Institute of Digestive Diseases, Xijing Hospital, Forth Military Medical University, Xi＇an 710033， Shaanxi Province, China

Subject headingsstomach neoplasms/pathology; tumor cells, cultured; drug screening assays, antitumor; antibody, monoclonal; multidrug resistance

Abstract

AIMTo prepare gastric cancer multiple drug resistance (MDR) associated monoclonal antibody (mAb) MGr1, and to screen tumor drug resistance cell epitope library with MGr1, so as to obtain novel cDNA fragment associated with tumor MDR.

METHODSBy hybridoma technique, gastric cancer MDR associated mAb MGr1 was prepared using gastric cancer MDR cell strain SGC7901/ VCR as immunogen. Tumor drug resistance cell epitope library was screened and rescreened using MGr1 as a probe. The cDNA fragment brought by the positive recombinant was sequenced forward and backward, then, homology analysis was undertaken within GenBank. The expression level of positive cDNA fragment in several types of tissues and cells was determined with in situ hybridization and Northern blot.

RESULTSTwenty-six clones of hybridoma whose supernatant had the ability to bind SGC7901/ VCR were obtained from 3268 clones after 21 times cell fusing. And one mAb of interest, which was named MGr1, was selected by immunohistochemistry and MDR reversal test of mAb. MGr1 stain was stronger on SGC7901/ VCR than on SGC7901, and could enhance the cytotoxity of 5-FU and ADR on SGC7901/ VCR. Western blot showed that the Mr of MGr1 was 80 000-110 000, which could not be recognized by Pgp or MRP antibody. Therefore MGr1 could be regarded as gastric cancer MDR associated mAb. By library screening, five bacterial clones showed affinity to MGr1, and one clone was confirmed to have positive recombinant after being screened repeatedly. The cDNA fragment brought by the positive recombinant, which was named MGr1-Ag, had 310 bp and showed no homology with other genes in GeneBank. Results of in situ hybridization suggested that the normal tissues of liver, esophagus, stomach, colon did not express the MGr1-Ag. Results of Northern blot suggested that the MGr1-Ag was expressed in SGC7901, SGC7901/ VCR, EC109 and HCC7402. The expression level of MGr1-Ag was very high in SGC7901/ VCR, but low in EC109 and even lower in SGC7901 and HCC7402.

CONCLUSIONGastric cancer MDR associated mAb MGr1 was successfully prepared; and a novel cDNA fragment associated with gastric cancer MDR, MGr1-Ag was obtained by screening tumor drug resistance cell epitope library with MGr1.

中国图书资料分类号R 979.1

摘要

目的制备胃癌多药耐药相关性单克隆抗体(mAb)，并以该抗体筛选肿瘤耐药细胞表位文库，以获得肿瘤耐药相关的新基因片段.

方法采用杂交瘤技术，以胃癌耐药细胞株SGC7901/ VCR为免疫原制备胃癌多药耐药相关性,mAbMGr1，并以MGr1作为探针对肿瘤耐药细胞表位文库反复进行筛选；对稳定阳性重组子所携带的cDNA片段进行正反向测序，并将测序结果输入GeneBank进行同源性分析；原位杂交与Northern blot检测阳性克隆在不同组织细胞中的表达.

结果经过21次融合获得了26株培养上清能与SGC7901/ VCR结合的杂交瘤克隆，经免疫组化和单克隆抗体逆转耐药实验发现其中一株mAb在SGC7901/ VCR膜表面及胞内的表达显著高于其相应的药物敏感细胞SGC7901，并能部分逆转SGC7901/ VCR对ADR和5-FU的耐药性. Western blot表明该mAb对应的抗原Mr 800 000～110 000，与Pgp和MRP不同，该株mAb被命名为MGr1. 以MGr1筛选文库，获得了5个能与MGr1抗体结合的携带阳性重组子的菌落，经反复筛选后，证实有一个稳定的阳性克隆；正反向测序结果表明该阳性cDNA片段长度为310 bp，GeneBank检索显示该片段无任何同源性；原位杂交表明该基因在肝、食管、胃、大肠的正常组织均未见表达，Northern blot证实该基因在胃癌细胞SGC7901，胃癌耐药细胞SGC7901/ VCR，食管癌细胞EC109，肝癌细胞HCC7402中均有表达，但在SGC7901/ VCR中的表达量显著高于其他细胞.

结论成功地制备了胃癌耐药相关性mAbMGr1. 以MGr1抗体筛选肿瘤耐药细胞表位文库获得了一个与胃癌耐药相关的新基因片段.

0引言

肿瘤细胞在接触化疗药物后，可以获得对结构和功能各不相同的多种药物的耐受性，即多药耐药性(multidrug resistance, MDR). 有些肿瘤细胞也可以天然地产生MDR. 目前认为，主要有3种机制介导MDR^［1］： ①细胞内药物蓄积减少；②细胞核内拓扑异构酶Ⅱ(topoisomerase Ⅱ, TOPOⅡ)活性减低；③细胞质中GST/ GSH解毒功能增强. 其中第一种机制的研究最为深入. 已经成功地发现和克隆3种具有药物外流泵功能的跨膜蛋白：P-糖蛋白(P-glycoprotein, Pgp)，多药耐药相关性蛋白(multidrug-resistance associated protein, MRP),肺耐药相关性蛋白(lung resistance-related protein, LRP). 它们的组织表达、表达调控、与患者预后的相关性以及相应的逆转措施被广泛地研究探讨. 但上述发现尚不能完满解释肿瘤MDR的产生及不同的耐药细胞在耐药机制和交叉耐药谱型上的差异. 为了深入研究肿瘤MDR现象和发现新的MDR机制，我们成功地诱导建立了人胃癌耐药细胞株SGC7901/ VCR，现利用SGC7901/ VCR作为免疫原制备胃癌MDR相关性单克隆抗体(mAb)MGr1，并以MGr1筛选由樊代明教授构建的肿瘤耐药细胞表位文库^［2］，以期获得与肿瘤MDR相关的新的基因片段.

1材料和方法

1.1材料胃癌细胞SGC7901，胃癌耐药细胞SGC7901/ VCR，食管癌细胞EC109，肝癌细胞HCC7402，小鼠骨髓瘤细胞SP2/ 0均为本所保存的细胞. ♀健康Balb/ c小鼠，4 wk～10 wk，由本校动物中心提供. 肝、食管、胃、大肠的正常组织及胃癌组织标本各30例均取自本院近期外科手术之新鲜标本. 辣根过氧化物酶(HRP)标记的大鼠抗小鼠IgG、小鼠抗人Pgp抗体、小鼠抗人MRP抗体、DAB、地高辛标记的dUTP(DIG-dUTP)、硝酸纤维素滤膜、蛋白酶K，DEPC，MOPS，MTT，PMSF均为Sigma公司产品. RPMI1640，HEPES，L-谷胺酰胺为Gibco公司产品. 探针标记试剂盒、异硫氰酸胍、限制性内切酶BamHⅠ,KpnⅠ，胎牛血清皆购于华美生物工程公司. ［^α-³²P］dATP购于北京亚辉公司. 高范围蛋白标准参照物(212 000，116 000， 97 000， 66 000， 57 000，40 000)为Promega公司产品.

1.2 方法

1.2.1mAb的制备SGC7901/ VCR 1×10⁷细胞与福氏完全佐剂ip Balb/ c小鼠，d 14以相同数量的细胞ip加强免疫，d 35以相同数量的细胞分别ip与iv强化免疫，3 d后小鼠脾脏分离淋巴细胞. 脾淋巴细胞与SP2/ 0骨髓瘤细胞的融合参照文献［3］进行. 采用免疫组织化学方法和酶联免疫吸附实验鉴定阳性克隆. 免疫组织化学选用胃正常组织和癌组织的石蜡切片及SGC7901和SGC7901/ VCR细胞扒片. 酶联免疫吸附实验选用SGC7901和SGC7901/ VCR分别包被的96孔板. 阳性克隆MGr1经有限稀释法3次亚克隆后，部分冻存细胞种，部分用以制备腹水.

1.2.2 免疫组织化学各组织切片常规脱蜡至水，封闭内源性酶，MGr1腹水(1∶500)作为一抗于37℃结合60min，HRP标记的大鼠抗小鼠IgG(1∶800)作为二抗于37℃结合30min，最后以DAB作为底物于37℃显色. 出现棕黄色颗粒为阳性，根据颜色的深浅分别记为弱阳性、阳性、强阳性. 制备SGC7901/ VCR细胞的超薄切片，在电子显微镜下观察MGr1相应的抗原在耐药细胞中的分布.

1.2.3 Western blot细胞蛋白粗提液的制备参照文献［4］，SDS-PAGE采用80 g/ L分离胶和50 g/ L积层胶. 蛋白电泳结束后，以电转移的方法将凝胶上的蛋白转移到硝酸纤维素滤膜上，并将滤膜置于50 g/ L脱脂奶粉于室温封闭30min. 之后，分别结合一抗(MGr1或Pgp及MRP)二抗(HRP标记的大鼠抗小鼠IgG)，最后以DAB显色阳性条带.

1.2.4 MTT实验参照文献［5］进行MTT实验，以确定MGr1能否增强ADR或5-FU对SGC7901/ VCR的细胞毒性.

1.2.5 文库筛选与复筛将携带文库的大肠杆菌涂于琼脂平板(主板)上并在37℃培养至肉眼可见的菌落出现，以粘贴法将主板上的菌落转移至硝酸纤维素滤膜上，并将主板置于37℃培养箱继续培养至菌落出现. 将滤膜贴于另一含IPTG(10 mol/ L)的琼脂平板并置42℃诱导培养3 h～4 h，氯仿固定滤膜上诱导的融合蛋白，并予裂解液裂解，再以50 g/ L脱脂奶粉封闭，常规结合一抗(MGr1)和二抗(HRP标记的大鼠抗小鼠IgG)，最后以DAB显色. 将主板与膜上显色的阳性斑点相对应的菌落挑选出来，并扩增培养. 将扩增后的细菌<