中华医学遗传学杂志
中華醫學遺傳學雜誌
중화의학유전학잡지
Chinese Journal of Medical Genetics
2015年
4期
462-467
,共6页
陈涛%邓益东%廖小平%赵建农%文国强%翁国虎%马飞%郑莹莹
陳濤%鄧益東%廖小平%趙建農%文國彊%翁國虎%馬飛%鄭瑩瑩
진도%산익동%료소평%조건농%문국강%옹국호%마비%정형형
α-synuclein基因%姜黄素%线粒体ATP敏感性钾离子通道%帕金森病
α-synuclein基因%薑黃素%線粒體ATP敏感性鉀離子通道%帕金森病
α-synuclein기인%강황소%선립체ATP민감성갑리자통도%파금삼병
α-synuclein gene%Curcumin%Mitochondrial ATP-sensitive potassium channel%Parkinson disease
目的 探讨姜黄素对α-突触核蛋白(α-synuclein)寡聚体形成、线粒体膜电位及线粒体ATP敏感性钾通道(mitochondrial ATP-sensitive potassium channels,mitoKATP)的影响.方法 构建野生型、A53T突变型α-synuclein真核表达质粒,用脂质体介导转染方式将构建好的质粒转入PC12细胞,并分别应用姜黄素(20 ìmol/L)、5-羟基葵酸盐(5-hydroxydecanoate,5-HD)进行干预;48 h后应用免疫印迹、斑点杂交检测细胞α-synuclein寡聚体形成;应用JC-1荧光探针及乳酸脱氢酶(lactate dehydrogenase,LDH)检测线粒体膜电位及细胞膜毒性损伤;并在姜黄素干预处理前15 min给予5-HD预处理,免疫印迹检测mitoKATP蛋白的功能亚基Kir6.2的改变,细胞膜片钳观察mitoKATP的改变.结果 将构建所得EGFP-α-synuclein-WT、EGFP-α-synuclein-A53T真核表达质粒转染PC12细胞,48 h后免疫印迹及斑点印迹结果显示,α-synuclein基因过表达或突变诱导均可促进α-synuclein寡聚体形成,姜黄素可明显减弱α-synuclein基因过表达或突变诱导的α-synuclein寡聚体形成;JC-1及LDH结果显示,与正常PC12细胞相比,转染野生型、A53T突变型组细胞LDH水平分别升高35.5％及42.3％(P＜0.05),JC-1红/绿荧光比率与正常PC12细胞相比下降60.44％、65.22％(P＜0.05),姜黄素干预后,LDH水平分别下降36.3％及23.5％(P＜0.05),红/绿荧光比率上升48.46％、50.33％(P＜0.05);转染野生型或A53T突变型α-synuclein融合表达载体组Kir6.2蛋白表达增强,早期通道电流有明显增高的趋势,随后降低,应用姜黄素干预后,α-synuclein基因过表达或突变所诱导的Kir6.2蛋白表达下调,但细胞mitoKATP通道电流增加,与转染组比较有统计学意义(P＜0.05).结论 姜黄素可抑制α-synuclein基因过度表达或突变诱导的α-synuclein寡聚体形成;姜黄素可能通过稳定线粒体膜电位阻断了α-synuclein基因过表达或突变所导致的细胞凋亡;mitoKATP通道的开放可能是α-synuclein基因过表达或A53T突变诱导细胞凋亡的自发始动保护机制,姜黄素可能通过开放mitoKATP通道拮抗α-synuclein基因过表达或突变所导致的细胞毒性,mitoKATP通道的开放程度与Kir6.2蛋白的表达不成正相关.
目的 探討薑黃素對α-突觸覈蛋白(α-synuclein)寡聚體形成、線粒體膜電位及線粒體ATP敏感性鉀通道(mitochondrial ATP-sensitive potassium channels,mitoKATP)的影響.方法 構建野生型、A53T突變型α-synuclein真覈錶達質粒,用脂質體介導轉染方式將構建好的質粒轉入PC12細胞,併分彆應用薑黃素(20 ìmol/L)、5-羥基葵痠鹽(5-hydroxydecanoate,5-HD)進行榦預;48 h後應用免疫印跡、斑點雜交檢測細胞α-synuclein寡聚體形成;應用JC-1熒光探針及乳痠脫氫酶(lactate dehydrogenase,LDH)檢測線粒體膜電位及細胞膜毒性損傷;併在薑黃素榦預處理前15 min給予5-HD預處理,免疫印跡檢測mitoKATP蛋白的功能亞基Kir6.2的改變,細胞膜片鉗觀察mitoKATP的改變.結果 將構建所得EGFP-α-synuclein-WT、EGFP-α-synuclein-A53T真覈錶達質粒轉染PC12細胞,48 h後免疫印跡及斑點印跡結果顯示,α-synuclein基因過錶達或突變誘導均可促進α-synuclein寡聚體形成,薑黃素可明顯減弱α-synuclein基因過錶達或突變誘導的α-synuclein寡聚體形成;JC-1及LDH結果顯示,與正常PC12細胞相比,轉染野生型、A53T突變型組細胞LDH水平分彆升高35.5％及42.3％(P＜0.05),JC-1紅/綠熒光比率與正常PC12細胞相比下降60.44％、65.22％(P＜0.05),薑黃素榦預後,LDH水平分彆下降36.3％及23.5％(P＜0.05),紅/綠熒光比率上升48.46％、50.33％(P＜0.05);轉染野生型或A53T突變型α-synuclein融閤錶達載體組Kir6.2蛋白錶達增彊,早期通道電流有明顯增高的趨勢,隨後降低,應用薑黃素榦預後,α-synuclein基因過錶達或突變所誘導的Kir6.2蛋白錶達下調,但細胞mitoKATP通道電流增加,與轉染組比較有統計學意義(P＜0.05).結論 薑黃素可抑製α-synuclein基因過度錶達或突變誘導的α-synuclein寡聚體形成;薑黃素可能通過穩定線粒體膜電位阻斷瞭α-synuclein基因過錶達或突變所導緻的細胞凋亡;mitoKATP通道的開放可能是α-synuclein基因過錶達或A53T突變誘導細胞凋亡的自髮始動保護機製,薑黃素可能通過開放mitoKATP通道拮抗α-synuclein基因過錶達或突變所導緻的細胞毒性,mitoKATP通道的開放程度與Kir6.2蛋白的錶達不成正相關.
목적 탐토강황소대α-돌촉핵단백(α-synuclein)과취체형성、선립체막전위급선립체ATP민감성갑통도(mitochondrial ATP-sensitive potassium channels,mitoKATP)적영향.방법 구건야생형、A53T돌변형α-synuclein진핵표체질립,용지질체개도전염방식장구건호적질립전입PC12세포,병분별응용강황소(20 ìmol/L)、5-간기규산염(5-hydroxydecanoate,5-HD)진행간예;48 h후응용면역인적、반점잡교검측세포α-synuclein과취체형성;응용JC-1형광탐침급유산탈경매(lactate dehydrogenase,LDH)검측선립체막전위급세포막독성손상;병재강황소간예처리전15 min급여5-HD예처리,면역인적검측mitoKATP단백적공능아기Kir6.2적개변,세포막편겸관찰mitoKATP적개변.결과 장구건소득EGFP-α-synuclein-WT、EGFP-α-synuclein-A53T진핵표체질립전염PC12세포,48 h후면역인적급반점인적결과현시,α-synuclein기인과표체혹돌변유도균가촉진α-synuclein과취체형성,강황소가명현감약α-synuclein기인과표체혹돌변유도적α-synuclein과취체형성;JC-1급LDH결과현시,여정상PC12세포상비,전염야생형、A53T돌변형조세포LDH수평분별승고35.5％급42.3％(P＜0.05),JC-1홍/록형광비솔여정상PC12세포상비하강60.44％、65.22％(P＜0.05),강황소간예후,LDH수평분별하강36.3％급23.5％(P＜0.05),홍/록형광비솔상승48.46％、50.33％(P＜0.05);전염야생형혹A53T돌변형α-synuclein융합표체재체조Kir6.2단백표체증강,조기통도전류유명현증고적추세,수후강저,응용강황소간예후,α-synuclein기인과표체혹돌변소유도적Kir6.2단백표체하조,단세포mitoKATP통도전류증가,여전염조비교유통계학의의(P＜0.05).결론 강황소가억제α-synuclein기인과도표체혹돌변유도적α-synuclein과취체형성;강황소가능통과은정선립체막전위조단료α-synuclein기인과표체혹돌변소도치적세포조망;mitoKATP통도적개방가능시α-synuclein기인과표체혹A53T돌변유도세포조망적자발시동보호궤제,강황소가능통과개방mitoKATP통도길항α-synuclein기인과표체혹돌변소도치적세포독성,mitoKATP통도적개방정도여Kir6.2단백적표체불성정상관.
Objective To investigate the effect of curcumin on oligomer formation and mitochondrial ATP-sensitive potassium channels (mitoKATP) induced by overexpression or mutation of α-synuclein.Methods Recombinant plasmids α-synuclein-pEGFP-A53T and α-synuclein-pEGFP-WT were transfected into PC12 cells by lipofectamin method,and intervened by application of curcumin (20 ìmol/L) and 5-hydroxydecanoate (5-HD).Oligomer formation in the cultured cells was identified by Western blotting and Dot blotting.Cytotoxicity and apoptosis of the PC12 cells were measured by lactate dehydrogenase (LDH) and JC-1 assays.mitoKATP were identified by Western blotting and whole cell patch clamp.Results Curcumin has significantly reduced the oligomer formation induced by overexpression or mutation of α-synuclein in the cultured cells.LDH has decreased by 36.3％ and 23.5％,and red / green fluorescence ratio of JC-1 was increased respectively by 48.46 ％ and 50.33 ％ after application of curcumin (P＜0.05).Protein expression of Kir6.2 has decreased and mitoKATP channel current has significantly increased (P＜ 0.05).Conclusion Curcumin can inhibit α-synuclein gene overexpression or mutation-induced α-synuclein oligomers formation.It may block apoptosis induced by wild-type overexpression or mutation of α-synuclein.By stabilizing mitochondrial membrane potential.Opening of mitoKATP channel may have been the initiating protective mechanism of apoptosis induced by wild-type overexpression or mutation of α-synuclein.Curcumin may antagonize above cytotoxicity through further opening the mitoKATP channel.
