摘要目的 探讨人14-3-3 γ基因转移对鱼藤酮诱导的多巴胺能细胞损伤的保护作用.方法用腺病毒载体携带人14-3-3 γ基因(Ad/14-3-3 γ)感染PC12细胞,基因转移成功后,用鱼藤酮处理细胞.四甲基偶氮唑盐法(MTT)测定细胞活性,DAP1染色法检测细胞凋亡,高效液相色谱检测细胞的分泌功能,激光共聚焦显微镜观察α-突触核蛋白(α-synuclein)的聚集.结果 Ad/14-3-3 γ组细胞吸光度A570 (0.46 ±0.09),高于Ad-null组(0.19 ±0.08)和鱼藤酮组(0.16±0.07),但低于正常对照组(0.63±0.11),分别与各组比较,差异均有统计学意义(均P＜ 0.01)；Ad/14-3-3 γ感染组细胞培养液中多巴胺( 189±11) ng/ml和去甲肾上腺素(55±8)ng/ml,含量高于Ad-null感染组(79±12,38±7)ng/ml和鱼藤酮组(81 ±13,39±7)ng/ml(均P＜ 0.01)；DAPI染色法显示Ad/14-3-3 γ组凋亡率(27％±6％),高于正常对照组(10％±5％),但明显低于Ad-null组(53％±10％)和鱼藤酮组(56％±12％),分别与各组比较,差异均有统计学意义(均P＜ 0.01)；激光共聚焦显微镜下观察Ad/14-3-3 γ组细胞胞质内颗粒状α-synuclein聚集体较Ad-null组和鱼藤酮组显著减少.结论 腺病毒介导的14-3-3 γ基因转移对鱼藤酮诱导的多巴胺能细胞损伤具有保护作用.

abstractsObjective To explore the protective effect of human 14-3-3 γ gene transfer on dopaminergic cells against rotenone-induced injury.Methods Adenovirus vector carrying the gene of 14-3-3 γ (Ad/14-3-3 γ) was employed to transfect PC12 cells.Then the cells were exposed to rotenone as a model of Parkinson's disease.Methyl thiazolyl tetrazolium (MTT) was used to assay the viability of PC12 cells.The 4',6-diamidino-2-phenylindole (DAPI) staining was used to analyze the apoptotic ratio of PC12 cells among the groups of control,Ad/14-3-3 γ,Ad-null and Rotenone. And high performance liquid chromatography ( HPLC ) was performed to detect the secreting functions of PC12 cells.The aggregates of α-Synuclein protein were detected under confocal microscopy.Results MTT showed that the cell absorbance A570 of Ad/14-3-3 γ group ( 0.46 ± 0.09 ) was higher than that of Ad-null group (0.19 ± 0.08 ) and rotenone group (0.16 ± 0.07 ),but lower than that of normal control ( 0.63 ± 0.11 ),( all P ＜ 0.01 ) ;HPLC-ECD showed that the levels of dopamine (189 ± 11 ) ng/ml and noradrenalin (55 ±8) ng/ml in the culture fluid of Ad/14-3-3 γ group were higher than those of Ad-null group(79 ± 12,38 ± 7 )ng/ml and rotenone group(81 ± 13,39 ± 7) ng/ml ( all P ＜ 0.01 ).DAPI staining showed that cell apoptosis ratio of group Ad/14-3-3 γ (27％ ±64％ ) was lower than that of group Ad-null (53％ ± 10％ ) and rotenone group (56％ ± 12％,P ＜ 0.01 ),but higher than that of control group ( 10％ ±5％,P ＜ 0.01 ).Under confocal microscopy,the aggregates of o-synuclein protein in PC12 cells were detected more in Ad-null group and rotenone group than that in Ad/14-3-3 γ group.Conclusion Gene transfer of Ad/14-3-3 γ has a protective effect on dopaminergic cells against rotenone-induced injury.