【摘要】 目的 :将单纯疱疹病毒胸苷激酶和粒细胞 单核细胞集落刺激因子融合基因 (Lxpsp hGM CSF Hytk)转导人恶性神经母细胞瘤株SH SY5Y ,观察外源融合基因的表达及其对肿瘤细胞的杀伤作用。方法 :FuGENE6介导将含融合基因的逆转录病毒表达载体和仅含潮霉素磷酸转移酶基因的对照载体分别转染逆转录病毒包装细胞系PA317。以适量病毒上清转导SH SY5Y ,RT PCR方法检测融合基因的存在 ;细胞因子依赖株TF 1的生长状况及丙氧鸟苷 (GCV)杀伤实验鉴定融合基因的表达。结果 :将重组病毒产生细胞PA317 GM CSF Hytk和PA317 Hy的病毒上清 2ml分别转导SH SY5Y ,6 0mg·L-1的潮霉素进行筛选 ,得到了稳定传代的阳性细胞集落SY5Y GMHytk ,SY5Y Hy。RT PCR检测阳性细胞集落中有外源的GM CSF存在。用阳性细胞集落上清培养TF 1细胞 ,细胞成活。SY5Y ,SY5Y GMHytk ,SY5Y Hy 3种细胞的生长曲线无明显差别 ,但用不同浓度GCV分别作用于此 3种细胞显示 :0 .0 3～ 30 0mg·L-1的GCV对SY5Y GMHytk有明显的杀伤作用 (IC50 =3mg·L-1) ,而对另外两种细胞几乎无毒性作用 (IC50 >30 0mg·L-1)。 30mg·L-1GCV作用 96h后可杀死 80 %以上的SY5Y GMHytk细胞。结论 :应用逆转录病毒介导 ,将融合基因GMCSF tk成功地导入了SH SY5Y肿瘤细胞株 ,并观察?更多还原

【Abstract】 Objective: To investigate the effectiveness of combination antitumoral therapy. Methods: A retroviral mediated fusion gene (Lxpsp hGM CSF Hytk)was transferred into human neuroblastoma cell line SH SY5Y. The recombinant vectors were transferred into a package cell line PA317 by FuGENE6, then the virus was used to infect SH SY5Y. Hygromycin B selected the positive cell colonies. RT PCR and a cytokine dependent cell line TF 1 were used to test the transduction and the secretion of bioactive GM CSF respectively. Results: RT PCR demonstrated the existence of the external GM CSF gene and the bioactivity of hGM CSF was tested exactly. The therapeutic efficacy of HSV tk followed by ganciclovir (GCV) administration was studied. The cytotoxic effect on SY5Y GMHytk cells exposed to GCV (0.03-300 mg·L -1 ) was obvious (IC 50 =3 mg·L -1 ), while GCV almost had not any cytotoxic effect on SH SY5Y and SY5Y Hy cells (IC 50 >300 mg·L -1 ). 30 mg·L -1 GCV killed over 80% (volume fraction) of SY5Y GMHytk after 96 hours. Conclusion : A retroviral mediated fusion gene was successfully transferred into the SH SY5Y , and the antitu moraleffect in vitro was observed.更多还原