肝癌导向治疗研究13年体会

本文总结1980年-1983年，1985年-1996年间有关肝癌导向治疗研究的简况。研究包括：抗AFP抗体的制备和提纯；标记方法的探讨；载体和弹头的筛选；标记物在动物、人体上的定位、显像；产品临床前质量和安全性鉴定；临床治疗方法的探讨等。一、抗AFP抗体的制备、纯化和标记方法起始用的抗体是从市场上用作诊断的抗体，经纯化后用于临床，1989年完成了用广西矮马制备抗人AFP抗体血清。先用盐析法粗提AFP，再经亲和层析法纯化的AFP免疫马后，收集马抗人AFP抗体血清，用盐析法和亲和层析法提纯抗体，抗体达聚丙烯酰胶凝胶电泳纯（效价在1…     

靶向抗PD-1/CD19双特异性抗体的表达与活性鉴定

目的:重组表达和纯化靶向抗PD-1/CD19双特异性抗体(bispecific antibody, BsAb)并验证其活性.方法:以pCAR1为载体,利用分子克隆技术构建抗PD-1/CD19 BsAb真核表达载体,通过PEI试剂转染哺乳动物细胞株CHO-S瞬时表达抗体.利用亲和层析法对BsAb进行纯化,用SDS-PAG...     

人源DKK1真核悬浮分泌表达系统的建立及其特异性抗体的制备和鉴定

目的:利用哺乳动物细胞悬浮培养表达系统分泌表达人源Dickkopf-1(DKK1)蛋白,纯化蛋白并制备特异性抗体。方法:构建DKK1真核表达载体pCMVcStrep-DKK1,并借助脂质体将载体瞬时转染入Free-Style293-F细胞(无血清培养),ELISA和蛋白印迹法检测DKK1蛋白表达量。利用亲和层析法纯化DKK1蛋白,Wnt信号通路荧光素酶报告系统检测其生理活性。以纯化的DKK1蛋白免疫BALB/c小鼠获得相应抗体,并初步鉴定其抗原特异性。结果:DKK1蛋白分泌表达在Free-Style293-F细胞培养液中,蛋白浓度在转染后第5天达最高(20mg/L)。所纯化的DKK1蛋白能够抑制Wnt3a蛋白诱导的报告基因的表达。抗DKK1小鼠抗体能特异性识别细胞内源性DKK1蛋白。结论:建立了在哺乳动物细胞悬浮培养系统中高效分泌表达人源DKK1重组蛋白的表达系统,并获得相应的特异性抗体,为其下一步结构与功能研究及在肿瘤中的应用奠定了实验基础。     

贵州民族药提取物A-3抗H22肿瘤细胞生长作用机制的初步研究

目的：对贵州民族药提取物A-3抗肿瘤作用机制进行初步研究。方法：研究A-3对小鼠皮肤迟发型超敏反应、肿瘤细胞凋亡、肿瘤血管生成及微管蛋白聚合解聚活性等四个方面的影响,初步探讨药物的抗肿瘤作用机制。结果：A-3能明显刺激正常小鼠和荷瘤小鼠的脾脏及胸腺增生,促进DTH反应。每天给予50mg／kg和100mg／kgA-3能见到明显促进H22肿瘤细胞凋亡的作用,但两剂量对H22肿瘤血管生成均无显著影响。浓度为0．1mmol／L时,A-3对兔脑微管蛋白体外的聚合解聚平衡反应无明显影响。结论：A-3可能通过提高机体免疫功能及促进肿瘤细胞凋亡等作用来抑制肿瘤生长。     

牛蛙核糖核酸酶的表达和鉴定及功能检测

目的：在大肠埃希菌中高效表达牛蛙核糖核酸酶（rana catesbeiana ribonuclease，RC-RNase），并对其纯化产物进行噻唑蓝（tetrazolium blue，MTT）实验，观察对肝癌细胞的抑制效果。方法：用异丙基硫代-D-半乳糖苷（isopropylthio-D-galactoride，IPTG）诱导原核表达质粒PET32a—RC-RNase在大肠埃希菌F．cali B121（DE3）plysS中表达。茴体经超声、离心后，用Ni—NTA Agarose亲和层析法对包涵体进行纯化。采用MTT法检测纯化的目的蛋白对体外培养的肝癌细胞的杀伤活性。结果：纯化的目的蛋白经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium doecyls ulphatepolyacrylamide gel eletrophoresis，SDSPAGE）蛋白电泳表明，RC-RNase在大肠埃希菌中是以包涵体的形式表迭。用MXT法的结果中可以看出，纯化的目的蛋白对体外培养的肝癌细胞具有一定的抑制效果，其IC50值（半抑制浓度）是22．44μg／mL，。结论：牛蛙核糖核酸酶在大肠埃希菌中得到大量表达，为进一步研究其生物学特性以及功能打下了良好的基础。     

HAbl8G／CDl47拮抗肽的筛选及对肝癌细胞侵袭力的抑制

目的 筛选能与HAbl8G／CD147相结合的多肽，从中寻找该因子的肽类拮抗剂，抑制肝癌的恶性转移。方法 利用亲和层析法获得HAbl8G／CD147抗原，在此基础上通过筛选噬菌体12肽库得到与HAbl8G／CDl47相结合的多肽，以固相法合成这些多肽并用质谱仪进行鉴定。最后利用侵袭力抑制实验从中筛选拮抗肝癌细胞转移的功能多肽。结果 经纯化的HAbl8G／CD147分子量为65000，Western blot验证结果阳性。用此纯化分子筛选噬菌体肽库，得到9条多肽。经体外功能验证，9条肽抑制肝癌细胞侵袭力的活性不一，其中3条有显著活性(P＜0．01)，活性最好的一条肽能使穿过Boyden小室膜的肝癌细胞数量较对照减少90．1％。结论 以亲和层析法和噬菌体随机肽库法相结合，筛选获得了有抗肝癌细胞侵袭力功能的多肽，此结果为抗肝癌转移肽类药物的研制奠定了基础。     

人参皂甙Rg3对小鼠肝癌H22腹水瘤的治疗研究

目的：研究人参皂甙Rg3治疗小鼠恶性腹腔积液的疗效。方法：100只雌、雄各半昆明种小鼠随机分为5组：Ⅰ组生理盐水组（0．9％NS）；Ⅱ组顺铂组（DDP0．5mg／kg）；Ⅲ组低剂量人参皂甙Rg3组（LPD0．3mg／kg）；Ⅳ组中剂量人参皂甙Rg3组（MPD1．0mg／kg）；Ⅴ组高剂量人参皂甙Rg3组（HPD3．0mg／kg）。所有小鼠肝癌H22腹水瘤模型建立后24小时开始分别腹腔注射0．2ml／只治疗，每日1次，共14天，同时观察小鼠的生活状态和体重。治疗结束后24小时，处死各组半数小鼠测各项指标，剩余小鼠停止治疗并观察生存时间。结果：各用药组较生理盐水组在腹水量（P〈0．05）、腹水中瘤细胞数和瘤细胞存活率方面都下降（P〈0．05）；随着人参皂甙Rg3给药浓度的增加，腹水中瘤细胞数及瘤细胞存活率下降（P〈0．05）；人参皂甙Rg3高剂量组腹水中瘤细胞数及瘤细胞存活率低于DDP组（P〈0．05）。各实验用药组较生理盐水组小鼠寿命延长，其中中剂量人参皂甙Rg3组延长最明显（P〈0．05）。结论：人参皂甙Rg3对小鼠恶性腹腔积液的形成具有抑制作用，这为临床应用提供了实验依据。     

抗人骨唾液蛋白单克隆抗体的制备及鉴定

目的制备高亲和力、高特异性的抗人骨唾液蛋白（BSP）单克隆抗体（mAb）,并对其生物学特性进行鉴定,为BSP作为乳腺癌骨转移临床诊断靶点的研究奠定基础。方法重组BSP蛋白免疫BALB／c小鼠,取其脾细胞与Sp2／0细胞融合,经筛选建立可稳定分泌抗BSPmAb细胞株,制备腹腔积液,ProteinG纯化。ELISA检测抗体的效价和亲和力,并用亚型鉴定试纸条鉴定mAb的亚型。应用Western blotting检测mAb的特异性,进一步利用纯化的mAb经免疫组织化学法检测乳腺癌细胞MDA-MB-231中BSP的表达情况。结果获得9株抗BSPmAb细胞株,选择其中2株（D001和D002）进一步鉴定,其上清效价分别为1：5120和1：10240,腹腔积液效价分别为1：25600和1：51200。2株抗体亚型均为IgG1型,轻链均为K型。Western blotting结果均在预期位置出现阳性条带。利用此2株细胞株所得抗体均可在乳腺癌细胞MDA-MB-231中检测到BSP的表达。结论成功制备能特异性识别BSP的mAb,为进一步研究BSP的生物学功能以及对BSP作为乳腺癌骨转移的标志物进行临床评价奠定基础。     

分泌抗人甲胎蛋白并对肝癌细胞有特异性亲和力的单克隆抗体杂交瘤细胞株的建立和鉴定

采用杂交瘤技术将小鼠骨髓瘤SP_2/O细胞与用人甲胎蛋白抗原免疫的BALB/c小鼠脾细胞融合,经多次筛选,获得7株分泌抗人甲胎蛋白并对肝癌细胞有特异性亲和力的单克隆抗体杂交瘤细胞株,其培养液上清和腹水效价分别为10~(-2)和10~(-6～9-)。杂交瘤腹水经硫酸铵盐析,DEAE纤维素层析,或免疫吸附亲和层析法纯化,每ml腹水单克隆抗体的产量为1～2mg。用ELISA,免疫双扩散、聚丙烯酰胺凝胶电泳、间接免疫荧光法鉴定了单克隆抗体的特异性、亲和力、稳定性及IgG亚类。     

一种经济高效的人外周血NK细胞纯化方法的建立

目的：建立一种经济并且纯化率高的人外周血NK细胞分离方法。方法：对用RosetteSep法直接从全血分离NK细胞的方法进行改进,先从全血中获得单个核细胞（PBMC）,与红细胞按比例混合后再进行分离,利用流式细胞仪检测纯度,并且用CCK-8法检测NK细胞对K562细胞的杀伤活性。结果：改进后分离每毫升血所需RosetteSep抗体复合物用量仅为直接分离法的1／50;NK细胞的纯度由纯化前的（11．02±3．15）％提高到（96．52±2．42）％,细胞回收率为（70．24±10．51）％;纯化的NK对靶细胞K562有较强的杀伤作用,与直接分离法比较杀伤活性差异无统计学意义（P〉0．05）。结论：改进后的方法可以获得高纯度的NK细胞,该分离方法不影响NK的杀伤活性,同时极大的降低了分离成本。初步建立了一种经济高效,快速简便,并且纯化率高的人外周血NK细胞纯化方法。     

Specific monoclonal antibody against human trypsin

A monoclonal antibody (MAb) directed against human trypsin-1 has been produced by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. Antibodies were screened by ultramicro enzymelinked immunosorbent assay (UMELISA). MAb was purified by affinity chromatography on protein A-Sepharose, and MAb had a high affinity for trypsin-1 with the affinity constant equal 1.79 x 10(9) L/mol. Specificity was studied by UMELISA using cross-reactant proteins; MAb gave a positive reaction with native trypsinogen-1 and with the same protein after reduction. Antibody appeared to be directed against sequential epitope. One-step purification is described. The method evolved the adsorption of the enzyme onto a Sepharose-MAb(3H9) affinity column. The collected fraction was characterized and is available for immunization of BALB/c mice and for the preparation of a standard for immunoenzymatic assay.     

RADIOIMMUNOLOCALIZATION OF XENOGRAFTED HUMAN GASTRIC ADENOCARCINOMA WITH ~(131)I-LABELED MONOCLONAL ANTIBODY RWS_(4) IN NUDE MICE

The monoclonal antibody (MAb) RWS4 specific to membrane-associated antigen of human gastric adenocarcinoma was purified by protein A-Sepharose 4B affinity chromatography and labeled with 131I by chloramine-T method. 131-RWS,, was injected (65 μCi/10μg/0.2 ml, intraperitoneally) into the stomach cancer-bearing nude mice (solid tumor about 1 cm in diameter), and its biodistribution was studied by SPECT and gamma-counter over a peroid of 7 days. A clear image of transplanted tumor was observed on the 4th day, and the image became more clear on the 6th day. After SPECT scanning, the animals were killed on the 3rd to 7th day separately and radioactivity was detected in various organs. The ratios of T/NT were calculated. The results were shown as follows: tumor/blood, was 3.41±0.29 on the 6th day and the tumor/other organs (liver, spleen, stomach, lung, heart, kidney and brain etc.) were>3. The specificity of the 131I-RWS4 was 7.74±0.65.     

Preparation of a novel monoclonal antibody specific for WIG-1 and detection of its expression pattern in human esophageal carcinoma

We have efficiently generated the first monoclonal antibody (MAb) against the human WIG-1 (wild-type p53-induced gene 1) protein, an apoptosis-related protein consisting of three zinc finger domains. Protein A affinity chromatography was performed to purify MAb from mice production ascites. Hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA). One MAb designated GW-3E4 (IgG1), effective in detecting the nuclear protein in human esophageal squamous cell carcinoma (ESCC) tissues and EC109 cell line, was characterized by ELISA and Western immunoblotting. Thus, it binds to native WIG-1 protein and should be useful in studies of WIG-1 protein function and expression. By Western immunoblotting analysis of 20 patients with ESCC using the MAb, we found that the expression of WIG-1 protein in tumor tissue was significantly higher than in incision margin. The results showed that WIG-1 might be a novel modifier in esophageal carcinogenesis, and the WIG-1 MAb should be useful in further study of the mechanism in WIG-1-related physiological reactions.     

Monoclonal antibody against human trypsin: production,characterization, and use for diagnosis

A monoclonal antibody (MAb) directed against human immunoaffinity purified trypsinogen has been produced by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. Antibodies were screened by ultramicro-enzyme-linked immunosorbent assay (UMELISA). The MAb was purified by affinity chromatography on protein A-sepharose, and MAb had a high affinity for trypsinogen with the affinity constant equal 2.06 x 10(9) L/mol. Specificity was studied by UMELISA using cross-reactant proteins; MAb gave a positive reaction with native trypsinogen-1 but did not react with the same protein after reduction. The antibody seem to be directed against conformational epitope. The MAb obtained was characterized immunologically and used to develop UMELISA for detection Trypsin. This monoclonal assay enabled the detection of 2.8 ng/mL.     

Purification of monoclonal antibody 3H11 against gastric cancer for in vivo use

Monoclonal antibody (McAb) 3H11 against gastric cancer was grown in the mouse ascites system. To acquire a clinical grade product for cancer radioimmuno-imaging was purified by two step high performance liquid chromatography (HPLC) protocol using protein A and high-performance hydroxylapatite (HPHT). An analysis of data reported shows the two step HPLC method to be the best purification procedure. This protocol satisfies purity and immunoreactivity requirement, and provides an sample sterility, free-pyrogens, free-mycoplasma and non-specific IgG contamination. This procedure described was capable of generating large amounts of clinical grade monoclonal antibody.     

Monoclonal antibody against free beta-subunit of human chorionic gonadotropin

Spleen cells from BALB/c mice immunized with free native human chorionic gonadotropin hormone beta-subunit (beta hCG) were fused with mouse myeloma cells (P3/X63-Ag8) and one hybridoma secreting monoclonal antibodies (MAbs), was obtained. This hybridoma specifically recognizes beta hCG and does not cross-react with other human glycoprotein hormones, such as luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and human chorionic gonadotropin (hCG). The MAb was of the IgG(1) subclass and ascitic fluid from this hybridoma was purified by affinity chromatography on Protein A-Sepharose CL-4B column to isolate the IgG(1) active fraction. The affinity constant of this MAb was 1.5 x 10(10)M(-1).     

PURIFICATION OF MONOCLONAL ANTIBODY 3H11 AGAINST GASTRIC CANCER FOR IN VIVO USE

Monoclonal antibody (McAb) 3H11 against gastric cancer was grown in the mouse ascites system. To acquire a clinical grade product for cancer radioimmuno-imaging was purified by two step high performance liquid chromatography (HPLC) protocol using protein A and high-performance hydroxylapatite (HPHT). An analysis of data reported shows the two step HPLC method to be the best purification procedure. This protocol satisfies purity and immunoreactivity requirement, and provides an sample sterility,free-pyrogens, free-mycoplasma and non-specific IgG contamination. This procedure described was capable of generating large amounts of clinical grade monoclonal antibody.     

抗人双特异性蛋白磷酸化酶18单克隆抗体的制备及鉴定

目的制备高亲和力、高特异性的抗人双特异性磷酸化酶18（Dusp18）单克隆抗体,并对其生物学特性进行鉴定,为Dusp18功能的研究奠定基础。方法重组Dusp18免疫BALB／c小鼠,取其脾细胞与SP2／0细胞融合,经筛选建立可稳定分泌抗Dusp18的单抗细胞株,制备腹腔积液,ProteinG纯化所得腹腔积液,ELISA及Westernblotting检测抗体的效价和亲和力,用亚型鉴定试纸条鉴定单克隆抗体的亚型。应用免疫组织化学检测Dusp18在肝癌组织中的表达情况。结果获得两株（F003和F004）单抗,上清效价分别为1：5120和1：10240,腹腔积液效价分别为1：25600和1：51200,Westernblotting结果均在预期位置出现阳性条带。利用两株抗体均可在肝癌组织中检测到Dusp18的表达。两株抗体亚型均为IgG1型,轻链均为k型。结论成功制备能特异性识别Dusp18的单克隆抗体,为进一步研究Dusp1的生物学功能,揭示其与肿瘤发生、发展之间的关系奠定了基础。     

A monoclonal antibody against human FXYD6

Previous research has shown that FXYD6 (FXYD domain-containing ion transport regulator 6) is highly increased in bile duct tumor. However, the biological function of FXYD6 is unclear. We aim to prepare and identify a monoclonal antibody against FXYD6, which will be used in diagnostics and as a tool in understanding the role of FXYD6 in pathogenesis of hepatobiliary cancer. In this study, hybridoma cell fusion technology is used for production of FXYD6 monoclonal antibody. BALB/c mice are immunized with FXYD6 synthetic peptide fragment. Hybridoma clones are screened using indirect enzyme-linked immunosorbent assay (ELISA). FXYD6 monoclonal antibody is produced by ascites revulsion. Protein A affinity chromatography is used for the purification of FXYD6 monoclonal antibody. Titer and specificity of monoclonal antibody are assessed by ELISA. Expression of FXYD6 in pancreatic cancer is detected by immunohistochemistry. As a result, one stable hybridoma cell clone producing FXYD6 monoclonal antibody has been established. 2.86?mg monoclonal antibody against FXYD6 with high specificity is prepared with titer of 1:5400. Immunohistochemistry shows that FXYD6 positive staining occurs in the cell membrane of pancreatic cancer, which results in an advantage in investigating the tissue distribution and biological function of FXYD6.