摘要
目的:观察抗CD20单链抗体(single chain variable fragment,scFv)-CD8-TCRζ融合基因转染的T淋巴细胞在体内外的抗人B细胞淋巴瘤作用,探讨利用CD20介导自体T淋巴细胞杀伤人B细胞淋巴瘤的可行性。方法:构建包含抗CD20scFv、CD8分子和CD3信号转导链ζ的融合基因,将其克隆入载体pcDNA3中,酶切鉴定正确后电转染入人外周血T淋巴细胞,诱导其表达抗CD20 scFv-CD8-TCRζ融合蛋白。流式细胞仪检测基因修饰后人T淋巴细胞结合CD20蛋白的功能;细胞杀伤试剂盒检测基因修饰后T淋巴细胞体外杀伤人B细胞淋巴瘤Raji细胞的能力;并在BALB/c裸鼠体内观察其对人B细胞淋巴瘤移植瘤的抑制效应。结果:成功构建、转染了抗CD20 scFv-CD8-TCRζ融合基因,并在人T淋巴细胞表面成功表达;流式细胞仪和细胞杀伤试剂盒检测结果表明经基因修饰后的人T淋巴细胞能特异性结合CD20抗原,并能特异性地杀伤人B细胞淋巴瘤Raji细胞,在裸鼠体内显著地抑制人B细胞淋巴瘤移植瘤的生长。结论:抗CD20 scFv-CD8-TCRζ融合基因转染的T淋巴细胞在体内外均能杀伤人B细胞淋巴瘤细胞,为进一步应用人T淋巴细胞杀伤作用治疗人B细胞淋巴瘤奠定了基础。
Objective：To observe the anti-tumor effect of T lymphocytes harboring anti-CD20 scFv-CD8-TCRζ fusion gene on human B lymphomas in vitro and in vivo, so as to explore the feasibility of CD20-mediated autogenous T lymphocytes in killing B-cell lymphomas. Methods, A fusion gene containing anti-CD20 scFv, CD8 molecule and CD3ζ chain was constructed and was cloned into pcDNA3. After confirmed by restriction endonuclease analysis, the fusion gene was used to transfect the human peripheral T lymphocytes through electroporation and expression of anti-CD20 scFv-CD8-TCRζ fusion protein was induced. The CD20 antigen-recognition ability of transfected T cells was determined by flow cytometry. The killing effect of transfected T cells on B cell lymphomas-Raji cells was tested in a cytotoxicity assay. The anti-tumor efficacy of this gene-modified T cell against the Raji tumor cell line was also evaluated in BALB/c nude mice. Results： We successfully constructed the anti-CD20 scFv-CD8-TCRζ fusion gene and expressed its protein on T cells. Flow cytometry and cytotoxicity assay demonstrated that the gene engineered T cells specifically recognized CD20 antigen and specifically inhibited B-cell lymphomas Raji cells. Furthermore, the T cells significantly inhibited the transplanted Raji cells in irradiated BALB/c nude mice. Conclusion： T lymphocytes transfected with anti-CD20 scFv-CD8-TCRζ fusion gene have antigen-specific anti-tumor effect in vitro and in vivo, which lays a foundation for utilizing human T lymphocytes to treat human B-cell lymphomas.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2006年第7期745-749,共5页
Academic Journal of Second Military Medical University
