSA封闭液(PBS配制),37℃1h。PBS清洗3次,每次5min。
(h)加入一抗:CELSR3抗体浓度:1:500,4℃冰箱放置16h后取出,室温复温15min,然后PBS洗4次,每次5min。
(i)滴加二抗,所述的二抗为辣根过氧化物酶标记羊抗兔IgG(购自福州迈新试剂公司,即用型,无需稀释),37℃45min。PBS洗4次,每次5min。
(j)PBS洗3次,每次5min。DAB(DAB显色试剂盒,购自上海生工)显色2-10min,镜下观察;双蒸水洗止显色,苏木素复染10s,用自来水冲洗浸泡。
(k)脱水。依次浸泡:75%乙醇:2min;80%乙醇:2min;90%乙醇:2min;无水乙醇:2min。
(L)用电吹风吹干,加入中性树胶,盖玻片覆盖。
(2)利用显微镜和成像装置随机选取肝癌组织和癌旁组织3个视野拍摄,利用Aperio Image Scope软件对组织样本的相片进行扫描,扫描后采用该软件的Algorithms(Positive Pixel Count V9)程序对每个样本进行阳性强度计算,计算数据如下:
(3)每个组织样本的免疫组织化学评分计算为Positivity×Log10[255/Iavg],其中Positivity=NPositive/NTotal,即阳性率,计算方法为阳性象素数量/显色总数量;Iavg=(Iwp+Ip+Isp)/(Nwp+Np+Nsp),即阳性平均强度,计算方法为阳性平均强度=(弱阳性象素总强度+阳性象素总强度+强阳性象素总强度)/(弱阳性象素数量+阳性象素数量+强阳性象素数量),即为该组织的免疫组化评分,用于后续分析。CELSR3高低表达标准以358例肝癌组织中CELSR3表达评分的中位数(0.462)为界。
(4)采用SPSS18.0进行统计分析,检验指标与临床资料之间的计数资料采用Pearson卡方检验,计量资料采用t检验。检测指标与临床预后的分析采用KaPlan-Meier生存分析,对数秩和检验(log-ranktest)比较生存曲线的差别。
按照上述方法,本发明在358例肝癌病人的肿瘤组织中检测结果如图1-3所示:CELSR3在1年内无复发转移组中弱表达;在复发转移组中高表达(图2);在癌旁组织中弱表达(图3)。
CELSR3与肝癌患者预后的关系:
通过Kaplan-Meier生存曲线分析,CELSR3的表达程度与肝癌患者的预后相关,如图4所示,CELSR3高表达的患者平均生存期(存活率)低于CELSR3低表达的患者。
实施例3
取某肝癌术后肿瘤样本进行石蜡包埋切片,并利用以上所述的免疫组织化学方法进行检测,经计算,其癌组织的CELSR3免疫组化组织评分为0.632。经过术后随访发现,该患者在术后第9个月发生肝癌复发转移,术后十八个月死亡。
实施例4
取某肝癌术后肿瘤样本进行石蜡包埋切片,并利用以上所述的免疫组织化学方法进行检测,经计算,其癌组织的CELSR3免疫组化组织评分为0.212。经过术后随访发现,该患者在术后1年均未发现转移复发,健在。
由以上试验结果可知,通过采用免疫组化的方法检测CELSR3蛋白分子相对表达量能预测肝癌远处转移风险以及患者术后的生存或死亡。当癌组织CELSR3的免疫组化评分高于0.462时,肝癌易出现复发转移,肝癌患者术后易死亡。显然CELSR3蛋白与肝癌具有相关性,因此,以CELSR3蛋白作为蛋白质分子标记对其表达量进行检测能预测肝癌手术后复发转移等事件,并判断预后。
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<110> 福建师范大学
<120> CELSR3蛋白在制备肝癌术后预后评估试剂盒中的应用、肝癌预后评估试剂盒及方法
<130> 1
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3312
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Met Ala Arg Arg Pro Pro Trp Arg Gly Leu Gly Gly Arg Ser Thr
1 5 10 15
Pro Ile Leu Leu Leu Leu Leu Leu Ser Leu Phe Pro Leu Ser Gln Glu
20 25 30
Glu Leu Gly Gly Gly Gly His Gln Gly Trp Asp Pro Gly Leu Ala Ala
35 40 45
Thr Thr Gly Pro Arg Ala His Ile Gly Gly Gly Ala Leu Ala Leu Cys
50 55 60
Pro Glu Ser Ser Gly Val Arg Glu Asp Gly Gly Pro Gly Leu Gly Val
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Arg Glu Pro Ile Phe Val Gly Leu Arg Gly Arg Arg Gln Ser Ala Arg
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Asn Ser Arg Gly Pro Pro Glu Gln Pro Asn Glu Glu Leu Gly Ile Glu
100 105 110
His Gly Val Gln Pro Leu Gly Ser Arg Glu Arg Glu Thr Gly Gln Gly
115 120 125
Pro Gly Ser Val Leu Tyr Trp Arg Pro Glu Val Ser Ser Cys Gly Arg
130 135 140
Thr Gly Pro Leu Gln Arg Gly Ser Leu Ser Pro Gly Ala Leu Ser Ser
145 150 155 160
Gly Val Pro Gly Ser Gly Asn Ser Ser Pro Leu Pro Ser Asp Phe Leu
165 170 175
Ile Arg His His Gly Pro Lys Pro Val Ser Ser Gln Arg Asn Ala Gly
180 185 190
Thr Gly Ser Arg Lys Arg Val Gly Thr Ala Arg Cys Cys Gly Glu Leu
195 200 205
Trp Ala Thr Gly Ser Lys Gly Gln Gly Glu Arg Ala Thr Thr Ser Gly
210 215 220
Ala Glu Arg Thr Ala Pro Arg Arg Asn Cys Leu Pro Gly Ala Ser Gly
225 230 235 240
Ser Gly Pro Glu Leu Asp Ser Ala Pro Arg Thr Ala Arg Thr Ala Pro
245 250 255
Ala Ser Gly Ser Ala Pro Arg Glu Ser Arg Thr Ala Pro Glu Pro Ala
260 265 270
Pro Lys Arg Met Arg Ser Arg Gly Leu Phe Arg Cys Arg Phe Leu Pro
275 280 285
Gln Arg Pro Gly Pro Arg Pro Pro Gly Leu Pro Ala Arg Pro Glu Ala
290 295 300
Arg Lys Val Thr Ser Ala Asn Arg Ala Arg Phe Arg Arg Ala Ala Asn
305 310 315 320
Arg His Pro Gln Phe Pro Gln Tyr Asn Tyr Gln Thr Leu Val Pro Glu
325 330 335
Asn Glu Ala Ala Gly Thr Ala Val Leu Arg Val Val Ala Gln Asp Pro
340 345 350
Asp Ala Gly Glu Ala Gly Arg Leu Val Tyr Ser Leu Ala Ala Leu Met
355 360 365
Asn Ser Arg Ser Leu Glu Leu Phe Ser Ile Asp Pro Gln Ser Gly Leu
370 375 380
Ile Arg Thr Ala Ala Ala Leu Asp Arg Glu Ser Met Glu Arg His Tyr
385 390 395 400
Leu Arg Val Thr Ala Gln Asp His Gly Ser Pro Arg Leu Ser Ala Thr
405 410 415
Thr Met Val Ala Val Thr Val Ala Asp Arg Asn Asp His Ser Pro Val
420 425 430
Phe Glu Gln Ala Gln Tyr Arg Glu Thr Leu Arg Glu Asn Val Glu Glu
435 440 445
Gly Tyr Pro Ile Leu Gln Leu Arg Ala Thr Asp Gly Asp Ala Pro Pro
450 455 460
Asn Ala Asn Leu Arg Tyr Arg Phe Val Gly Pro Pro Ala Ala Arg Ala
465 470 475 480
Ala Ala Ala Ala Ala Phe Glu Ile Asp Pro Arg Ser Gly Leu Ile Ser
485 490 495
Thr Ser Gly Arg Val Asp Arg Glu His Met Glu Ser Tyr Glu Leu Val
500 505 510
Val Glu Ala Ser Asp Gln Gly Gln Glu Pro Gly Pro Arg Ser Ala Thr
515 520 525
Val Arg Val His Ile Thr Val Leu Asp Glu Asn Asp Asn Ala Pro Gln
530 535 540
Phe Ser Glu Lys Arg Tyr Val Ala Gln Val Arg Glu Asp Val Arg Pro
545 550 555 560
His Thr Val Val Leu Arg Val Thr Ala Thr Asp Arg Asp Lys Asp Ala
565 570 575
Asn Gly Leu Val His Tyr Asn Ile Ile Ser Gly Asn Ser Arg Gly His
580 585 590
Phe Ala Ile Asp Ser Leu Thr Gly Glu Ile Gln Val Val Ala Pro Leu
595 600 605
Asp Phe Glu Ala Glu Arg Glu Tyr Ala Leu Arg Ile Arg Ala Gln Asp
610 615 620
Ala Gly Arg Pro Pro Leu Ser Asn Asn Thr Gly Leu Ala Ser Ile Gln
625 630 635 640
Val Val Asp Ile Asn Asp His Ile Pro Ile Phe Val Ser Thr Pro Phe
645 650 655
Gln Val Ser Val Leu Glu Asn Ala Pro Leu Gly His Ser Val Ile His
660 665 670
Ile Gln Ala Val Asp Ala Asp His Gly Glu Asn Ala Arg Leu Glu Tyr
675 680 685
Ser Leu Thr Gly Val Ala Pro Asp Thr Pro Phe Val Ile Asn Ser Ala
690 695 700
Thr Gly Trp Val Ser Val Ser Gly Pro Leu Asp Arg Glu Ser Val Glu
705 710 715 720
His Tyr Phe Phe Gly Val Glu Ala Arg Asp His Gly Ser Pro Pro Leu
725 730 735
Ser Ala Ser Ala Ser Val Thr Val Thr Val Leu Asp Val Asn Asp Asn
740 745 750
Arg Pro Glu Phe Thr Met Lys Glu Tyr His Leu Arg Leu Asn Glu Asp
755 760 765
Ala Ala Val Gly Thr Ser Val Val Ser Val Thr Ala Val Asp Arg Asp
770 775 780
Ala Asn Ser Ala Ile Ser Tyr Gln Ile Thr Gly Gly Asn Thr Arg Asn
785 790 795 800
Arg Phe Ala Ile Ser Thr Gln Gly Gly Val Gly Leu Val Thr Leu Ala
805 810 815
Leu Pro Leu Asp Tyr Lys Gln Glu Arg Tyr Phe Lys Leu Val Leu Thr
820 825 830
Ala Ser Asp Arg Ala Leu His Asp His Cys Tyr Val His Ile Asn Ile
835 840 845
Thr Asp Ala Asn Thr His Arg Pro Val Phe Gln Ser Ala His Tyr Ser
850 855 860
Val Ser Val Asn Glu Asp Arg Pro Met Gly Ser Thr Ile Val Val Ile
865 870 875 880
Ser Ala Ser Asp Asp Asp Val Gly Glu Asn Ala Arg Ile Thr Tyr Leu
885 890 895
Leu Glu Asp Asn Leu Pro Gln Phe Arg Ile Asp Ala Asp Ser Gly Ala
900 905 910
Ile Thr Leu Gln Ala Pro Leu Asp Tyr Glu Asp Gln Val Thr Tyr Thr
915 920 925
Leu Ala Ile Thr Ala Arg Asp Asn Gly Ile Pro Gln Lys Ala Asp Thr
930 935 940
Thr Tyr Val Glu Val Met Val Asn Asp Val Asn Asp Asn Ala Pro Gln
945 950 955 960
Phe Val Ala Ser His Tyr Thr Gly Leu Val Ser Glu Asp Ala Pro Pro
965 970 975
Phe Thr Ser Val Leu Gln Ile Ser Ala Thr Asp Arg Asp Ala His Ala
980 985 990
Asn Gly Arg Val Gln Tyr Thr Phe Gln Asn Gly Glu Asp Gly Asp Gly
995 1000 1005
Asp Phe Thr Ile Glu Pro Thr Ser Gly Ile Val Arg Thr Val Arg Arg
1010 1015 1020
Leu Asp Arg Glu Ala Val Ser Val Tyr Glu Leu Thr Ala Tyr Ala Val
1025 1030 1035 1040
Asp Arg Gly Val Pro Pro Leu Arg Thr Pro Val Ser Ile Gln Val Met
1045 1050 1055
Val Gln Asp Val Asn Asp Asn Ala Pro Val Phe Pro Ala Glu Glu Phe
1060 1065 1070
Glu Val Arg Val Lys Glu Asn Ser Ile Val Gly Ser Val Val Ala Gln
1075 1080 1085
Ile Thr Ala Val Asp Pro Asp Glu Gly Pro Asn Ala His Ile Met Tyr
1090 1095 1100
Gln Ile Val Glu Gly Asn Ile Pro Glu Leu Phe Gln Met Asp Ile Phe
1105 1110 1115 1120
Ser Gly Glu Leu Thr Ala Leu Ile Asp Leu Asp Tyr Glu Ala Arg Gln
1125 1130 1135
Glu Tyr Val Ile Val Val Gln Ala Thr Ser Ala Pro Leu Val Ser Arg
1140 1145 1150
Ala Thr Val His Val Arg Leu Val Asp Gln Asn Asp Asn Ser Pro Val
1155 1160 1165
Leu Asn Asn Phe Gln Ile Leu Phe Asn Asn Tyr Val Ser Asn Arg Ser
1170 1175 1180
Asp Thr Phe Pro Ser Gly Ile Ile Gly Arg Ile Pro Ala Tyr Asp Pro
1185 1190 1195 1200
Asp Val Ser Asp His Leu Phe Tyr Ser Phe Glu Arg Gly Asn Glu Leu
1205 1210 1215
Gln Leu Leu Val Val Asn Gln Thr Ser Gly Glu Leu Arg Leu Ser Arg
1220 1225 1230
Lys Leu Asp Asn Asn Arg Pro Leu Val Ala Ser Met Leu Val Thr Val
1235 1240 1245
Thr Asp Gly Leu His Ser Val Thr Ala Gln Cys Val Leu Arg Val Val
1250 1255 1260
Ile Ile Thr Glu Glu Leu Leu Ala Asn Ser Leu Thr Val Arg Leu Glu
1265 1270 1275 1280
Asn Met Trp Gln Glu Arg Phe Leu Ser Pro Leu Leu Gly Arg Phe Leu
1285 1290 1295
Glu Gly Val Ala Ala Val Leu Ala Thr Pro Ala Glu Asp Val Phe Ile
1300 1305 1310
Phe Asn Ile Gln Asn Asp Thr Asp Val Gly Gly Thr Val Leu Asn Val
1315 1320 1325
Ser Phe Ser Ala Leu Ala Pro Arg Gly Ala Gly Ala Gly Ala Ala Gly
1330 1335 1340
Pro Trp Phe Ser Ser Glu Glu Leu Gln Glu Gln Leu Tyr Val Arg Arg
1345 1350 1355 1360
Ala Ala Leu Ala Ala Arg Ser Leu Leu Asp Val Leu Pro Phe Asp Asp
1365 1370 1375
Asn Val Cys Leu Arg Glu Pro Cys Glu Asn Tyr Met Lys Cys Val Ser
1380 1385 1390
Val Leu Arg Phe Asp Ser Ser Ala Pro Phe Leu Ala Ser Ala Ser Thr
1395 1400 1405
Leu Phe Arg Pro Ile Gln Pro Ile Ala Gly Leu Arg Cys Arg Cys Pro
1410 1415 1420
Pro Gly Phe Thr Gly Asp Phe Cys Glu Thr Glu Leu Asp Leu Cys Tyr
1425 1430 1435 1440
Ser Asn Pro Cys Arg Asn Gly Gly Ala Cys Ala Arg Arg Glu Gly Gly
1445 1450 1455
Tyr Thr Cys Val Cys Arg Pro Arg Phe Thr Gly Glu Asp Cys Glu Leu
1460 1465 1470
Asp Thr Glu Ala Gly Arg Cys Val Pro Gly Val Cys Arg Asn Gly Gly
1475 1480 1485
Thr Cys Thr Asp Ala Pro Asn Gly Gly Phe Arg Cys Gln Cys Pro Ala
1490 1495 1500
Gly Gly Ala Phe Glu Gly Pro Arg Cys Glu Val Ala Ala Arg Ser Phe
1505 1510 1515 1520
Pro Pro Ser Ser Phe Val Met Phe Arg Gly Leu Arg Gln Arg Phe His
1525 1530 1535
Leu Thr Leu Ser Leu Ser Phe Ala Thr Val Gln Gln Ser Gly Leu Leu
1540 1545 1550
Phe Tyr Asn Gly Arg Leu Asn Glu Lys His Asp Phe Leu Ala Leu Glu
1555 1560 1565
Leu Val Ala Gly Gln Val Arg Leu Thr Tyr Ser Thr Gly Glu Ser Asn
1570 1575 1580
Thr Val Val Ser Pro Thr Val Pro Gly Gly Leu Ser Asp Gly Gln Trp
1585 1590 1595 1600
His Thr Val His Leu Arg Tyr Tyr Asn Lys Pro Arg Thr Asp Ala Leu
1605 1610 1615
Gly Gly Ala Gln Gly Pro Ser Lys Asp Lys Val Ala Val Leu Ser Val
1620 1625 1630
Asp Asp Cys Asp Val Ala Val Ala Leu Gln Phe Gly Ala Glu Ile Gly
1635 1640 1645
Asn Tyr Ser Cys Ala Ala Ala Gly Val Gln Thr Ser Ser Lys Lys Ser
1650 1655 1660
Leu Asp Leu Thr Gly Pro Leu Leu Leu Gly Gly Val Pro Asn Leu Pro
1665 1670 1675 1680
Glu Asn Phe Pro Val Ser His Lys Asp Phe Ile Gly Cys Met Arg Asp
1685 1690 1695
Leu His Ile Asp Gly Arg Arg Val Asp Met Ala Ala Phe Val Ala Asn
1700 1705 1710
Asn Gly Thr Met Ala Gly Cys Gln Ala Lys Leu His Phe Cys Asp Ser
1715 1720 1725
Gly Pro Cys Lys Asn Ser Gly Phe Cys Ser Glu Arg Trp Gly Ser Phe
1730 1735 1740
Ser Cys Asp Cys Pro Val Gly Phe Gly Gly Lys Asp Cys Gln Leu Thr
1745 1750 1755 1760
Met Ala His Pro His His Phe Arg Gly Asn Gly Thr Leu Ser Trp Asn
1765 1770 1775
Phe Gly Ser Asp Met Ala Val Ser Val Pro Trp Tyr Leu Gly Leu Ala
1780 1785 1790
Phe Arg Thr Arg Ala Thr Gln Gly Val Leu Met Gln Val Gln Ala Gly
1795 1800 1805
Pro His Ser Thr Leu Leu Cys Gln Leu Asp Arg Gly Leu Leu Ser Val
1810 1815 1820
Thr Val Thr Arg Gly Ser Gly Arg Ala Ser His Leu Leu Leu Asp Gln
1825 1830 1835 1840
Val Thr Val Ser Asp Gly Arg Trp His Asp Leu Arg Leu Glu Leu Gln
1845 1850 1855
Glu Glu Pro Gly Gly Arg Arg Gly His His Val Leu Met Val Ser Leu
1860 1865 1870
Asp Phe Ser Leu Phe Gln Asp Thr Met Ala Val Gly Ser Glu Leu Gln
1875 1880 1885
Gly Leu Lys Val Lys Gln Leu His Val Gly Gly Leu Pro Pro Gly Ser
1890 1895 1900
Ala Glu Glu Ala Pro Gln Gly Leu Val Gly Cys Ile Gln Gly Val Trp
1905 1910 1915 1920
Leu Gly Ser Thr Pro Ser Gly Ser Pro Ala Leu Leu Pro Pro Ser His
1925 1930 1935
Arg Val Asn Ala Glu Pro Gly Cys Val Val Thr Asn Ala Cys Ala Ser
1940 1945 1950
Gly Pro Cys Pro Pro His Ala Asp Cys Arg Asp Leu Trp Gln Thr Phe
1955 1960 1965
Ser Cys Thr Cys Gln Pro Gly Tyr Tyr Gly Pro Gly Cys Val Asp Ala
1970 1975 1980
Cys Leu Leu Asn Pro Cys Gln Asn Gln Gly Ser Cys Arg His Leu Pro
1985 1990 1995 2000
Gly Ala Pro His Gly Tyr Thr Cys Asp Cys Val Gly Gly Tyr Phe Gly
2005 2010 2015
His His Cys Glu His Arg Met Asp Gln Gln Cys Pro Arg Gly Trp Trp
2020 2025 2030
Gly Ser Pro Thr Cys Gly Pro Cys Asn Cys Asp Val His Lys Gly Phe
2035 2040 2045
Asp Pro Asn Cys Asn Lys Thr Asn Gly Gln Cys His Cys Lys Glu Phe
2050 2055 2060
His Tyr Arg Pro Arg Gly Ser Asp Ser Cys Leu Pro Cys Asp Cys Tyr
2065 2070 2075 2080
Pro Val Gly Ser Thr Ser Arg Ser Cys Ala Pro His Ser Gly Gln Cys
2085 2090 2095
Pro Cys Arg Pro Gly Ala Leu Gly Arg Gln Cys Asn Ser Cys Asp Ser
2100 2105 2110
Pro Phe Ala Glu Val Thr Ala Ser Gly Cys Arg Val Leu Tyr Asp Ala
2115 2120 2125
Cys Pro Lys Ser Leu Arg Ser Gly Val Trp Trp Pro Gln Thr Lys Phe
2130 2135 2140
Gly Val Leu Ala Thr Val Pro Cys Pro Arg Gly Ala Leu Gly Ala Ala
2145 2150 2155 2160
Val Arg Leu Cys Asp Glu Ala Gln Gly Trp Leu Glu Pro Asp Leu Phe
2165 2170 2175
Asn Cys Thr Ser Pro Ala Phe Arg Glu Leu Ser Leu Leu Leu Asp Gly
2180 2185 2190
Leu Glu Leu Asn Lys Thr Ala Leu Asp Thr Met Glu Ala Lys Lys Leu
2195 2200 2205
Ala Gln Arg Leu Arg Glu Val Thr Gly His Thr Asp His Tyr Phe Ser
2210 2215 2220
Gln Asp Val Arg Val Thr Ala Arg Leu Leu Ala His Leu Leu Ala Phe
2225 2230 2235 2240
Glu Ser His Gln Gln Gly Phe Gly Leu Thr Ala Thr Gln Asp Ala His
2245 2250 2255
Phe Asn Glu Asn Leu Leu Trp Ala Gly Ser Ala Leu Leu Ala Pro Glu
2260 2265 2270
Thr Gly Asp Leu Trp Ala Ala Leu Gly Gln Arg Ala Pro Gly Gly Ser
2275 2280 2285
Pro Gly Ser Ala Gly Leu Val Arg His Leu Glu Glu Tyr Ala Ala Thr
2290 2295 2300
Leu Ala Arg Asn Met Glu Leu Thr Tyr Leu Asn Pro Met Gly Leu Val
2305 2310 2315 2320
Thr Pro Asn Ile Met Leu Ser Ile Asp Arg Met Glu His Pro Ser Ser
2325 2330 2335
Pro Arg Gly Ala Arg Arg Tyr Pro Arg Tyr His Ser Asn Leu Phe Arg
2340 2345 2350
Gly Gln Asp Ala Trp Asp Pro His Thr His Val Leu Leu Pro Ser Gln
2355 2360 2365
Ser Pro Arg Pro Ser Pro Ser Glu Val Leu Pro Thr Ser Ser Ser Ile
2370 2375 2380
Glu Asn Ser Thr Thr Ser Ser Val Val Pro Pro Pro Ala Pro Pro Glu
2385 2390 2395 2400
Pro Glu Pro Gly Ile Ser Ile Ile Ile Leu Leu Val Tyr Arg Thr Leu
2405 2410 2415
Gly Gly Leu Leu Pro Ala Gln Phe Gln Ala Glu Arg Arg Gly Ala Arg
2420 2425 2430
Leu Pro Gln Asn Pro Val Met Asn Ser Pro Val Val Ser Val Ala Val
2435 2440 2445
Phe His Gly Arg Asn Phe Leu Arg Gly Ile Leu Glu Ser Pro Ile Ser
2450 2455 2460
Leu Glu Phe Arg Leu Leu Gln Thr Ala Asn Arg Ser Lys Ala Ile Cys
2465 2470 2475 2480
Val Gln Trp Asp Pro Pro Gly Leu Ala Glu Gln His Gly Val Trp Thr
2485 2490 2495
Ala Arg Asp Cys Glu Leu Val His Arg Asn Gly Ser His Ala Arg Cys
2500 2505 2510
Arg Cys Ser Arg Thr Gly Thr Phe Gly Val Leu Met Asp Ala Ser Pro
2515 2520 2525
Arg Glu Arg Leu Glu Gly Asp Leu Glu Leu Leu Ala Val Phe Thr His
2530 2535 2540
Val Val Val Ala Val Ser Val Ala Ala Leu Val Leu Thr Ala Ala Ile
2545 2550 2555 2560
Leu Leu Ser Leu Arg Ser Leu Lys Ser Asn Val Arg Gly Ile His Ala
2565 2570 2575
Asn Val Ala Ala Ala Leu Gly Val Ala Glu Leu Leu Phe Leu Leu Gly
2580 2585 2590
Ile His Arg Thr His Asn Gln Leu Val Cys Thr Ala Val Ala Ile Leu
2595 2600 2605
Leu His Tyr Phe Phe Leu Ser Thr Phe Ala Trp Leu Phe Val Gln Gly
2610 2615 2620
Leu His Leu Tyr Arg Met Gln Val Glu Pro Arg Asn Val Asp Arg Gly
2625 2630 2635 2640
Ala Met Arg Phe Tyr His Ala Leu Gly Trp Gly Val Pro Ala Val Leu
2645 2650 2655
Leu Gly Leu Ala Val Gly Leu Asp Pro Glu Gly Tyr Gly Asn Pro Asp
2660 2665 2670
Phe Cys Trp Ile Ser Val His Glu Pro Leu Ile Trp Ser Phe Ala Gly
2675 2680 2685
Pro Val Val Leu Val Ile Val Met Asn Gly Thr Met Phe Leu Leu Ala
2690 2695 2700
Ala Arg Thr Ser Cys Ser Thr Gly Gln Arg Glu Ala Lys Lys Thr Ser
2705 2710 2715 2720
Ala Leu Thr Leu Arg Ser Ser Phe Leu Leu Leu Leu Leu Val Ser Ala
2725 2730 2735
Ser Trp Leu Phe Gly Leu Leu Ala Val Asn His Ser Ile Leu Ala Phe
2740 2745 2750
His Tyr Leu His Ala Gly Leu Cys Gly Leu Gln Gly Leu Ala Val Leu
2755 2760 2765
Leu Leu Phe Cys Val Leu Asn Ala Asp Ala Arg Ala Ala Trp Met Pro
2770 2775 2780
Ala Cys Leu Gly Arg Lys Ala Ala Pro Glu Glu Ala Arg Pro Ala Pro
2785 2790 2795 2800
Gly Leu Gly Pro Gly Ala Tyr Asn Asn Thr Ala Leu Phe Glu Glu Ser
2805 2810 2815
Gly Leu Ile Arg Ile Thr Leu Gly Ala Ser Thr Val Ser Ser Val Ser
2820 2825 2830
Ser Ala Arg Ser Gly Arg Thr Gln Asp Gln Asp Ser Gln Arg Gly Arg
2835 2840 2845
Ser Tyr Leu Arg Asp Asn Val Leu Val Arg His Gly Ser Ala Ala Asp
2850 2855 2860
His Thr Asp His Ser Leu Gln Ala His Ala Gly Pro Thr Asp Leu Asp
2865 2870 2875 2880
Val Ala Met Phe His Arg Asp Ala Gly Ala Asp Ser Asp Ser Asp Ser
2885 2890 2895
Asp Leu Ser Leu Glu Glu Glu Arg Ser Leu Ser Ile Pro Ser Ser Glu
2900 2905 2910
Ser Glu Asp Asn Gly Arg Thr Arg Gly Arg Phe Gln Arg Pro Leu Cys
2915 2920 2925
Arg Ala Ala Gln Ser Glu Arg Leu Leu Thr His Pro Lys Asp Val Asp
2930 2935 2940
Gly Asn Asp Leu Leu Ser Tyr Trp Pro Ala Leu Gly Glu Cys Glu Ala
2945 2950 2955 2960
Ala Pro Cys Ala Leu Gln Thr Trp Gly Ser Glu Arg Arg Leu Gly Leu
2965 2970 2975
Asp Thr Ser Lys Asp Ala Ala Asn Asn Asn Gln Pro Asp Pro Ala Leu
2980 2985 2990
Thr Ser Gly Asp Glu Thr Ser Leu Gly Arg Ala Gln Arg Gln Arg Lys
2995 3000 3005
Gly Ile Leu Lys Asn Arg Leu Gln Tyr Pro Leu Val Pro Gln Thr Arg
3010 3015 3020
Gly Ala Pro Glu Leu Ser Trp Cys Arg Ala Ala Thr Leu Gly His Arg
3025 3030 3035 3040
Ala Val Pro Ala Ala Ser Tyr Gly Arg Ile Tyr Ala Gly Gly Gly Thr
3045 3050 3055
Gly Ser Leu Ser Gln Pro Ala Ser Arg Tyr Ser Ser Arg Glu Gln Leu
3060 3065 3070
Asp Leu Leu Leu Arg Arg Gln Leu Ser Arg Glu Arg Leu Glu Glu Ala
3075 3080 3085
Pro Ala Pro Val Leu Arg Pro Leu Ser Arg Pro Gly Ser Gln Glu Cys
3090 3095 3100
Met Asp Ala Ala Pro Gly Arg Leu Glu Pro Lys Asp Arg Gly Ser Thr
3105 3110 3115 3120
Leu Pro Arg Arg Gln Pro Pro Arg Asp Tyr Pro Gly Ala Met Ala Gly
3125 3130 3135
Arg Phe Gly Ser Arg Asp Ala Leu Asp Leu Gly Ala Pro Arg Glu Trp
3140 3145 3150
Leu Ser Thr Leu Pro Pro Pro Arg Arg Thr Arg Asp Leu Asp Pro Gln
3155 3160 3165
Pro Pro Pro Leu Pro Leu Ser Pro Gln Arg Gln Leu Ser Arg Asp Pro
3170 3175 3180
Leu Leu Pro Ser Arg Pro Leu Asp Ser Leu Ser Arg Ser Ser Asn Ser
3185 3190 3195 3200
Arg Glu Gln Leu Asp Gln Val Pro Ser Arg His Pro Ser Arg Glu Ala
3205 3210 3215
Leu Gly Pro Leu Pro Gln Leu Leu Arg Ala Arg Glu Asp Ser Val Ser
3220 3225 3230
Gly Pro Ser His Gly Pro Ser Thr Glu Gln Leu Asp Ile Leu Ser Ser
3235 3240 3245
Ile Leu Ala Ser Phe Asn Ser Ser Ala Leu Ser Ser Val Gln Ser Ser
3250 3255 3260
Ser Thr Pro Leu Gly Pro His Thr Thr Ala Thr Pro Ser Ala Thr Ala
3265 3270 3275 3280
Ser Val Leu Gly Pro Ser Thr Pro Arg Ser Ala Thr Ser His Ser Ile
3285 3290 3295
Ser Glu Leu Ser Pro Asp Ser Glu Val Pro Arg Ser Glu Gly His Ser
3300 3305 3310

Claims (6)

1.CELSR3蛋白在制备肝癌术后预后评估试剂盒中的应用。
2.根据权利要求1所述的CELSR3蛋白在制备肝癌术后预后评估试剂盒中的应用,其特征在于:以CELSR3蛋白作为分子标记,利用人源CELSR3单克隆抗体或人源CELSR3多克隆抗体,结合免疫组化实验试剂,检测CELSR3蛋白在肝癌组织中的相对表达量。
3.根据权利要求2所述的CELSR3蛋白在制备肝癌术后预后评估试剂盒中的应用,其特征在于:所述人源CELSR3多克隆抗体由序列为SEQ ID NO.1所示的CELSR3蛋白免疫兔子获得。
4.应用于肝癌术后预后评估的试剂盒,其特征在于:所述试剂盒包括用于对肝癌组织中的CELSR3蛋白的相对表达量进行检测的人源CELSR3单克隆抗体或人源CELSR3多克隆抗体,以及免疫组化实验试剂。
5.根据权利要求4所述的应用于肝癌术后预后评估试剂盒,其特征在于:所述免疫组化实验试剂包括:二甲苯、乙醇、3%H2O2、3%BSA封闭液、DAB显色试剂、苏木素、辣根过氧化物酶、PBS以及0.01M EDTA修复液。
6.一种基于权利要求4所述的试剂盒进行肝癌术后预后评估的方法,其特征在于:其包括以下步骤:
(1)取肝癌患者病理标本,利用人源CELSR3单克隆抗体或人源CELSR3多克隆抗体,以及免疫组化实验试剂,进行免疫组化染色;
(2)利用显微镜和成像装置随机选取肝癌组织和癌旁组织3个视野拍摄为数码照片;
(3)利用Aperio Image Scope软件对组织样本进行扫描,扫描后采用该软件的Algorithms程序对每个样本进行阳性强度计算,换算为该样品的免疫组织化学评分;
(4)通过所得到的评分评估患者术后的预后情况。
CN201710759221.0A 2017-08-30 2017-08-30 Celsr3蛋白在制备肝癌术后预后评估试剂盒中的应用、肝癌预后评估试剂盒及方法 Active CN107632160B (zh)

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