自杀基因治疗神经母细胞瘤的实验研究

Experimental study on suicide gene therapy of neuroblastoma

目的 探讨自杀基因疗法对神经母细胞瘤的作用 ,为临床应用打下基础。方法 利用含胸苷激酶基因 (tk)与潮霉素磷酸转移酶基因 (hy)的融合基因的真核表达载体及仅含hy的空载体 ,以脂质体为介导 ,将这两种质粒分别转染神经母细胞瘤细胞系SH SY5Y。利用潮霉素药物敏感实验筛选SH SY5Y ,用不同浓度的丙氧鸟苷 (GCV)分别作用于SH SY5Y、SH SY5Y hytk及SH SY5Y hy。 结果 利用潮霉素药物敏感实验筛选SH SY5Y ,12d时SH SY5Y全部死亡的最低浓度为6 0 μg/ml,30 μm丙氧鸟苷可最大限度杀死SH SY5Y hytk。杀伤效应为 96h死亡率 >80 %。仅含有18%的SH SY5Y hytk就可使周围的 2 0 %的SH SY5Y细胞死亡 ,总死亡率为 38%。 结论 6 0 μg/ml潮霉素是筛选SH SY5Y hytk及SH SY5Y hy阳性克隆的最佳剂量。 30 μM丙氧鸟苷是作用于SH SY5Y hytk的最佳剂量。tk/GCV系统大于 18%SH SY5Y hytk具有旁观者效应 ,可极大地提高疗效。

Objective In order to understand the effect of tk suicide gene therapy on neuroblas- toma. Methods Hygromycin phosphotransferase tk fusion gene and hygromycin phosphotransferase carrier were transduced separately into two groups of neuroblastoma cell line. Different ganciclovir (GCV) dosage was given to SH-SY5Y, SH-SY5Y-hy and SH-SY5Y-hytk separately. Results The cytotoxic effects of GCV on SH-SY5Y-hy-tk cells was obvious (IC 50 = 0.03 *!μM) and killed over 80% of cells after 96 hours with the dosage of 30*!μM GCV. The lowest concentration of hygromycin was 60μg/ml, There was no difference in the growth curve of SH-SY5Y, SH-SY5Y-hy and SH-SY5Y-hytk. Conclusions The best dosage of hygromycin is 60*!μg/ml for selecting the SH-SY5Y-hytk. The best dosage of GCV to kill SH-SY5Y-hytk is 30*!μM . SH-SY5Y-hytk has the bystander effect, if it is greater than 18% in the mixture cells of SH-SY5Y-hytk in the tk/GCV system.