TARGET CELL SPECIFIC GENE TRANSFER BY ANTIFECTION AND REGULATION OF PROLIFERATION OF CANCER CELLS

通过抑制和调节癌细胞增殖进行靶细胞特异性基因转移

• 批准号: 10470173
• 负责人: TSUCHIYA Shigeru
• 金额: $ 7.49万
• 依托单位: TOHOKU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10470173/
• 关键词: Antifection; Anti-CD3 antibody; pEGFP; WASP遺伝子野性型; WASP遺伝子変異型; GFP融合蛋白; フローサイトメーター

Purpose : To develop an antifection method, a new technique of antibody-mediated targeted gene transfection, we determined optimal conditions of the gene transfer.Materials and Methods : OKT3, the antibody (Ab) against CD3 was chemically conjugated using benzoquinone to pEGFP, plasmid DNA containing green fluorescent protein (GFP) gene. The Ab-DNA conjugate was enriched by layering the mixture on a 40% sucrose solution followed by an ultracentrifugation. Coupling between DNA and Ab was determined by a 1% agarose gel electrophoresis followed by western blotting by anti-mouse IgG antibody. Jurkat T cells expressing CD3 were incubated with the Ab-DNA conjugate, and then determined the expression of GFP by flowcytometry.Results and Discussion : Agarose gel electrophoresis showed a change of the electrophoretic mobility in the Ab-DNA conjugate, as compared to plasmid DNA alone. The western blot revealed that anti-mouse IgG antibody reacted against the observed band in the Ab-DNA reaction mixture, indicating the existence of the Ab-DNA conjugate. Successful transfection of the Ab-DNA conjugate into Jurkat cells was observed by coupling lipids (FuGENE) to the Ab-DNA conjugate. Chloroquine failed to confer successful transfection due to its cytotoxicity. Antifection could be an useful method for gene therapy.

目的：为了开发一种抗体方法，一种抗体介导的靶向基因转染的新技术，我们确定了基因转移的最佳条件。材料和方法：OKT3，针对CD3的抗体（AB）使用苯甲酰酮对PEGFP，含有绿色荧光蛋白（gfpp pegfp）化学结合CD3。通过将混合物分层在40％的蔗糖溶液上，然后进行超速离心，从而富含AB-DNA结合物。 DNA和AB之间的耦合由1％琼脂糖凝胶电泳，然后通过抗小鼠IgG抗体进行蛋白质印迹。将表达CD3的Jurkat T细胞与AB-DNA偶联物孵育，然后通过流胞度仪确定GFP的表达。分析和讨论：与单独质粒DNA相比，AB-DNA共轭物中电泳迁移率的变化显示。蛋白质印迹显示，抗小鼠IgG抗体在AB-DNA反应混合物中与观察到的带反应，表明AB-DNA结合物的存在。通过将脂质（Fugene）耦合到AB-DNA偶联物，可以成功转染AB-DNA结合物转染到Jurkat细胞中。由于其细胞毒性，氯喹未能成功转染成功转染。抗分裂可能是基因治疗的有用方法。

项目成果

Kumaki S et al.: "Prolonged secretion of IL-15 in patients with severe forms of graft-versus-host disease after allogeneic bone marrow transplantation in children" International J Hematology. 67. 307-312 (1998)

Kumaki S 等人：“儿童同种异体骨髓移植后患有严重移植物抗宿主病的患者 IL-15 分泌延长”《国际血液学杂志》。

Kumaki S, Ishii N, Minegishi M, Tsuchiya S, Cosman D, Sugamura K and Konno T: "Functional role of interleukin-4(IL-4) and IL-7 in the development of X-linked severe combined immunodeficiency"Blood. 93. 607-612 (1999)

Kumaki S、Ishii N、Minegishi M、Tsuchiya S、Cosman D、Sugamura K 和 Konno T：“白细胞介素 4 (IL-4) 和 IL-7 在 X 连锁严重联合免疫缺陷发展中的功能作用”血液。

Suzuki T.et al.: "Bilateral pneumothoraces with multiple bullae in a patient with asymptomatic bronchiolitis obliterance 10 years bone marrow transplantation"Bone Marrow Transplantation. 23. 829-831 (1999)

Suzuki T.等人：“无症状性细支气管炎闭塞患者双侧气胸伴多发大疱10年骨髓移植”骨髓移植。

Kawai S, Sasahara Y, Minegishi M, Tsuchiya S, Fujie H, Ohashi Y, Kumaki S, Konno T: "Immunological reconstitution by allogeneic bone marrow transplantation in a child with the X-linked hyper-IgM syndrome"Eur J Pediatr. 158. 394-397 (1999)

Kawai S、Sasahara Y、Minegishi M、Tsuchiya S、Fujie H、Ohashi Y、Kumaki S、Konno T：“X 连锁高 IgM 综合征儿童通过同种异体骨髓移植进行免疫重建”Eur J Pediatr。

Kawai S. et al.: "Immunological reconstitution by allogeneic bone marrow transplantation in a child with the X-linked hyper-lgM syndrome"Eur J Pediatr. 158. 394-397 (1999)

Kawai S.等人：“通过同种异体骨髓移植对患有X连锁高IgM综合征的儿童进行免疫重建”Eur J Pediatr。