CN102391992A - 抗人非小细胞肺癌单克隆抗体及其应用 - Google Patents
抗人非小细胞肺癌单克隆抗体及其应用 Download PDFInfo
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Abstract
本发明属于生物技术领域，公开了抗人非小细胞肺癌单克隆抗体及其应用。一株分泌抗人非小细胞肺癌单克隆抗体的杂交瘤细胞株NM001-1，2011年8月31日保藏于中国典型培养物保藏中心，保藏号为CCTCC NO：C201172。杂交瘤细胞株NM001-1分泌的抗人非小细胞肺癌的单克隆抗体NJ001-1。本发明杂交瘤(CCTCC NO：C201172)所分泌的单抗NJ001-1产量多、效价高，对肺腺癌及鳞癌细胞系有特异性反应。体内外研究结果显示：单克隆抗体NJ001-1在体内外对肺腺癌有显著的抑制作用。因此该单抗可以在制备治疗人非小细胞肺癌的药物中应用。
Description
技术领域
本发明属于生物技术领域，涉及抗人非小细胞肺癌单克隆抗体及其应用。
背景技术
肺癌是世界范围内最常见的恶性肿瘤，其发病率和死亡率占各种恶性肿瘤的首位，已成为严重危害人类健康和生命的主要疾病之一。传统的治疗方法对肺癌的治疗效果均不理想。手术治疗对多发、转移或复发的肺癌患者无能为力，且易复发；化疗对相当数量的患者疗效不佳，并且存在广泛的全身毒副作用；由于受到胸腔重要脏器的限制，放疗的治疗作用也很有限。近年来，靶向抗肿瘤生物治疗逐渐成为肺癌临床治疗的研究热点，因其在提高疗效的同时，还能大幅度降低副作用的发生风险，是提高肺癌疗效的一条重要途径。目前已研制出一些肺癌分子靶向药物，但这些药物对肺癌靶向治疗的临床疗效尚需进一步提高。因此，发现更多有效的治疗靶位和靶向药物对肺癌病人的诊断、治疗都具有重要意义。
肺癌预后差，患者的5年生存率低于15％，其中非小细胞肺癌(NSCLC)占所有肺癌病例的80％～85％。肺癌预后差的主要原因是难以早期发现、早期诊断从而进行早期治疗。肺癌特异性的单克隆抗体不仅可用于肿瘤诊断研究，在肿瘤治疗方面也是当前医药研究的热点。肺癌相关单抗的研究虽有很多[2-3]，但筛选针对人肺癌不同靶点的单抗的研究较少。
发明内容
本发明的目的是针对现有技术的上述缺陷，提供一株分泌抗人非小细胞肺癌单克隆抗体的杂交瘤细胞株。
本发明的另一目的是提供人非小细胞肺癌的单克隆抗体。
本发明的又一目的是提供该人非小细胞肺癌的单克隆抗体的应用。
本发明的目的可通过如下技术方案实现：
一株分泌抗人非小细胞肺癌单克隆抗体的杂交瘤细胞株NM001-1，2011年8月31日保藏于中国典型培养物保藏中心，保藏号为CCTCC NO：C201172。
由所述的保藏号为CCTCC NO：C201172的杂交瘤细胞株分泌的抗人非小细胞肺癌的单克隆抗体NJ001-1。
所述的保藏号为CCTCC NO：C201172的杂交瘤细胞株在制备治疗人非小细胞肺癌的药物中的应用。
所述的保藏号为CCTCC NO：C201172的杂交瘤细胞株在制备诊断人非小细胞肺癌的检测试剂中的应用。
所述的单克隆抗体NJ001-1在制备治疗人非小细胞肺癌的药物中的应用。
所述的单克隆抗体NJ001-1在制备诊断人非小细胞肺癌的检测试剂中的应用。
有益效果：
本发明以人肺腺癌细胞系SPCA1为免疫原，用杂交瘤技术，筛选到一株能够稳定分泌抗人非小细胞肺癌单克隆抗体的杂交瘤细胞株NM001-1(CCTCC NO：C201172)。该杂交瘤(CCTCC NO：C201172)所分泌的单抗NJ001-1产量多、效价高，对肺腺癌及鳞癌细胞系有特异性反应，与正常肺细胞、健康人PBMC及其他一些常见的肿瘤细胞系(肝癌、乳腺癌及结肠癌)无反应或低反应性。在免疫组化分析中，单抗NJ001-1与31例肺腺癌及24例肺鳞癌组织均呈阳性反应，其中肺腺癌组织染色呈强阳性；与20例肺炎性假瘤组织不反应；16例小细胞肺癌组织中仅2例表现为弱阳性；16例乳腺癌组织中仅3例表现为弱阳性。
本发明采用软琼脂克隆形成实验、裸鼠移植瘤模型研究了单抗NJ001-1在体内、体外实验中对肺腺癌的作用效应，结果表明单抗NJ001-1在体内外对肺腺癌有显著的抑制作用。本发明进一步研究了单抗NJ001-1的抑癌机制，结果表明单抗NJ001-1通过诱导细胞凋亡抑制肿瘤的发生、发展，可较好地直接干预或辅助治疗肺癌。
附图说明
图1、分泌单抗NJ001-1的杂交瘤细胞NM001-1的染色体分析图
图2、间接细胞ELISA法分析单抗NJ001-1与多种肿瘤细胞及非肿瘤细胞的反应性
图3、单抗NJ001-1的间接免疫荧光反应结果图(×400)
蓝色荧光：Hoechst核染结果；绿色荧光：FITC标记二抗染色结果；
A：肺腺癌细胞SPCA1，B：乳腺癌细胞ZR-75-30，C：肝癌细胞HepG2，D：人胚肺成纤维细胞WI-38。
图4、单抗NJ001-1与SPCA1裂解液的Western blot分析
图5、单抗NJ001-1与不同病理组织反应的免疫组化分析(×200)
A：非小细胞肺癌(腺癌)，B：非小细胞肺癌(鳞癌)，C：乳腺癌，D：肺炎性假瘤，E：小细胞肺癌。
图6、SPCA1在软琼脂上形成的克隆(×100)
A-F依次为对照组、100、200、300、400、500μg/ml单抗NJ001-1组。
图7、裸鼠接种部位肌肉组织HE染色结果(×200)
A：单抗NJ001-1孵育组，接种部位肌肉组织HE染色结果，B：对照组，接种部位肿瘤组织HE染色结果。
图8、裸鼠经单抗NJ001-1治疗后瘤重变化
*P＜0.05，与对照组相比；^P＜0.05，与200μg单抗组相比。
图9、单抗NJ001-1作用不同时间SPCA1细胞的形态变化(×100)
A：24h对照组；B：24h单抗组；C：48h对照组；D：48h单抗组。
图10、单抗NJ001-1诱导SPCA1细胞凋亡的流式细胞检测结果
A：单抗NJ001-1诱导SPCA1细胞凋亡的流式细胞分析图；
B：单抗NJ001-1诱导SPCA1细胞凋亡的流式细胞分析结果统计图，Annexin V+/PI-和Annexin V+/PI+细胞百分率分别表示早、晚期凋亡率。
生物材料样品的保藏信息
杂交瘤细胞株NM001-1于2011年8月31日保藏于中国典型培养物保藏中心，保藏地址为中国武汉，武汉大学，保藏号为CCTCC NO：C201172。
具体实施方式
1、以下实施例中所涉及的细胞及主要试剂
人肺癌细胞系SPCA1、A549、NCI-H460、NCI-H520，人肝癌细胞系HepG2，人乳腺癌细胞系ZR-75-30，人结肠癌细胞系Colo205及人胚肺成纤维细胞系WI-38购自中国科学院细胞库；SP2/0小鼠骨髓瘤细胞系由本实验室保存；6～8周龄和8～10周龄雌性BALB/c小鼠购自上海斯莱克实验动物公司。RPMI 1640、DMEM、胎牛血清、0.25％胰蛋白酶、聚乙二醇、次黄嘌呤胸腺嘧啶核苷(HT)、氨基喋呤次黄嘌呤胸腺嘧啶核苷(HAT)和石蜡油购自美国Gibco Invitrogen公司；辣根过氧化物酶(HRP)标记羊抗鼠IgG、FITC标记的羊抗鼠IgG及可溶型单组分TMB底物溶液购自北京天根公司；小鼠Ig亚型测定试剂盒购自美国Santa Cruz公司；Western blot显色剂购自美国Cell Signaling公司；肺炎性假瘤组织、肺癌组织及乳腺癌组织由江苏省人民医院病理科提供；Model 550型酶标仪购自Bio-Rad公司。PRMI 1640、胎牛血清购自公司。琼脂糖购自Promega公司。Annexin V-FITC细胞凋亡检测试剂盒购自凯基生物技术有限公司。
2、以下实施例中所涉及的细胞培养方法为
上述细胞分别生长于含10％胎牛血清、青霉素、链霉素各100U/mL的DMEM或RPMI1640培养液中，37℃、5％CO2恒温培养箱中培养。人外周血单个核细胞(PBMC)获自健康献血者，采用常规淋巴细胞分离液分离。
3、统计学分析
采用SPSS 16.0统计学软件对数据进行统计分析。定性资料的组间比较采用Fisher’s确切概率法，P＜0.05时具有统计学意义；定量资料的组间比较采用单因素方差分析。齐性方差的两两比较用LSD法，P＜0.05时差异有统计学意义；非齐性方差用Dunnett’s C法，以0.05作为两两比较时的显著性水平。
实施例1杂交瘤的获得
1.1动物免疫 取6～8周雌性BALB/c小鼠，每只用2×106SPCA1细胞/次腹腔注射免疫4次，每次间隔3周。每次免疫前均进行小鼠内眦采血，间接细胞ELISA法检测小鼠血清抗体效价，待免疫小鼠血清抗体滴度达到最大并不再升高时取鼠脾细胞进行融合，融合前3d加强免疫1次。
1.2间接细胞ELISA试验 于96孔板上接种2×105/孔的SPCA1细胞，至细胞生长融合达80％，95％乙醇固定，PBS洗3次，0.2％Triton-X-100通透20min，再用50g/L BSA 37℃封闭2h，依次加入不同稀释度的免疫小鼠血清100μL，37℃温育1h，再经PBS洗3次后，加入1∶1000稀释的HRP标记的羊抗鼠IgG 100μL，37℃温育45min，经PBS洗涤后，加入TMB显色液，37℃温育10min后终止反应，用酶标仪测定450nm时吸光度(A)值，以未免疫小鼠血清(1∶1000)作为阴性对照。
1.3细胞融合 取免疫小鼠脾脏研磨制成细胞悬液，与处于对数生长期的骨髓瘤细胞SP2/0进行融合(姚晓玲，柳晓燕，吴强，等.人肺癌相关单克隆抗体的制备及其抗原的纯化[J].中国免疫学杂志，2006，22(12)：1140-1145.)，首次融合960孔，融合1周后出现细胞克隆，有800孔生长杂交瘤细胞，融合率约为83％。按照1.2中的方法进行间接细胞ELISA试验筛选阳性杂交瘤细胞(将1.2间接细胞ELISA试验中的免疫小鼠血清替换为杂交瘤细胞培养上清)，进行转种和4次有限稀释法亚克隆，获得稳定分泌抗SPCA1单抗且阳性最强的2株杂交瘤细胞株NM001-1及NM001-2。
实施例2单抗腹水的制备及纯化
取8～10周龄雌性BALB/c小鼠腹腔注射0.5mL石蜡油，10d后分别腹腔注射生长良好的杂交瘤细胞NM001-1及NM001-2约1×106/只，1～2周后抽吸腹水，37℃1h后，4℃过夜，次日分别将腹水离心，经Protein G亲和层析柱纯化，得到纯化的单克隆抗体NJ001-1及NJ001-2。
实施例3单抗的鉴定
3.1单抗Ig亚类的鉴定：纯化单抗用PBS 1∶10000稀释，按照测定试剂盒说明书操作。单抗NJ001-1及NJ001-2的亚类均为IgG，轻链为κ链。
3.2单抗效价的测定：分别将纯化的单抗NJ001-1及NJ001-2用PBS倍比稀释，分别取100μL加入包被有SPCA1细胞的96孔板中，用间接细胞ELISA法测定A450值，能与包被细胞发生免疫反应的单抗最大稀释度即为其效价。单克隆抗体NJ001-1效价为4×106，单克隆抗体NJ001-2效价为1.6×106。将杂交瘤细胞株NM001-1于2011年8月31日保藏于中国典型培养物保藏中心，保藏地址为武汉，武汉大学，保藏号为CCTCC NO：C201172。
3.3染色体鉴定：将对数生长的杂交瘤细胞NM001-1秋水仙碱处理8h，再收集细胞，经离心甩片在玻片上，用0.075mol/L KCl低渗处理，用甲醇冰醋酸固定液固定，经10％Giemsa染色10min，镜下检测染色体。杂交瘤细胞的染色体数目范围为100～106条，因为小鼠细胞的染色体数目为40条，而SP2/0细胞染色体数目平均为62～68条，证明杂交瘤细胞中的染色体来自免疫小鼠的脾细胞和小鼠骨髓瘤细胞SP2/0，均属杂交瘤细胞染色体核型，见图1。
3.4单抗特异性的鉴定：用纯化的单抗NJ001-1与上述8种细胞系及健康人PBMC分别做间接细胞ELISA分析，观察有无阳性反应。结果显示，单抗NJ001-1仅对肺癌细胞(SPCA1、A549、NCI-H520、NCI-H460)抗原具有较强反应，对其他肿瘤细胞(HepG2、Colo 205、ZR-75-30)抗原、正常人胚肺细胞(WI-38)抗原及健康人PBMC均无反应(见图2)。
实施例4间接免疫荧光试验
6孔板内放置无菌盖玻片，将上述细胞系置于板内培养，待细胞贴壁生长时取出盖玻片，95％乙醇固定细胞，PBS洗3次后，0.2％Triton-X-100通透20min，再用50g/L BSA 37℃封闭2h，然后加入1∶100稀释的纯化单抗NJ001-1，37℃温育1h，再经PBS洗5次后，加入1∶80稀释的FITC标记的羊抗鼠IgG，37℃温育1h，经PBS洗涤后，Hoechest染色10min，PBS洗涤，甘油封闭，LSM710型激光共聚焦显微镜(德国zeus公司)观察，结果见图3。图3显示单抗NJ001-1仅对SPCA1细胞抗原具有较强反应，即在其细胞膜产生明显的黄绿色荧光，表明单抗NJ001-1识别的抗原定位于细胞膜。单抗NJ001-1对ZR-75-30、HepG2、WI-38无反应，表现为无绿色荧光。
实施例5 Western blot检测单抗特异识别抗原
将SPCA1细胞裂解液进行SDS-PAGE电泳，再转移到硝酸纤维膜上，50g/L脱脂奶粉中室温封闭2h，加入1∶300稀释的单抗NJ001-1，4℃过夜。PBS-T洗3次后，加入1∶1000稀释的HRP标记的羊抗鼠IgG，37℃温育45min，最后以ECL发光液显色。Western blot显示，胶片上可见一清晰条带，其分子量为70 000，见图4。
实施例6免疫组化染色
取人肺炎性假瘤组织、肺腺癌组织、肺鳞癌组织、小细胞肺癌组织及乳腺癌组织，石蜡包埋、切片，以纯化单抗NJ001-1为一抗，SPCA1免疫小鼠血清作一抗的阳性对照，用PBS分别代替一抗、二抗作为阴性对照，煮沸法进行抗原修复，用Cell Signaling公司显色试剂显色，苏木素复染，镜下观察。免疫组化染色法结果见表1。单抗NJ001-1与肺腺癌及肺鳞癌组织抗原有强阳性反应，且其识别的相应抗原主要分布在肺癌组织细胞的细胞膜；而与小细胞肺癌组织、肺炎性假瘤组织以及乳腺癌组织呈无反应或弱阳性，见图5。非小细胞肺癌与小细胞肺癌组织的胞膜抗原表达率有显著差异(P＜0.001)；非小细胞肺癌与肺炎性假瘤组织、乳腺癌组织的胞膜抗原表达率也有统计学差异(P＜0.001，P＜0.001)；肺腺癌和肺鳞癌组织抗原表达率没有差别，但肺鳞癌组织染色强度略低于肺腺癌。
表1 单抗NJ001-1与不同病理组织反应的免疫组化结果
备注：“-”阴性；“+”弱阳性；“++”中等阳性；“+++”强阳性
实施例7软琼脂克隆形成实验
用生理盐水制备3％浓度的琼脂糖溶液，高压蒸汽灭菌。在6孔细胞培养板中制备双层凝胶琼脂(Hong KW，Kim CG，Lee SH，et al.A novel anti-EGFR monoclonal antibody inhibitingtumor cell growth by recognizing different epitopes from cetuximab[J].J Biotechnol，2010，145(1)：84-91.)。底层为支持层，将含10％胎牛血清的RPMI 1640完全培养基与3％琼脂糖按5∶1比例混合，配制成含0.5％琼脂糖的培养基，2mL/孔加入6孔板中，室温冷却凝固。收集处于对数生长期的SPCA1，用完全培养基制成单细胞悬液，37℃保温，取适量的单细胞悬液与3％琼脂糖溶液、不同浓度单克隆抗体NJ001-1溶液充分混合，2mL/孔铺板制成含0.3％琼脂糖的上层琼脂，每孔含有2×104个细胞，抗体终浓度分别为0、100、200、300、400、500、1000和2000μg/mL，每剂量设3个平行样。常温下待琼脂凝固后，将培养板移入37℃、5％CO2培养箱中培养2周。倒置显微镜下计数，将≥50个细胞的集落判定为一个克隆，计算克隆形成率、抑制率。克隆形成率＝(克隆数/接种细胞数)×100％；抑制率＝(1-单抗组克隆形成率/对照组克隆形成率)×100％。实验重复3次。
软琼脂克隆形成试验表明：单抗NJ001-1能够有效抑制肺腺癌细胞SPCA1在软琼脂中的克隆形成，并且其抑制程度与抗体的作用浓度正相关(表2)。单抗NJ001-1作用后的细胞形成集落显著减少，且集落远小于对照组，部分细胞不能在软琼脂中生长形成集落，呈单个散在的细胞(图6)。
表2 单抗NJ001-1对SPCA1软琼脂克隆形成的影响
实施例8裸鼠移植瘤模型实验
8.1裸鼠预实验
将2×106个SPCA1细胞与单抗NJ001-1于37℃孵育2h接种于5只裸鼠右侧腋部皮下，建立单抗孵育组。对照组5只裸鼠于相同部位接种等量未经单抗NJ001-1作用的SPCA1细胞。接种后3周，颈椎脱臼处死裸鼠，解剖。单抗孵育组裸鼠在接种部位均未形成肉眼可见的肿块，对照组接种部位形成明显的肿块。分别取单抗孵育组和对照组接种部位组织10％甲醛固定，石蜡包埋、切片，常规HE染色，显微镜下观察组织病理学改变，结果见图7。单抗孵育组接种部位肌肉组织HE染色显示无肿瘤细胞生长；对照组肿块组织做病理切片染色显示出恶性肿瘤的组织学特点。
8.2裸鼠移植瘤模型实验
另外25只裸鼠随机分成5组，分别为未经单抗NJ001-1治疗对照组(简称对照组)、200μg、300μg、400μg、500μg单抗NJ001-1治疗组(简称单抗组)，每组5只，经右侧腋部皮下注射法，每只接种2×106个SPCA1细胞，200μL/只。接种当天开始接受单抗治疗，单抗组腹腔注射200μL不同浓度的单抗溶液(分别含有200、300、400、500μg单抗NJ001-1)，前5天每天注射1次，之后每4天注射1次，连续2周(累计注射9次)。对照组以等体积的生理盐水代替，注射时间和途径相同。每日观察小鼠活动、精神状况、进食情况及肿块出现时间等并记录。自皮下结节出现起，游标卡尺测量移植瘤长径(a)及短径(b)，移植瘤体积V＝ab2/2[8]。治疗结束后，裸鼠颈椎脱臼处死，解剖分离肿瘤并称取瘤重、计算抑瘤率。抑瘤率＝(1-单抗组平均瘤重/对照组平均瘤重)×100％。
将SPCA1细胞皮下接种于裸鼠，同时腹腔注射生理盐水和不同剂量的单抗NJ001-13周。接种后第9天，种植区出现肉眼可见的结节，成瘤率100％。第13天起测量肿瘤体积，观察至第21天发现：第17天起300μg、400μg、500μg单抗组与对照组相比，肿瘤体积的增长速度明显延缓，差异持续至实验结束(P＝0.004，P＝0.003，P＝0.003，表3)。治疗结束后分离肿瘤并称量肿块重量，结果见图8。对照组、200μg、300μg、400μg、500μg单抗组平均瘤重分别为(1.71±0.27)g，(1.50±0.20)g，(1.03±0.44)g，(0.91±0.19)g和(0.88±0.21)g；四组小鼠肿块重量具有统计学差异(F＝4.303，P＝0.043)；300μg、400μg、500μg单抗组平均瘤重显著低于对照组(P＝0.036，P＝0.032，P＝0.014)；200μg单抗组与500μg单抗组间也存在显著差异(P＝0.043)。200μg、300μg、400μg、500μg单抗组抑瘤率分别为12.28％、39.77％、46.78％和48.54％。结果表明：单抗NJ001-1在体内能显著抑制人肺腺癌移植瘤的生长。
表3裸鼠经单抗NJ001-1治疗后瘤体积变化
*P＜0.05，与对照组相比。
实施例9细胞凋亡检测
收集处于对数生长期的SPCA1制成单细胞悬液，以1×105细胞/孔加入12孔板中，过夜培养，吸弃培养上清，并用D-Hank’s液500μL/孔洗一遍。抗体组(24h、48h两组)：每孔加入500μL单抗NJ001-1溶液，单抗浓度为300μg/mL；对照组(24h、48h两组)：每孔加入500μL培养液。干预后分别培养24h、48h，倒置显微镜下观察拍照，结果见图9；收集细胞，流式细胞仪检测细胞凋亡。每组设3个平行孔，实验重复3次。流式细胞仪检测细胞凋亡方法如下：0.25％胰酶消化细胞，细胞悬液2000rpm×5min离心；PBS洗涤细胞两次，收集细胞；加入500μL的Binding Buffer悬浮细胞；加入5μL Annexin V-FITC混匀后，再加入5μLPropidium Iodide混匀；室温、避光、反应5-15min；1h内进行流式细胞仪的观察和检测，激发波长Ex＝488nm；发射波长Em＝530nm。Annexin V-FITC的绿色荧光通过FITC通道(FL1)检测；PI红色荧光通过PI通道(FL3)检测。Annexin V+/PI-(％)和Annexin V+/PI+(％)分别表示早、晚期细胞凋亡率，总凋亡率则为早期和晚期凋亡率之和。
图9显示对照组细胞形态正常，生长旺盛；300μg/mL单抗NJ001-1作用于SPCA1细胞24h、48h后，细胞逐渐呈现胞核固缩、胞浆减少等凋亡改变，与对照组形态差别显著。凋亡检测结果(图10)显示：24h、48h单抗组细胞总凋亡率达45.36％和69.00％，与对照组相比有显著升高(P＝0.001，P＜0.001)；48h、24h单抗组总凋亡率比较存在统计学差异(P＝0.003)。24h、48h单抗组晚期凋亡率与对照组相比也有显著差异(P＝0.002，P＝0.001)且48h单抗组比24h单抗组明显增高(P＝0.004)。表明随着作用时间的延长，SPCA1细胞的凋亡率逐渐升高，提示单抗NJ001-1诱导SPCA1的凋亡作用存在时间依赖性。
Claims (6)
1.一株分泌抗人非小细胞肺癌单克隆抗体的杂交瘤细胞株NM001-1，2011年8月31日保藏于中国典型培养物保藏中心，保藏号为CCTCC NO：C201172。
2.由权利要求1所述的保藏号为CCTCC NO：C201172的杂交瘤细胞株分泌的抗人非小细胞肺癌的单克隆抗体NJ001-1。
3.权利要求1所述的保藏号为CCTCC NO：C201172的杂交瘤细胞株在制备治疗人非小细胞肺癌的药物中的应用。
4.权利要求1所述的保藏号为CCTCC NO：C201172的杂交瘤细胞株在制备诊断人非小细胞肺癌的检测试剂中的应用。
5.权利要求2所述的单克隆抗体NJ001-1在制备治疗人非小细胞肺癌的药物中的应用。
6.权利要求2所述的单克隆抗体NJ001-1在制备诊断人非小细胞肺癌的检测试剂中的应用。
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| CN103439509A (zh) * | 2013-09-06 | 2013-12-11 | 潘世扬 | 特异性循环非小细胞肺癌细胞标志物的应用 |
| CN105126098A (zh) * | 2015-07-31 | 2015-12-09 | 南京壬辰生物科技有限公司 | 单克隆抗体nj001-1在制备抑制nsclc侵袭及转移的药物中的应用 |
| CN107417785A (zh) * | 2016-10-28 | 2017-12-01 | 南京麦科林生物医药科技有限公司 | 一种肿瘤特异性抗原tsp70及其应用 |
| CN113087801A (zh) * | 2021-04-07 | 2021-07-09 | 北京欣颂生物科技有限公司 | 一种用核酸和抗体联合检测肺癌的试剂盒 |
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| 《南京医科大学学报（自然科学版）》 20110731 王芳等 抗人非小细胞肺癌单克隆抗体对肺腺癌抑制作用的实验研究 第940-944页 1-6 第31卷, 第7期 * |
| 《肿瘤》 19930930 潘世扬等 肺癌单克隆抗体制备及研究 第227-229页 1-6 第13卷, 第5期 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102944681A (zh) * | 2012-11-21 | 2013-02-27 | 潘世扬 | 非小细胞肺癌胸水肿瘤标志物的应用 |
| CN103439509A (zh) * | 2013-09-06 | 2013-12-11 | 潘世扬 | 特异性循环非小细胞肺癌细胞标志物的应用 |
| CN105126098A (zh) * | 2015-07-31 | 2015-12-09 | 南京壬辰生物科技有限公司 | 单克隆抗体nj001-1在制备抑制nsclc侵袭及转移的药物中的应用 |
| CN107417785A (zh) * | 2016-10-28 | 2017-12-01 | 南京麦科林生物医药科技有限公司 | 一种肿瘤特异性抗原tsp70及其应用 |
| CN113087801A (zh) * | 2021-04-07 | 2021-07-09 | 北京欣颂生物科技有限公司 | 一种用核酸和抗体联合检测肺癌的试剂盒 |
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