CEA-重组痘苗病毒对CEA阳性肿瘤疗效的实验研究

目的:为探讨CEA-重组痘苗病毒对CEA阳性肿瘤的治疗作用.方法:构建CEA阳性小鼠Hepa肝癌细胞模型(Hepa-CEA);对4组实验鼠进行颈背部皮下注射CEA阳性或CEA阴性同源鼠Hepa肝癌细胞,再接种痘苗病毒(野生型W-VV或重组型CEA-rV)3次:观察动物反应,并在接种癌细胞后每周测量一次皮下肿瘤大小.结果:4组实验鼠颈背部皮下均可见瘤块形成,但3个对照组肿瘤生长迅速,组间差异无统计学意义(P>0.05),而接种Hepa-CEA/CEA-rV组小鼠皮下肿瘤生长缓慢,瘤块较小,与3个对照组比较有统计学上的显著差异(P<0.01),整个实验过程中接种鼠未表现有毒副反应,结论:CEA-rV可能通过诱导机体免疫系统产生CEA特异性的细胞或体液免疫应答反应,对CEA阳性肿瘤的生长明显产生抑制作用.     

CEA-重组痘苗病毒预防CEA阳性肿瘤的动物实验研究

目的：探讨ＣＥＡ－重组痘苗病毒对ＣＥＡ阳性肿瘤的预防作用。方法：构建ＣＥＡ阳性小鼠Ｈｅｐａ肝癌细胞模型。将实验鼠分为４组,先分别皮下接种痘苗病毒３次,再分别颈背部皮下注射ＣＥＡ阳性或ＣＥＡ阴性同源鼠Ｈｅｐａ肝癌细胞。观察动物反应并在接种癌细胞后每周测量一次肿瘤大小。结果：３个对照组各级部皮下均可见有瘤块形成,并且以相近速度快速增长,３组之间无统计学上的显著差异,而接种ＣＥＡ－ｒＶ／Ｈｅｐａ－ＣＥＡ     

荷CEA-rV的DC增强CD3AK对CEA阳性肿瘤特异杀伤作用的研究

目的 观察荷CEA-rV的DC增强CD3AK细胞对CEA阳性肿瘤细胞的特异性杀伤功能。方法 用脐血制备脐血单个核细胞（UBMC），培养3h后，收集悬浮细胞制备CD3AK，重悬贴壁细胞制备DC。DC培养3d时加入CEA-rV，制备CEA-vV＋DC；在CD3AK培养8d时，加入CEA-rV＋DC混合培养，制备CEA-rV＋DC＋CD3AK。用MTT法测效应细胞UBMC，CD3AK，DC＋CD3AK，CEA-rV＋DC＋CD3AK，分别对CEA阳性靶细胞：LOVO，A549和CEA阴性靶细胞：肝癌细胞株BEL-7402，红白血病细胞株K652的杀伤活性。结果 ①用CEA兔抗人单克隆抗体对四种靶细胞株的鉴定结果表明，LOVO和A549确是CEA阳性细胞株，BEL．7402和K562确是CEA阴性细胞株。②培养10d的DC流式细胞仪检测结果示：高表达MHCI，Ⅱ类分子CD86（82．7％），CD80（51．1％），CD83（57．5％），CD40（69.4％），低表达CD123分子，光学和电子显微镜观察，DC细胞呈不规则形，有大量突起和伪足，成熟DC直径15-20um，胞浆线粒体，内质网丰富。证明为成熟DC。③CD3AK细胞和单纯UBMC细胞的流式细胞仪的免疫分子表型结果是，无论CD3，CD4，CD8和CD28，在CD3AK细胞组中的比率都是UBMC组的二倍以上。说明CD3AK组中成熟T淋巴细胞量明显高于UBMC细胞组。④三种效应细胞CEA-rV＋DC＋CD3AK，DC＋CD3AK和CD3AK对四种靶细胞的杀伤率均比单纯UBMC组明显提高（P〈0.01），而且CEA-rV＋DC＋CD，AK组〉DC＋CD3AK组〉CD3AK组〉UBMC组。说明细胞因子，DC和CEA-rV都有进一步提高UBMC广谱的杀伤肿瘤细胞活性的作用。⑤CEA-rV＋DC＋CD3AK和CD，AK相比较，对CEA阳性细胞LOVO和A549的杀伤活性提高的显著性（P〈0．01）明显优于对CEA阴性细胞K562和BEL-7402显著性（P〈0．05）。说明CEA-rV＋DC能显著提高CD3AK细胞对CEA阳性肿瘤的特异杀伤活性。CEA-rV＋DC＋CD3AK组和DC＋CD3AK组相比，对CEA阴性细胞株的杀伤活性无显著差异（P〉0．05），而对CEA阳性细胞株的杀伤活性确能明显提高（P〈0．01）。进一步证实了CEA-rV能明显提高CD3AK对CEA阳性肿瘤的特异杀伤作用。结论 CEA-rV＋DC能明显提高CD3AK细胞对CEA阳性细胞株杀伤作用，单纯DC对提高CD3AK杀伤CEA阳性细胞的作用不显著，结果表明CEA-rV在提高CD3AK对CEA阳性肿瘤的特异杀伤作用中起着关键作用。     

荷CEA-rV的DC诱导的CIK对CEA阳性原代肿瘤细胞的特异杀伤作用

目的：探讨携带CEA重组基因痘苗病毒（CEA-rV）抗原的树突状细胞（DC）诱导的杀伤细胞（CIK）对CEA阳性原代肿瘤细胞的特异性杀伤作用。方法：用脐血制备脐血单个核细胞（UBMC）、DC、CIK、DC＋CIK、CEArV＋DC＋CIK和CEA-rV＋DC＋UNBC细胞。取新鲜切除的食管癌组织和肺癌组织，制备原代培养的CEA阳性肿瘤细胞作为靶细胞。用MTT法测5组效应细胞对两种靶细胞的杀伤活性。结果：1）用CEA兔抗人单克隆抗体鉴定证实两种原代培养靶细胞系CEA阳性细胞。2）流式细胞仪测定DC的分子表型，证实为DC。3）流式细胞仪测定CIK的CD3^＋CD56^＋双阳性细胞达75．4％。4）DC＋CIK和CIK相比，对两种CEA阳性肿瘤细胞的杀伤率差异无统计学意义，P〉0．05。5）CE-rV4-DC＋CIK对两种CEA阳性肿瘤细胞的杀伤率（78．80％）明显高于DC＋CIK（52．94％），P％0．05。结论：CEA—rV＋DC能明显提高CIK细胞对原代培养的CEA阳性肿瘤细胞的杀伤活性，而单纯DC不能提高CIK的杀伤活性，说明CEA—rV在提高CIK对CEA阳性肿瘤细胞的特异杀伤作用中起重要作用。     

荷CEA-rV的DC增强CD3AK对CEA阳性肿瘤的特异杀伤作用

癌胚抗原（carcino embryonic antigen，CEA）是一种常见的肿瘤相关抗原，是目前国际上公认的肿瘤标记物。在人体上皮细胞恶性肿瘤中，包括90％的胃肠道肿瘤、胰腺癌、80％以上的非小细胞肺癌和50％左右的乳腺癌，都呈CEA阳性。人们称这类肿瘤为CEA阳性肿瘤。罗超权等心。用牛痘病毒构建的CEA-重组基因痘苗病毒（CEA-rV）既有CEA抗原的高表达，又可刺激机体产生CEA抗体。     

微波固化治疗肝癌抗肿瘤再生长的实验研究

探讨微波固化治疗肝癌对肿瘤再生长的抑制作用。方法：制作 Ｈ２２鼠肝癌皮下移植模型,对肿瘤分别进行微波固化（Ａ组）、手术切除（Ｂ组）及不治疗（Ｃ组）３种处理,然后对３组动物分别再次皮下接种不 量的Ｈ２２癌细胞。观察再接种癌灶的发癌情况。结果：再接种癌细胞后两个月内发癌率依再接种癌细胞数量而不同。接种１０＾７细胞的Ａ、Ｂ、Ｃ组发     

通过痘苗病毒介导的IL-4基因的表达在小鼠体内可以抑制肿瘤的形成

为了探讨通过重组痘苗病毒将白细胞介素4(IL-4)基因转染到体内表达的IL-4的抗肿瘤作用,本研究构建了表达IL-4基因的重组痘苗病毒VV_1/IL-4。小鼠皮下接种10′pfu的VV_1/IL-4后1至7d,在其皮肤发现10~5pfu/cm~2的痘苗病毒并在肝、肾内也发现少量病毒。这表明重组痘苗病毒可以有效地将细胞因子基因转染至皮肤。作者进一步探讨了这一重组痘苗病毒感染后对体内肿瘤形成的作用。C3H/Hen小鼠腿部接种10~3NCTC2472肿瘤细胞后给以VV_1/IL-4痘苗病毒治疗,结果表明,无论是用VV_1/IL-4治疗还是用对照病毒VV2/βgal或是PBS对照治疗均在3周内形成肿瘤。但用VV_1/IL-4治疗组肿瘤形成延缓且肿瘤生长速度缓慢。当用VV_1/IL-4每周治疗一次,连续治疗8周,93％小鼠未形成肿     

荷CEA-rV的DC增强CD3AK对CEA阳性肿瘤特异性杀伤作用的研究

Objective： To survey the special killing activity of CD3AK on anti-CEA-positive tumor enhanced by umbilical cord blood dendritic cell （DC） loaded with CEA recombinant vaccinia virus （CEA-rV）. Methods： Freshly isolated umbilical blood mononuclear calls （UBMC） were cultivated for 3 h. Suspension cells and attached calls were used to induce CD3AK calls and DC separately. DC was loaded with CEA-rV on the 3rd day to prepare CEA-rV＋DC. CD3AK cells were co-cultured with CEA- rV＋DC on the 8th day, to prepare CEA-rV＋DC＋CD3AK. The killing activity of each effector＇s cell, which included UBMC, CD3AK, DC＋CD3AK and CEA-rV＋DC＋CD3AK, was measured respectively by MTT reduction assay. Results： （1） 4 target cells were con- firmed by CEA monoclonal antibody of rabbit anti-human. Lovo and A549 were really CEA positive cell lines, while Bel-7402 and K562 were CEA negative cell lines. （2） It was showed by flow-cytometry that the mature DC cultured at 10th day expressed MHC I, II molecules such as CD86, CDS0, CD83 and CD40 highly, but CD123 lowly. The expression rates of CD86, CDS0, CD83 and CD40 was 82.7%, 51.1%, 57.5% and 69.4%, respectively. The appearances and intra-cellular structures of DC were observed through light and electron microscope. The diameter of mature DC was 15-20 μm presented the irregular morphologic appearanca, much prominences and pseudopodium. There were abundant mitochondria and endoplasmic reticulum in DC endochylema. （3） The rates of CD3, CD4, CD8 and CD28 in CD3AK cells group were 2 folds higher than that in UBMC group by FACS. It was said that the numbers of the mature T lymphocyte in CD3AK cells group were much greater than that in UBMC group. （4） The killing activities to 4 target cells of 3 effector＇s cells, which included CEA-rV＋DC＋CD3AK, DC＋CD3AK and CD3AK, were much greater than that of UBMC （P〈0.01）. Moreover, comparing with the killing activities of 4 effector＇s： CEA-rV＋DC＋CD3AK group 〉 DC＋CD3AK group 〉 CD3AK group 〉 UBMC group. It showed that, cytokine, DC and CEA-rV could efficiently elevate the killing activity of UBMC on broad-spectrum tumor cells. （5） Comparing with the killing activities of CEA-rV＋DC＋CD3AK and CD3AK cells to CEA positive and negative cells, the killing activities of CEA-rV＋DC＋CD3AK to CEA positive tumor calls, Lovo and A549 calls （P〈0.01） were remarkably better than that to CEA negative tumor cells BEL-7402 and K562 cells （P〈0.05）. It was said that the CEA-rV＋DC could obviously enhance the killing activity of CD3AK on CEA positive tumor cells. Comparing with the killing activities of CEA-rV＋DC＋CD3AK and DC＋CD3AK cells, the killing activity of CD3AK on CEA negative tumor cells was no statistical difference （P〉0.05）. However, the killing activity to CEA positive cells of CEA-rV＋DC＋CD3AK group was notably higher than that of DC＋CD3AK group. Namely, CEA-rV could distinctly promote the special killing activity to CEA positive tumor cells of CD3AK, but could not do it to CEA negative tumor cells. Conclusion： CEA-rV＋DC could obviously enhance the special killing activity of CD3AK on CEA positive tumor cell lines, while the DC only couldn＇t. The results indicated that the CEA-rV played an important role during the special killing activity of CD3AK cells to CEA positive tumor cells.     

重组人MUC1-MBP融合蛋白的抗肿瘤作用

目的： 研究重组人MUC1MBP的抗肿瘤作用。方法：用重组人MUC1MBP皮下注射途径免疫健康鼠，采用MTT法测定小鼠抗MUC1特异的CTL活性；通过免疫鼠成瘤及荷瘤鼠治疗实验检测其对肿瘤的预防和治疗作用。结果：MUC1MBP免疫鼠CTL对MCF7及Lewis肺癌细胞的杀伤率分别为(47.7±4.3) %和(67.5±6.5) %；免疫鼠成瘤结果免疫组小鼠共长5个肿瘤，存活时间明显延长，对照组共长51个，平均生存时间23 d；荷瘤鼠治疗结果显示治疗组小鼠肿瘤平均体积386 mm3，对照组小鼠肿瘤平均体积4 000 mm3。结论：重组人MUC1MBP具有明显的治疗、预防肿瘤和抑制肿瘤转移的作用,有希望发展成人类抗肿瘤疫苗。     

荷CEA-rV的DC增强CD3AK对CEA阳性肿瘤的特异杀伤作用

癌胚抗原(carcino embryonic antigen,CEA)是一种常见的肿瘤相关抗原,是目前国际上公认的肿瘤标记物.在人体上皮细胞恶性肿瘤中,包括90%的胃肠道肿瘤、胰腺癌、80%以上的非小细胞肺癌和50%左右的乳腺癌,都呈CEA阳性.人们称这类肿瘤为CEA阳性肿瘤[1].罗超权等[2]用牛痘病毒构建的CEA-重组基因痘苗病毒(CEA-rV)既有CEA抗原的高表达,又可刺激机体产生CEA抗体.     

Immunohistochemical demonstration of carcinoembryonic antigen (CEA) and its correlation with grading and staging on tissue sections of urinary bladder carcinomas

In 150 transitional cell carcinomas (TCC) of the urinary bladder, 50 each of Grades 1, 2, and 3, the content of carcinoembryonic antigen (CEA) was examined immunocytochemically and correlated to grading and staging. Fifty-seven percent of the TCC contained CEA-positive tumor cells. Their distribution in tumor tissue is described. They were found in 24% of Grade 1 cases, in 72% of Grade 2 carcinomas, and in 76% of Grade 3 tumors. None of the Grade 1 cases contained more than 10% CEA-positive cells, whereas 34% and 40% of the Grade 2 and 3 tumors, respectively, revealed more than 10% CEA-positive tumor cells. According to the correlation found between grading and staging, the percentage of TCC containing CEA-positive tumor cells was 34% in pTA, 59% in pT1, and 80% in pT2/3 tumors. The results show a correlation between CEA content of tumor tissue and histopathologic malignancy in TCC of urinary bladder.     

Immunohistochemical study of carcinoembryonic antigen (CEA) in gastric tumors: correlation with preoperative serum levels, histologic type, and grade of anaplasia of the tumor

The occurrence of carcinoembryonic antigen (CEA) was studied in 45 cases of gastric tumors by the immunoperoxidase technique. CEA-positive staining was found in 48.8% of tumors. A correlation was found between preoperative CEA values and tumor CEA staining. All patients with serum CEA values below 2.5 ng/ml showed CEA-negative staining of tumor. In patients with serum CEA values between 2.6 and 10 ng/ml, the tumors showed a minority of CEA-positive cells; but in patients with serum CEA values above 10 ng/ml, the tumors contained a majority of CEA-positive cells. CEA-positive staining was found in 34.4% of tumors of the diffuse type, and in 75% of tumors of the intestinal type. A high percentage of CEA positivity was seen in well-differentiated tumors (87.7%) compared to the moderately differentiated (69.2%), and to the undifferentiated (28.7%). A faint CEA-positivity was observed in intestinal metaplasia, while normal gastric mucosa was CEA-negative.     

CEA-rV负荷DC诱导CIK对CEA+肿瘤细胞的杀伤研究

  目的 用CEA-rV负荷脐血来源树突状细胞（DC）后，再诱导CEA抗原特异的细胞因子诱导的杀伤细胞，研究其对CEA阳性肿瘤细胞株杀伤活性的作用。方法 用淋巴细胞分离液分离脐带血单核细胞，分别诱导DC、CIK细胞及CEA-rV负载DC后，再与CIK细胞混合培养诱导CEA特异的细胞毒性T淋巴细胞。分别用以上不同类型细胞杀伤CEA+和CEA-的肿瘤细胞株。结果 CEA-rV诱导的特异性CIK对CEA阳性肿瘤细胞的杀伤活性，Lovo为58.2 %、A549为62.4 %，较之DC-CIK组对CEA阳性肿瘤细胞Lovo为44.8 % 、A549为50.2 %，具有更高的杀伤活性，差异有统计学意义（P＜0.05）。CEA-rV诱导的特异性CIK对CEA阴性肿瘤细胞的杀伤活性K562为79.7 %、Bel7402为70.1 %，DC-CIK组对CEA阴性肿瘤细胞K562为78.7 %、Bel7402为67.8 % ，两组间无明显区别，差异无统计学意义（P＞0.05）。用CEA-rV诱导的特异性CIK对CEA阳性肿瘤细胞Lovo杀伤提高了89.4 %，A549提高了83.1 %；而对于CEA阴性肿瘤细胞K562杀伤提高了32.5 %，Bel7402提高了21.7 %，两组间差异有统计学意义（P＜0.05）。结论 负载CEA-rV基因重组痘苗的DCs（CEA-rV-DCs）诱导的细胞毒性T淋巴细胞（CEA-rV-DCs-CIK）对CEA阳性肿瘤细胞有特异性杀伤能力。     

Carcinoembryonic antigen (CEA)-specific T-cell activation in colon carcinoma induced by anti-CD3 x anti-CEA bispecific diabodies and B7 x anti-CEA bispecific fusion proteins.

Two bispecific recombinant molecules, an anti-CD3 x anti-carcinoembryogenic antigen (CEA) diabody and a B7 x anti-CEA fusion protein, were tested for their capacity to specifically activate T cells in the presence of CEA-expressing colon carcinoma cells. T-cell activation by the anti-CD3 x anti-CEA diabody required close contact to CEA-positive cells and resulted in diabody-mediated cytotoxicity against the target cells. Additionally, CD28-mediated costimulation in combination with anti-CD3 x anti-CEA diabodies induced activation of autologous T cells in CEA-positive primary colon carcinoma specimens, as determined by flow cytometry. The high specificity of the bispecific diabody approach could be further enhanced by the use of B7 x anti-CEA fusion proteins because the costimulatory CD28-signaling to the T cells strictly depended on the expression of CEA on the target cells. We demonstrate that displaying engagement sites for the T-cell antigens CD3 and CD28 on the surface of colon carcinoma cells is a suitable way to activate and retarget T cells in a highly tumor-specific manner. For clinical purposes, B7 x anti-tumor-associated antigen (TAA) fusion proteins, which are equally effective but more specific compared with anti-CD28 monoclonal anti-bodies, thus may improve the tumor specificity of anti-CD3 x anti-TAA bispecific antibodies. Furthermore, B7-negative tumors can be converted into B7-positive tumors by B7 x anti-TAA fusion proteins without the need for B7 gene transfer to the malignant cells.     

The use of carcinoembryonic antigen for identification of human tumor cells in malignant effusions

A specific anti-carcinoembryonic antigen (CEA) antiserum was used to identify CEA-positive tumor cells in peritoneal and pleural effusions obtained from patients with various malignant neoplasia. In 7 out of 10 fluids in which tumor cells were detected by cytological examinations, cytoplasmic CEA-positive cells were also detected by an indirect immunofluorescence test. In addition, out of 11 fluid samples cytologically negative for tumor cells, CEA-positive cells were found in 8 cases. When both staining for cytoplasmic CEA and soluble fluid CEA levels were considered in combination, 81% of the samples were found to be positive by either one or both of these markers, whereas only 54% were positive by using cytological criteria. The data suggest that CEA marker may be used to identify tumor cells and to assess malignancy in pleural and peritoneal effusions in patients with certain types of cancer. The CEA marker was also used for identifying tumor cells grown in tissue cultures and for separating viable CEA-positive and CEA-negative cells from a mixed cell population using the fluorescence-activated cell sorter.     

A new human ovarian carcinoma cell line: establishment and analysis of tumor-associated markers

In the present study we describe the establishment and characteristics of a new human tumor cell line (OV-1063) positive for carcinoembryonic antigen (CEA) originating from ovarian metastatic tumor cells. Analysis of the cultured cells during their in vitro adaptation period revealed while the primary culture exhibited a low proportion of CEA-positive cells, this proportion increased with culture passages and eventually more than 90% of the cells in the established line were CEA-positive. Thus, during the period of adaptation to in vitro growth, a selection for CEA-positive cells took place but the amount of CEA secreted per each positive cell seemed to be constant. Several tumor-associated characteristics were found positive on the established OV-1063 cell line. The in vitro growing cell line exhibited an abnormal chromosome pattern with a near-trisomy karyotype for some chromosomes, colony formation in soft agar as well as positive staining with a monoclonal antibody B38.1. Culture supernatants of the OV-1063 cells contained significant amounts of CEA as well as CA-125 antigen which is an ovarian-carcinoma-associated antigen.     

Carcinoembryonic antigen expression of resurgent human colon carcinoma after treatment with therapeutic doses of 90Y-alpha-carcinoembryonic antigen monoclonal antibody

We have previously shown that the colon carcinoma (LS174T) xenografts that emerged shortly after radioimmunotherapy with 90Y-labeled anti-CEA monoclonal antibody (MAb) ZCE025 lacked significant expression of CEA in comparison with the untreated tumors. The present study was designed to establish if the immunophenotype of the treated tumors was the result of CEA specific therapy and if the effect was permanent. Athymic mice bearing LS174T tumors were treated either with 120 mu Ci of 90Y-ZCE025, an equal dose of 90Y-96.5 (nonspecific MAb), or received no treatment. When the treated tumors grew to approximately 1.5 cm in diameter (6 weeks after therapy), they were resected and aliquoted to be transplanted to other mice, plated in tissue culture, fixed in formalin, and homogenized for CEA quantitation. The procedure was repeated 3 times (a total of 4 months after treatment). The CEA content was evaluated 2 and 6 weeks after therapy and when the tumors were transplanted. We confirmed a 4-fold decrease of CEA in the resurgent tumors 6 weeks after specific 90Y-ZCE025 therapy, which was twice the decrease experienced by the tumors treated with nonspecific 90Y-96.5, indicating substantial and specific killing of CEA-expressing cells. The CEA content slowly but progressively increased with each new pass of the tumor in the mice, reaching approximately one-half the value of the controls at the end of the study. The resurgent tumors were also studied by immunohistochemistry with MAbs detecting different epitopes of CEA, keratin, TAG-72, and epithelial membrane antigen to evaluate possible additional immunophenotypic changes induced by radioimmunotherapy. Only the expression of TAG-72 (recognized by MAb B72.3) increased immediately after therapy, but it returned to the original levels by the end of the study. These results suggest that: (a) specific radioimmunotherapy with 90Y-ZCE025 selectively kills cells that express higher levels of CEA; (b) the immunophenotype of the surviving fraction of the tumor appears to slowly revert to its original form; and (c) other tumor markers unrelated to CEA can also be affected. These observations have important implications for the design of radioimmunotherapy trials.