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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?
OBJECTIVE: To transduce herpes simplex virus thymidine kinase and granulocyte monocyte-colony stimulating factor fusion gene (Lxpsp hGM CSF Hytk) into the human malignant neuroblastoma cell line SH SY5Y and observe the expression of the foreign fusion gene and its effect on tumor cells. The killing effect. METHODS : FuGENE6 was used to transfect the retroviral packaging cell line PA317 with the retroviral expression vector containing the fusion gene and the control vector containing the hygromycin phosphotransferase gene alone. SH SY5Y was transduced with a suitable amount of virus supernatant, and the presence of the fusion gene was detected by RT PCR. The growth status of the cytokine-dependent strain TF 1 and the gancopene (GCV) killing assay were used to identify the expression of the fusion gene. RESULTS: The recombinant virus-producing cells, PA317 GM CSF Hytk and PA317 Hy, were transduced with SH SY5Y and 60 mg·L-1 hygromycin, respectively, and screened for the stable passage of positive cell colonies SY5Y GMHytk SY5Y. Hy. RT PCR detected the presence of exogenous GM CSF in positive cell colonies. TF 1 cells were cultured with positive cell colony supernatants and the cells survived. The growth curves of SY5Y, SY5Y GMHytk and SY5Y Hy cells showed no significant difference, but with different concentrations of GCV acting on these 3 cells respectively, the GCV of 0. 0 3 to 30 0 mg·L -1 had obvious effect on SY5Y GMHytk. Killing effect (IC50 = 3 mg·L-1) and almost no toxicity to the other two cells (IC50 >300 mg·L-1). 30mg·L-1GCV can kill more than 80% of SY5Y GMHytk cells after 96 hours. Conclusion: The retrovirus-mediated fusion gene GMCSF tk was successfully introduced into the SH SY5Y tumor cell line and observed.