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Inhibition of Growth and Metastasis of Gastric Cancer by Transfection of B7-1

Author: KangJian
Tutor: XiaJianGuo
School: Nanjing Medical University
Course: Surgery
Keywords: B7-1 SGC-7901 lymph node metastasis immunogene therapy
CLC: R735.2
Type: Master's thesis
Year: 2009
Downloads: 6
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Abstract

Objective: To construct eukaryotic expressing plasmid of B7-1 gene, then transfect the plasmid steadily into SGC-7901 which is a cell line of gastric cancer, finally investigate the influence on the growth and metastasis of gastric cancer by transfection of B7-1.Methods: Total RNA was extracted from peripheral blood monouclear cells which were isolated from peripheral blood of the healthy volunteers. B7-1 gene was acquired by RT-PCR and the sites of restriction enzymes were brought into the product. The B7-1 gene and pcDNA3.1(+) were cut by restriction enzymes; then pured and connected by T4 ligase; eventually transfected into E.coli DH5α. The positive clone was selected and cultured, and then plasmids were obtained and verified. The sequence was confirmed and the recombined plasmid was named B7-1pcDNA3.1(+). Plasmids B7-1pcDNA3.1(+) and pcDNA3.1(+) were transfected into SGC-7901,then selected by G418 to establish the stable cell lines S-B and S-P. To detect the expression of B7-1 from levels of mRNA and protein, then test the distribution of cell cycle. Human peripheral blood mononuclear cells (PBMC) were separated and co-cultured with gastric cancer cell lines whose adhesion and cytotoxicity were tested; Apoptosis condition of gastric cancer cells were also monitored. Gastric cancer cells were inoculated into the great curve of BALB/c nude mouse close to the gastric antrum; Growth and metastasis of the gastric cancer were observed.Results: B7-1pcDNA3.1(+) was constructed successfully and stable cell lines called S-B and S-P were selected perfectly. B7-1 was high expressed in S-B, but wasn’t expressed in S-P and S. S-B had the significant difference with S-P and S in distribution of cell cycle. Adhesion and cytotoxicity of PBMC to S-B were stronger than S-P and S, but no significant difference between S-P and S. Tumorigenesis and lymph node metastasis of S-B were both lower than S; NK cells were intensively infiltrated in the S-B tumors, however, were scarcely observed in the lesions associated with S.Conclusion: The transfection of B7-1 slowed the process of cell cycle which was arrested in S stage so that inhibit the cell proliferation. Besides, it enhanced both the adhesion and cytotoxity to SGC-7901 of PBMC and the immunogenicity of SGC-7901 so that prevent its growth and lymph node metastasis.

Inhibition of Growth and Metastasis of Gastric Cancer by Transfection of B7-1

Abstract

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