中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2012年
1期
55-59
,共5页
陈小武%王埮%袁昆雄%王源江%王淑荣%孙圣刚%陈志斌
陳小武%王埮%袁昆雄%王源江%王淑榮%孫聖剛%陳誌斌
진소무%왕담%원곤웅%왕원강%왕숙영%손골강%진지빈
14-3-3蛋白质类%腺病毒科%基因疗法%细胞保护%基因转移技术
14-3-3蛋白質類%腺病毒科%基因療法%細胞保護%基因轉移技術
14-3-3단백질류%선병독과%기인요법%세포보호%기인전이기술
14-3-3 proteins%Adenoviridae%Gene therapy%Cytoprotection%Gene transfer techniques
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.
目的 探討人14-3-3 γ基因轉移對魚籐酮誘導的多巴胺能細胞損傷的保護作用.方法用腺病毒載體攜帶人14-3-3 γ基因(Ad/14-3-3 γ)感染PC12細胞,基因轉移成功後,用魚籐酮處理細胞.四甲基偶氮唑鹽法(MTT)測定細胞活性,DAP1染色法檢測細胞凋亡,高效液相色譜檢測細胞的分泌功能,激光共聚焦顯微鏡觀察α-突觸覈蛋白(α-synuclein)的聚集.結果 Ad/14-3-3 γ組細胞吸光度A570 (0.46 ±0.09),高于Ad-null組(0.19 ±0.08)和魚籐酮組(0.16±0.07),但低于正常對照組(0.63±0.11),分彆與各組比較,差異均有統計學意義(均P＜ 0.01)；Ad/14-3-3 γ感染組細胞培養液中多巴胺( 189±11) ng/ml和去甲腎上腺素(55±8)ng/ml,含量高于Ad-null感染組(79±12,38±7)ng/ml和魚籐酮組(81 ±13,39±7)ng/ml(均P＜ 0.01)；DAPI染色法顯示Ad/14-3-3 γ組凋亡率(27％±6％),高于正常對照組(10％±5％),但明顯低于Ad-null組(53％±10％)和魚籐酮組(56％±12％),分彆與各組比較,差異均有統計學意義(均P＜ 0.01)；激光共聚焦顯微鏡下觀察Ad/14-3-3 γ組細胞胞質內顆粒狀α-synuclein聚集體較Ad-null組和魚籐酮組顯著減少.結論 腺病毒介導的14-3-3 γ基因轉移對魚籐酮誘導的多巴胺能細胞損傷具有保護作用.
목적 탐토인14-3-3 γ기인전이대어등동유도적다파알능세포손상적보호작용.방법용선병독재체휴대인14-3-3 γ기인(Ad/14-3-3 γ)감염PC12세포,기인전이성공후,용어등동처리세포.사갑기우담서염법(MTT)측정세포활성,DAP1염색법검측세포조망,고효액상색보검측세포적분비공능,격광공취초현미경관찰α-돌촉핵단백(α-synuclein)적취집.결과 Ad/14-3-3 γ조세포흡광도A570 (0.46 ±0.09),고우Ad-null조(0.19 ±0.08)화어등동조(0.16±0.07),단저우정상대조조(0.63±0.11),분별여각조비교,차이균유통계학의의(균P＜ 0.01)；Ad/14-3-3 γ감염조세포배양액중다파알( 189±11) ng/ml화거갑신상선소(55±8)ng/ml,함량고우Ad-null감염조(79±12,38±7)ng/ml화어등동조(81 ±13,39±7)ng/ml(균P＜ 0.01)；DAPI염색법현시Ad/14-3-3 γ조조망솔(27％±6％),고우정상대조조(10％±5％),단명현저우Ad-null조(53％±10％)화어등동조(56％±12％),분별여각조비교,차이균유통계학의의(균P＜ 0.01)；격광공취초현미경하관찰Ad/14-3-3 γ조세포포질내과립상α-synuclein취집체교Ad-null조화어등동조현저감소.결론 선병독개도적14-3-3 γ기인전이대어등동유도적다파알능세포손상구유보호작용.
Objective 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.
