【Abstract】 Objective To investigate the expression of tumor associated glycoprotein-72 （TAG-72） in human breast cancer cells. Methods Two breast cancer cell lines MCF-7 and Bcap-37 were cultured and prepared. TAG-72 expressions in MCF-7 and Bcap-37 were detected immunochemically with anti-TAG-72 single chain variable fragment (scFv). Results TAG-72 was positively expressed in MCF-7 cells but negatively expressed in Bcap-37 cells. Conclusion Tumor associated antigen TAG-72 is expressed in certain human breast cancer cells, which indicate TAG-72 may be used as a tumor maker and anti-TAG-72 scFv may play a role in the diagnosis and treatment of breast cancer.
Objective To study effect of carcinoembryonic antigen (CEA) positive targeted lymphocytes on gastric cancer cells in vitro and in vivo. Methods The peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy volunteers. The recombinant vector anti-CEA-scFv-CD3ζ-pcDNA3.0 was transfected into the PBMCs by lipofectamine 2000, by this means, the CEA special lymphocytes were obtained. Meanwhile, the PBMCs transfected with empty plasmid pcDNA3.0 were used as control (empty vector lymphocytes). The different lymphocytes and gastric cancer cells (CEA positive KATOⅢ gastric cancer cells and CEA negative BGC-823 gastric cancer cells) were co-cultured, then the ability to identify the gastric cancer cells and it’s effect on apoptosis of gastric cancer cells were observed at 24 h or 36 h later respectively. The CEA special lymphocytes and empty vector lymphocytes were injected by the tail vein of nude mice bearing gastric cancer cells, then it’s effect on the tumor was observed. Results ① The CEA special lymphocytes could strongly identify the KATOⅢ gastric cancer cells (identification rate was 72.3%), which could weakly identify the BGC-823 gastric cancer cells (identification rate was 7.8%). ② The apoptosis rate of the co-culture of CEA special lymphocytes and KATOⅢ gastric cancer cells was significantly higher than that of the co-culture of empty vector lymphocytes and KATOⅢ gastric cancer cells (P=0.032), which had no significant difference between the co-culture of CEA special lymphocytes and BGC-823 gastric cancer cells and the co-culture of empty vector lymphocytes and BGC-823 gastric cancer cells (P=0.118). ③ The tumor volume of the co-culture of CEA special lymphocytes and KATOⅢ gastric cancer cells was significantly smaller than that of the co-culture of empty vector lymphocytes and KATOⅢ gastric cancer cells (F=5.010, P<0.01) or the co-culture of CEA special lymphocytes and BGC-823 gastric cancer cells (F=4.982, P<0.01), which had no significant difference between the co-culture of CEA special lymphocytes and BGC-823 gastric cancer cells and the co-culture of empty vector lymphocytes and BGC-823 gastric cancer cells (F=1.210, P>0.05). Conclusion CEA special lymphocytes can promote cell apoptosis and inhabit tumor reproduction of CEA positive gastric cancer cells in vitro and in vivo.
Objective To investigate the effect of tumor associated glycoprotein-72 （TAG-72） redirected T lymphocytes on breast cancer cells. Methods Peripheral blood mononuclear cells （PBMCs）were isolated from healthy volunteers. The recombinant vector anti-TAG-72-scFv-CD3ζ-pcDNA 3.0 were transfected into PBMCs by lipofectamineTM2000 （transfection group）， PBMCs transfected with plasmid pcDNA 3.0 as control group. MCF-7 and Bcap37 cells were cocultivated with PBMCs of transfection group and control group， respectively， and antitumor response of G1 block was observed. Results G1 block rate of MCF-7 cells in transfection group was （82.3±6.9）%， which was significantly higher than that in control group 〔（43.4±3.9）%， P＜0.05〕. G1 block rate of Bcap37 cells in transfection group was （51.3±4.7）%， and not differed from that in control group 〔（45.6±2.5）%， P＞0.05〕. Conclusion TAG-72 redirected T lymphocytes can inhibit the cell proliferation of TAG-72 positive breast cancer cells， and it may provide valuable tools for the cellular immunotherapy.
Objective To construct a green fluorescent protein expression plasmid pEGFP-C3-anti-TAG72 scFv-CD28, containing anti-TAG72 single chain variable fragment (scFv) fused into the transmembrane and intracellular domain of the signal-transducing chain of CD28 gene, and to transfect it into peripheral blood mononuclear cells． Methods Recombinant transmembrane and intracellular domain of CD28 cDNA and anti-TAG72 scFv cDNA fragment was subcloned into pEGFP-C3 vector. Recombinant clones were selected by Kanamyein， and then identified by PCR， enzyme digestion analysis and DNA sequencing. The recombinant plasmid was transfected into peripheral blood mononuclear cells by means of lipofection. The recombinant protein expression was confirmed by immunocytochemistry, laser scanning confocal microscope, PCR and Western blot analysis. Results The fused gene fragment of anti-TAG72 scFv-CD28 was successfully inserted into pEGFP-C3 plasmid, and it was confirmed by enzyme digestion and DNA sequencing. The fused anti-TAG72 scFv-CD28 gene and its protein was identified in peripheral blood mononuclear cells． Conclusion The eukaryotic expression plasmid pEGFP-C3-anti-TAG72 scFv-CD28 was successfully constructed and transiently expressed in peripheral blood mononuclear cells， which would lay a foundation for further studies on the role of it to activate tumor-associated antigen-specific T lymphocyte, for generating of modified T lymphocytes targeting gastrointestinal tumors.
Objective To construct a mammalian vector encoding angiostatin kringle 5 (K5) under the control of αfetoprotein （AFP） enhancer and albumin promoter, and to observe the expression of angiostatin by introducting angiostatin gene into hepatocellular carcinoma cells through gene transfection. Methods Angiostatin cDNA was amplified from normal human eukaryotic cells by using RTPCR. Meanwhile, AFP enhancer and albumin promoter sequences were directed cloned and were inserted into vector pcDNA3.1. The recombinant vector of pcDNA3.1AFABangiostatin K5His was constructed, which contained the angiostatin K5 cDNA sequence that was under the control of the AFP enhancer and promoter. Angiostatin K5 cDNA was introduced into human AFP positive hepatocellular carcinoma cell lines with the transfected cultured cells that were mediated with Lipofectamine 2000. The expression of angiostatin K5 was analyzed by Western blot and the protein was dectected with antiHis antibody. Results The 500base pair of angiostatin K5 was in accordance with the expected sequence and the recombinant vector of pcDNA3.1AFABangiostatin K5His was also confirmed as the anticipated sequence. The expression of angiostatin K5 in AFP positive hepatocellular carcinoma cells was detected both by SDSPAGE and Western blot. Conclusion Efficient construction and expression of angiostatin K5 to AFP positive cells make it possible for antiangiogenesis therapy of human hepatocellular carcinomas, which may provide a promising approach.