抗人肝癌细胞膜单克隆抗体HCMP－1的制备及其生物学特性的初步鉴定

用超选择免疫法，诱导ＢＡＬＢ／ｃ小鼠对正常人肝细胞抗原产生选择性低免疫反应后，二步腹腔注射人肝癌细胞悬液，２～４周后用人肝癌细胞作强化免疫动物１次，取出脾细胞与ＳＰ２／０骨髓瘤细胞用ＰＥＧ法融合制备杂交瘤。采用细胞抗原ＥＬＩＳＡ法，同时测定培养上清对肝癌细胞及正常肝细胞的免疫反应，筛出一株能稳定分泌抗人肝癌细胞膜抗原的单克隆抗体（ＨＣＭＰ－１ＭＡｂ）的杂交瘤细胞株。ＨＣＭＰ－１ＭＡｂ是一种ＩｇＧ２ａ亚型的单抗，它与ＨＢｓＡｇ、ＨＢｃＡｇ、ＨＢｅＡｇ等乙型肝炎病毒相关抗原无交叉免疫反应，与ＡＦＰ－亦无阳性反应。免疫萤光法证实ＨＣＭＰ－１ＭＡｂ对ＱＧＹ－７７０３、ＢＥＬ－７４０２及ＳＭＭＣ－７７２１等人肝癌细胞株呈阳性反应。改良ＡＢＣ免疫组织化学证明ＨＣＭＰ－１ＭＡｂ与肝细胞癌组织呈阳性反应。说明ＨＣＭＰ－１ＭＡｂ对肝癌反应的阳性率较高，特导性较好。超选择免疫法，先联合应用免疫抑制药物使动物对正常肝细胞抗原产生低免疫反应性。再以肝癌细胞免疫时，有利于动物免疫系统对肿瘤相关抗原的识别及特异性抗体的产生。从而提高了杂交瘤制备的成功率。我们认为这一方法在单抗制备方面有较高的应用价值。     

抗人肝癌细胞膜单克隆抗体HCMP—1的制备及其生物学特性的初步鉴定

用超选择免疫法，诱导ＢＡＬＢ／ｃ小鼠对正常人肝细胞抗原产生选择性低兔疫反应后，二步腹腔注射人肝癌细胞悬液，２－４周后用人肝癌细胞作强化免疫动物１次，取出脾细胞与ＳＰ２／０骨髓瘤细胞用ＰＥＧ法融合制备杂交瘤。采用细胞抗原ＥＬＩＳＡ法，同时测定培养上清对肝癌细胞及正常肝细胞的免疫反应，筛出一株能稳定分泌抗人肝癌细胞膜抗原的单克隆抗体的杂交瘤细胞株，ＨＣＭＰ－１ＭＡｂ是一种ＩｇＧ２亚型的单抗，它与ＨＢｓ     

抗人肺腺鳞癌单克隆抗体的研制

将人肺腺癌细胞ＳＰＣ、Ａ５４９、Ａ４２７序贯免疫ＢＡＬＢ／Ｃ小鼠，取脾细胞与小鼠骨髓瘤细胞ＳＰ２／０融合。获得一株稳定分泌抗肺腺鳞癌单克隆抗体的杂交瘤细胞株──Ｓ５Ａ１０─２，制备了小鼠腹水抗体并进行了纯化，腹水效价为１．０２×１０７，亚型测定为ＩｇＧ１．免疫组化分析ｓ５Ａ１０－２单抗与肺腺、鳞癌和肺鳞癌有较好的选择反应性，与正常组织无交叉反应，ＷｅｓｔｅｒｎＢｌｏｔ测定Ｓ５Ａｌ０─２在ＳＰＣ细胞膜上的抗原分子量为４０Ｋｄ、５０Ｋｄ和８０Ｋｄ，在Ａ５４９，细胞膜上的抗原分子量为４３Ｋｄ，而与Ａ４２７细胞膜蛋白无明显反应。该抗体的亲和常数为８．４３×１０１０Ｍ－１，Ｓ５Ａ１０－２杂交瘤细胞株，经液氮冻存后复苏，其分泌抗体能力不受影响。     

介导多种胃癌杀伤效应的双功能抗体

作者设计了一种带有“通用接头”的ｂｓＭＡｂ，即制备抗胃癌及抗半抗原ＴＮＰ的ｂｓＭＡｂ，它可介导多种经ＴＮＰ化的肿瘤杀伤因子。采用分泌抗胃癌单克隆抗体（ＭｃＡｂ）的杂交瘤变异株３Ｈ１１—ＨＡＴｓ与经ＫＬＩ－ＴＮＰ免疫的Ｂａ１ｂ／ｃ小鼠脾细胞进行融合，经筛选及克隆化共获得１０株二次杂交瘤，它们均能分泌与胃癌靶细胞ＢＧＣ８２３及ＢＳＡ－ＴＮＰ呈双阳性反应的抗体。但桥联法测定表明，只有二次杂交瘤１Ｇ７（γ２ｂ，γ２ｂ）及６Ａ３（γ２ｂ，μ）分泌ｂｓＭＡｂ。实验表明，ｂｓＭＡｂ６Ａ３和１Ｇ７可介导不同性质的经ＴＮＰ化的肿瘤杀伤剂，如蓖麻毒素、人血清白蛋白与丝裂霉素的偶联物，及牛血清白蛋白与阿霉素的偶联物。研究结果还表明，桥联法在确认ｂｓＭＡｂ中具有重要意义。分泌双特异性抗体的二次杂交瘤上清中不一定含有ｂｓＭＡｂ。一般认为γ链与μ链不可能组合形成ｂｓＭＡｂ，但本研究通过亚类分析、桥联法测定及体外细胞毒试验证实，二次杂交瘤６Ａ３分泌的ｂｓＭＡｂ确是由μ链与γ２ｂ链组合而成，这是一个新的发现。     

阴阳莲提取物3,4‘,5—三羟基芪—3—β—单—D—葡萄糖苷对人大肠癌细胞？…

目的：研究蓼科植物阴阳莲提取物３,４’,５－三羟基芪－３－β－单－Ｄ－葡萄糖苷（３,４’,５,ｔｒｉｈｙｄｒｏｘｙｓｔｉｂｅｎｅ－３－β－ｍｏｎｏ－Ｄ－ｇｌｕｃｃｏｓｉｄｅ,ＴＨＭＧ）对大肠癌细胞的体外生长特性、粘附性及浸润力的影响;从细胞层次探讨ＴＨＭＧ抗转移作用的基本环节。方法：检测４种大肠癌细胞（ＨＲ８３４８,Ｈｃｅ８６９３,ＨＴ２９,ＬｏＶｏ）在ＴＨＭＧ０．４ｍｍｏｌ／Ｌ,０．８ｍｍｏｌ／     

导入H—ras癌基因后肝癌细胞转移特性的变化

目的探讨Ｈ－ｒａｓ癌基因与肝癌细胞转移行为的关系。方法应用磷酸钙转染法将Ｈ－ｒａｓ癌基因导入人肝癌细胞株ＳＭＭＣ７７２１,通过进行粘附试验、运动试验、Ⅳ型胶原酶（ｃⅣａｓｅ）的分泌、表皮生长因子受体（ＥＧＦＲ）的表达等转移相关指标的测定,观察转染前后细胞转移特性的变化。结果转染Ｈ－ｒａｓ的ＳＭＭＣ７７２１细胞对胞外基质成分层粘连蛋白（ＬＮ）、纤维粘连蛋白（ＦＮ）的粘附能力提高,细胞的运动能力增强,ｃⅣａｓｅ的分泌明显增加,ＥＧＦＲ表达有中等程度的增加,且上述变化与Ｈ－ｒａｓ癌基因的蛋白产物ｐ２１的表达有明确的相关关系。结论Ｈ－ｒａｓ癌基因能在体外诱导人肝癌细胞的转移表型的产生,提高肝癌细胞的转移潜能。     

人鼻咽癌细胞株中c—Ha—ras基因DNase—1超敏区的序列分析

ｃ－Ｈａ－ｒａｓ基因的活化与鼻咽癌的发生有关。为了进一步了解ｃ－Ｈａ－ｒａｓ基因在鼻咽癌中的调控模式和活化机制，本文用ＤＮａｓｅ－１超敏区分析方法对人鼻咽癌细胞株ＨＮＥ２和ＨＯＮＥ１的ｃ－Ｈｅ－ｒａｓ基因进行研究，发现ｃ－Ｈａ－ｒａｓ基因启动子部位存在一个重要的ＤＮａｓｅ－１超敏区，测序结果表明其ＤＮＡ序列与正常ｃ－Ｈａ－ｒａｓ基因的相应序列完全等同。提示鼻咽癌细胞中ｃ－Ｈａ－ｒａｓ基因的调控模式     

小鼠B16黑色素瘤细胞HACs的提纯及其抑瘤作用和机理的探讨

目的：从小鼠Ｂ１６黑色素瘤细胞胞浆中提纯热休克蛋白抗原肽复合物（ｈｅａｔ ｓｈｏｃｋ ｐｒｏｔｅｉｎ－ａｎｔｉｇｅｎ ｐｅｐｔｉｄｅ ｃｏｍｐｌｅｓｅｓ．ＨＡＣＣｓ）,并观察其抑瘤作用及探索其机制。方法：ＣＮＢｒ－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析法纯纯Ｂ１６ ＨＡＣｓ,应用体内免疫法检测ＨＡＣ的抑瘤作用,结晶紫染色法检测γ－ＩＦＮ分泌活性,ＣｏｎＡ诱导的淋巴母细胞增殖法检测ＩＬ－２分泌活性,＾３Ｈ－     

用^99mTc标记大肠癌单克隆抗体的放射免疫定位研究

目的：Ｈｂ３是一株针对大肠癌相关抗原ＣＡ－Ｈｂ３的ＩｇＭ型单克隆抗体，免疫组织化学民大肠癌组织反应的阳性率为８６．４％，与正常大肠组织无反应。为确定Ｈｂ３用于放射免疫定位诊断大肠癌的可能性和实用价值进行了本研究。方法：采用羟基磷灰石层析法纯化抗体，冷消化法制备片段Ｆ（ａｂ‘）２，标记方法采用ＳｎＣｌ２．２Ｈ２Ｏ为还原剂、直接法标记＾９９ｍＴｃ，所用放射性剂量为１５－４０ｍＣｉ，显像用ＳＰＥＣＴ，显     

抗上皮细胞黏附分子单克隆抗体的制备及应用

 目的　制备抗人类上皮细胞黏附分子（EpCAM）的单克隆抗体（mAb）并对其进行鉴定及初步功能研究。方法　将原核表达的EpCAM免疫BALB／c小鼠,按常规方法将小鼠脾细胞与小鼠骨髓瘤Sp2／0细胞进行融合,用酶联免疫吸附试验进行阳性克隆的筛选。免疫印迹法对制备的mAb进行鉴定,用免疫组织化学法对临床的3份大肠癌组织样本进行初步检测。结果　酶联免疫吸附试验筛选获得了3株分泌抗EpCAM的mAb杂交瘤细胞株1B10、3G2和4E11。免疫印迹检测结果表明,3株杂交瘤细胞株分泌的抗体均能与EpCAM呈阳性反应,与GST蛋白不发生反应。免疫酶组织化学染色结果显示3株mAb能使3份大肠肿瘤组织样本呈现阳性反应,效果优于对照mAb。结论　成功地制备了3株识别大肠癌细胞表面抗原的mAb,这些mAb在大肠癌的诊断中可能具有一定潜在的应用价值。     

Radioimmunodetection of human lung cancer xenografts by isotope labeling monoclonal antibody ALT-04 in nude mice

Three nude mouse models bearing the human lung cancer were established from two fresh surgical specimens and one cell line. Tumor histological structure and cell morphology were similar before and after transplantation. The monoclonal antibody ALT-04 (McAb ALT-04) against human lung cancer was labeled by 125I, 131I and 201Tl. Radioimmunodetection study showed that all the three kinds of xenografts in nude mice were specifically located by McAb ALT-04. Imaging examination confirmed the ability of isotope labeling McAb ALT-04 to detect the presence of transplanted human lung cancer tissues without the aid of background subtraction manipulations. It is suggested that McAb ALT-04 have the possibility of locating in the tumor diagnosis and guiding in the treatment.     

131I标记抗人成骨肉瘤单抗荷瘤裸鼠体内分布和放射免疫显像研究

目的 :研究13 1I标记抗人成骨肉瘤单克隆抗体在荷人成骨肉瘤裸鼠体内分布和放射免疫定位显像。方法 :采用Iodogen固相法标记制备13 1I HOSMcAb。 2 5只荷瘤裸鼠随机分为 5组 ,分别腹腔注射13 1I HOSMcAb后 ,于6、12、2 4、48和 72h 5个时间段进行裸鼠的体内分布研究 ;对 5只荷瘤裸鼠分别腹腔注射13 1I HOSMcAb后 ,于 6、12、2 4、48和 72h 5个时间段进行荷瘤裸鼠的放射免疫定位显像研究。结果 :在荷人成骨肉瘤裸鼠腹腔注射13 1I HOSMcAb后 ,12h肿瘤与血的T/NT比值为1 3 7,2 4h为 3 75 ,48h达到最高为 5 2 4。腹腔注射13 1I HOSMcAb后 ,12h肿瘤部位即可见明显放射性浓聚 ,48h本底明显降低 ,肿瘤呈放射性热区。结论 :13 1I HOSMcAb对成骨肉瘤定向性较好 ,对放射免疫定位显像有利 ,为进一步放射免疫治疗研究提供了理论基础     

Comparison between 131I and 111In as radiolabels for monoclonal antibodies in immunoscintigraphy of tumor bearing nude mice

F(ab')2 fragments of monoclonal antibody (MoAb) 19-9 with specificity for human colorectal adenocarcinomas were labeled with 111In or 131I and infused into nude mice bearing the human adenocarcinoma HT 29 in order to compare their preferential biodistribution according to the radiolabel used. Animal tissue distribution measured one day and five days after infusion showed that tumor accumulation was greater for 111In than for 131I. However, non specific binding of 111In labeled MoAb 19-9 was also greater in normal tissue than 131I labeled antibody, except in blood. Therefore, the tumor/normal tissue ratios were to the advantage of 131I MoAb 19-9 and a better contrast was obtained on imaging with 131I as compared to 111In labeled MoAb 19-9. Based on this experimental model 111In does not seem to be the optimal candidate for tumor imaging using radiolabeled MoAb.     

荷人前列腺癌裸鼠移植瘤模型的建立及其应用研究

目的 应用人前列腺癌细胞株LNCaP接种裸鼠,鼠间移植传代,建立人前列腺癌裸鼠移植瘤模型.方法 观察移植瘤大体形态和组织病理学检查,并应用免疫组织化学方法观察肿瘤组织细胞中前列腺特异膜抗原（PSMA）的表达以及125I标记抗PSMA单抗的荷瘤裸鼠体内放射免疫显像.结果 移植瘤的形态和功能特性与原发肿瘤基本相似,移植瘤的移植成功率为100%;放免显像显示标记抗体能浓聚于肿瘤部位,经尾静脉注射标记抗体后96 h,肿瘤显像清晰,肿瘤组织/非肿瘤组织放射性比值（T/NT）均大于2.8.结论 本动物模型的建立为今后前列腺癌放射免疫治疗的实验研究提供1个理想的模型.     

Caveolin-1 levels are down-regulated in human colon tumors, and ectopic expression of caveolin-1 in colon carcinoma cell lines reduces cell tumorigenicity

Caveolin-1 expression and function were investigated in human colon cancer. Low levels of caveolin-1 mRNA and protein were detected in several colon carcinoma cell lines. Moreover, caveolin-1 protein levels were significantly reduced in human tumor epithelial mucosa (3.6 +/- 1.4-fold) when compared with normal colon mucosa for a majority (10 of 15) of the patients characterized. To directly assess the role of caveolin-1 in tumor development, caveolin-1 was reexpressed in the HT29 and DLD1 colon carcinoma cells, and the resulting HT29-cav-1 or DLD1-cav-1 cells were tested for tumorigenicity in nude mice. In most experiments, tumor formation was either blocked or retarded for HT29-cav-1 cells (10 of 13 mice) and DLD1-cav-1 cells (5 of 7 mice), as compared with both mock-transfected and parental HT29 or DLD1 cells. Interestingly, basal caveolin-1 levels were significantly reduced in HT29-cav-1 and DLD1-cav-1 cells isolated from tumors. Likewise, endogenous caveolin-1 mRNA and protein levels were found to be reduced in NIH-3T3 cells recovered from tumors after injection into nude mice. Thus, reexpression of caveolin-1 in colon carcinoma lines reduced the probability of tumor formation in vivo, and when tumors did develop from either HT29-cav-1, DLD1-cav-1, or NIH-3T3 cells, lower basal levels of caveolin-1 were detected. Finally, evidence was obtained indicating that initial caveolin-1 down-regulation in colon cancer cells need not be an entirely irreversible process because cell survival on selection for either drug resistance or increased metastatic potential correlated with increased caveolin-1 expression levels.     

Reactivity of monoclonal antibodies ALT-01 and ALT-04 and identification of lung cancer-associated antigens

Two IgG1 type monoclonal antibodies ALT-01 and ALT-04 were prepared by two different immunization schedules. ALT-01 was generated by fusing murine myeloma NS-1 cells with splenocytes from a BALB/c mouse immunized by human lung squamous carcinoma cells, which were coated by antisera to mixed human lymphocytes. For preparation of ALT-04, human lung squamous carcinoma xenograft-bearing nude mice were injected I. P. with the spleen cells of normal BALB/c mice in order to acquire immunofunction. The spleen cells from these tumor-bearing nude mice were fused with NS-1 cells. Then, these hybridomas were screened and cloned for 3 times. Two antibodies were shown to recognize the surface antigen on human lung carcinoma cells and several kinds of tumor cell lines but not those on normal cell lines. ALT-01 reacted to neither human lung carcinoma tissue nor its xenograft. ALT-04 reacted to human lung carcinoma tissue, of which, reaction to adenocarcinoma was the strongest but not to various normal tissues. Immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis and autoradiography was used to detect the associated antigen in 35S-labeled human lung carcinoma cells. Antigens, reacting to ALT-01, show one band of Mr 38,000 but those to ALT-04 reveal two bands of Mr 48,000 and 36,000.     

Pemetrexed disodium combined with oxaliplatin,SN38, or 5-fluorouracil,based on the quantitation of drug interactions in human HT29 colon cancer cells

Premetrexed disodium (MTA) is a novel multitargeted antifolate that inhibits thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase. It exhibits a broad spectrum of activity against several human tumor types including colorectal cancer. Therefore, we evaluated the anti-proliferative potential of MTA combined with drugs known to exert therapeutic activity against colon cancer, including 5-fluorouracil, oxaliplatin, and SN38, the active metabolite of irinotecan. The effects of MTA, alone or combined with one of theses 3 drugs, were investigated in parental human HT29 colon cancer cells and in 5-fluorouracil-resistant counterparts HT29-5FU cells. These drugs were administered either simultaneously or sequentially. Functional interactions between MTA, 5-fluorouracil, oxaliplatin, and SN38 were evaluated using median-effect plot analysis. The drug combination and sequence with optimal effects were evaluated in athymic mice bearing human HT29 tumor cell xenografts. Combinations of MTA with 5-fluorouracil required high concentrations to achieved additive and/or synergistic effects. Simultaneous exposure to MTA and oxaliplatin led to synergistic activity in both parental and 5-fluorouracil-resistant human HT29 colon cancer cells, leading to additive antitumor effects and minimal toxicity in athymic mice bearing HT29 cell tumors. Synergism between MTA and SN38 was also observed in both parental and 5-fluorouracil-resistant HT29 cells. These results argue in favor of clinical trials of chemotherapy combining MTA with oxaliplatin or irinotecan (CPT-11), for the treatment of patients with colon cancer.     

Sialyl Lewis-X抗原在大肠癌肝转移中的作用研究及临床意义

目的探讨SialylLewis-X(SLeX)抗原在大肠癌肝转移中的作用及临床意义。方法采用大肠癌Lovo、HT29细胞与人脐静脉血管内皮及内皮细胞的粘附试验,分别用扫描电镜及透射电镜观察Lovo、HT29细胞与脐静脉血管内皮及内皮细胞的粘附性及SLeX单抗封闭大肠癌细胞后,这种粘附性的改变;采用实验性裸鼠大肠癌肝转移模型分别观察SLeX单抗封闭Lovo、HT29细胞前后对实验性裸鼠大肠癌肝转移的影响。结果高表达SLeX抗原的Lovo细胞与脐静脉血管内皮的粘附性较低表达SLeX抗原的HT29细胞强,Lovo细胞与脐静脉血管内皮细胞的连接方式与HT29细胞明显不同;高表达SLeX抗原的Lovo细胞引起裸鼠肝转移率高于低表达SLeX抗原的HT29细胞。结论大肠癌细胞表面SLeX抗原在大肠癌细胞与脐静脉血管内皮细胞的粘附及实验性裸鼠肝转移中起重要作用,SLeX单抗能有效地抑制肿瘤细胞与脐静脉血管内皮细胞的粘附,并能降低实验性裸鼠肝转移的形成。     

Aberrant expression of OX1 receptors for orexins in colon cancers and liver metastases: an openable gate to apoptosis

Resistance to apoptosis is a recurrent theme in colon cancer. We have shown previously that the 7-transmembrane spanning receptor OX1R for orexins promotes robust apoptosis in the human colon cancer cell line HT29 through an entirely novel mechanism involving phosphorylation of tyrosine-based motifs in OX1R. Here, we investigated the status of OX1R in a large series of human colorectal tumors and hepatic metastases. All primary colorectal tumors regardless of their localization and Duke's stages and all hepatic metastases tested expressed OX1R mRNA and/or protein. In sharp contrast, adjacent normal colonocytes or hepatocytes as well as control normal tissues were negative. Next, we showed that nine human colon cancer cell lines established from primary tumors or metastases expressed OX1R mRNA and underwent important apoptosis on orexin-A challenge. Most interestingly, orexin-A also promoted robust apoptosis in cells that are resistant to the most commonly used drug in colon cancer chemotherapy, 5-fluorouracil. When human colon cancer cells were xenografted in nude mice, orexin-A administered at day 0 strongly slowed the tumor growth and even reversed the development of established tumors when administered 7 days after cell inoculation. Orexin-A also acts by promoting tumor apoptosis in vivo because caspase-3 is activated in tumors on orexin treatment of nude mice. These findings support that OX1R is an Achilles heel of colon cancers, even after metastasis or chemoresistance. They suggest that OX1R agonists might be novel candidates for colon cancer therapy.     

188Re标记CEA嵌合人源化抗体在荷人结肠癌裸鼠的放射免疫显像研究

背景与目的：癌胚抗原（carcinoembbryonic antigen,CEA）在多种肿瘤,尤其是结肠癌中高表达,抗CEA抗体在人结肠癌的诊断治疗中具有良好的应用前景。本研究采用^188Re标记CEA嵌合抗体,研究其在荷人结肠癌裸鼠体内的生物分布及放射免疫显像。方法：用氯化亚锡还原法标记CEA嵌合抗体及其亲本鼠单抗C50,薄层层析法鉴定标记抗本的标记率、放化纯度及体外稳定性,ELISA鉴定标记后的免疫活性,研究^188Re-CEA嵌合抗体在荷人结肠癌裸鼠体内的分布及放射免疫显像效果,并与C50鼠单抗的显像效果进行比较。结果：^188Re-CEA嵌合抗体的放化纯度大于95%;比活为36MBq/mg;免疫活性为61%;薄层层析示其在体外稳定。^188Re-CEA嵌合抗体的生物学分布显示：注射后24h,肿瘤与肾脏、血液的放射性比值分别为0.75、0.99,与其余各脏器的放射性比值均大于1.78;48h,肿瘤与肾脏、血液的放射性比值分别为1.02、1.12,与其余各脏器的放射性比值均大于2.08。20h后,肿瘤显影清晰。CEA嵌合抗体的放射生物学特性及显像效果与C50鼠单抗基本相同。结论：^188Re-CEA嵌合抗体在裸鼠体内放射免疫显像显示出良好的肿瘤特异性,且显像速度较快,可显示的肿瘤最小可达0.5cm。CEA嵌合抗体降低了免疫原性,在人体内显像更优于其亲本鼠单抗C50。