中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2014年
8期
712-717
,共6页
余曼%陈波%张瑞帆%吴峥峥
餘曼%陳波%張瑞帆%吳崢崢
여만%진파%장서범%오쟁쟁
不饱和脂肪酸/生物合成%黄斑变性/遗传性%大鼠肾上腺嗜铬细胞瘤细胞株%气相色谱-质谱联用仪%免疫印迹法%实时定量PCR%ELOVL4基因%Stargardt黄斑营养不良
不飽和脂肪痠/生物閤成%黃斑變性/遺傳性%大鼠腎上腺嗜鉻細胞瘤細胞株%氣相色譜-質譜聯用儀%免疫印跡法%實時定量PCR%ELOVL4基因%Stargardt黃斑營養不良
불포화지방산/생물합성%황반변성/유전성%대서신상선기락세포류세포주%기상색보-질보련용의%면역인적법%실시정량PCR%ELOVL4기인%Stargardt황반영양불량
Unsaturated fatty acid/biosynthesis%Macular degeneration/genetics%PC12 cell/rat%Gas chromatography-mass spectrometry%Blotting,western%Real-time quantitative PCR%ELOVL4 gene%Stargardt macular dystrophy
背景 研究证实,超长链脂肪酸延伸酶4(ELOVL4)基因是Stargardt病的致病基因,且ELOVL4可能是C≥28的超长链脂肪酸的延伸酶,而常染色体显性遗传Stargardt病Ⅲ型(STGD3)的发病可能与视网膜中C≥28的超长链脂肪酸的代谢有关.探讨ELOVL4在STGD3发病中的作用及其与超长链脂肪酸代谢的关系有望对STGD3的治疗提供新的思路. 目的 探讨ELOVL4基因在STGD3发病和进展中的作用及机制.方法 将腺病毒(Ad)转染重组病毒质粒36 h的人胚肾293细胞(HEK293)以纯化病毒,构建携带ELOVL4基因和绿色荧光蛋白(GFP)的重组腺病毒载体并转入培养的大鼠肾上腺嗜铬细胞瘤细胞株(PC12),将培养的PC12细胞分成PC12组、PC12+ Ad5-GFP组和PC12+ Ad5-ELOVL4组,根据分组情况分别将Ad-GFP和Ad-ELOVL4病毒质粒按1×104～2×104噬斑形成单位(pfu/ml)的滴度加入含质量分数2％胎牛血清(FBS)的细胞培养液转导24 h,采用实时定量PCR法(qRT-PCR)定量分析各组细胞中ELOVL4 mRNA的相对表达量;采用Western blot法检测细胞中ELOVL4蛋白的表达.分别在各组培养基中加入花生四烯酸(AA或20:4n6)、二十碳五烯酸(EPA或20:5n3)和二十二碳六烯酸(DHA或22:6n3),孵育48 h后进行脂肪酸提取,通过气相色谱-质谱联用仪(GC-MS)分析各组细胞中超长链脂肪酸产物. 结果 PC12+Ad-ELOVL4组、PC 12+ Ad-GFP组和PC12组间细胞中ELOVL4 mRNA表达水平(ELOVL4 mRNA/RPL19 mRNA)分别为0.833±0.138、0.027±0.002、0.024±0.002,3个组的总体差异有统计学意义(F=102.700,P=0.000),其中PC 12+ Ad5-ELOVL4组PC12细胞中ELOVL4 mRNA表达水平是PC12组和PC12+Ad5-GFP组的30 ～ 40倍;Western blot法检测结果证实,PC12+Ad5-ELOVL4组PC12细胞中ELOVL4蛋白呈阳性表达.GC-MS法检测发现PC 12+ Ad5-ELOVL4组PC12细胞中有C28-C38多不饱和脂肪酸的合成,其中均以C34和C36多不饱和脂肪酸合成量最多.结论 ELOVL4蛋白酶可催化C28-C38多不饱和脂肪酸的合成,这个结论为STGD3患者的治疗提供了新的思路.
揹景 研究證實,超長鏈脂肪痠延伸酶4(ELOVL4)基因是Stargardt病的緻病基因,且ELOVL4可能是C≥28的超長鏈脂肪痠的延伸酶,而常染色體顯性遺傳Stargardt病Ⅲ型(STGD3)的髮病可能與視網膜中C≥28的超長鏈脂肪痠的代謝有關.探討ELOVL4在STGD3髮病中的作用及其與超長鏈脂肪痠代謝的關繫有望對STGD3的治療提供新的思路. 目的 探討ELOVL4基因在STGD3髮病和進展中的作用及機製.方法 將腺病毒(Ad)轉染重組病毒質粒36 h的人胚腎293細胞(HEK293)以純化病毒,構建攜帶ELOVL4基因和綠色熒光蛋白(GFP)的重組腺病毒載體併轉入培養的大鼠腎上腺嗜鉻細胞瘤細胞株(PC12),將培養的PC12細胞分成PC12組、PC12+ Ad5-GFP組和PC12+ Ad5-ELOVL4組,根據分組情況分彆將Ad-GFP和Ad-ELOVL4病毒質粒按1×104～2×104噬斑形成單位(pfu/ml)的滴度加入含質量分數2％胎牛血清(FBS)的細胞培養液轉導24 h,採用實時定量PCR法(qRT-PCR)定量分析各組細胞中ELOVL4 mRNA的相對錶達量;採用Western blot法檢測細胞中ELOVL4蛋白的錶達.分彆在各組培養基中加入花生四烯痠(AA或20:4n6)、二十碳五烯痠(EPA或20:5n3)和二十二碳六烯痠(DHA或22:6n3),孵育48 h後進行脂肪痠提取,通過氣相色譜-質譜聯用儀(GC-MS)分析各組細胞中超長鏈脂肪痠產物. 結果 PC12+Ad-ELOVL4組、PC 12+ Ad-GFP組和PC12組間細胞中ELOVL4 mRNA錶達水平(ELOVL4 mRNA/RPL19 mRNA)分彆為0.833±0.138、0.027±0.002、0.024±0.002,3箇組的總體差異有統計學意義(F=102.700,P=0.000),其中PC 12+ Ad5-ELOVL4組PC12細胞中ELOVL4 mRNA錶達水平是PC12組和PC12+Ad5-GFP組的30 ～ 40倍;Western blot法檢測結果證實,PC12+Ad5-ELOVL4組PC12細胞中ELOVL4蛋白呈暘性錶達.GC-MS法檢測髮現PC 12+ Ad5-ELOVL4組PC12細胞中有C28-C38多不飽和脂肪痠的閤成,其中均以C34和C36多不飽和脂肪痠閤成量最多.結論 ELOVL4蛋白酶可催化C28-C38多不飽和脂肪痠的閤成,這箇結論為STGD3患者的治療提供瞭新的思路.
배경 연구증실,초장련지방산연신매4(ELOVL4)기인시Stargardt병적치병기인,차ELOVL4가능시C≥28적초장련지방산적연신매,이상염색체현성유전Stargardt병Ⅲ형(STGD3)적발병가능여시망막중C≥28적초장련지방산적대사유관.탐토ELOVL4재STGD3발병중적작용급기여초장련지방산대사적관계유망대STGD3적치료제공신적사로. 목적 탐토ELOVL4기인재STGD3발병화진전중적작용급궤제.방법 장선병독(Ad)전염중조병독질립36 h적인배신293세포(HEK293)이순화병독,구건휴대ELOVL4기인화록색형광단백(GFP)적중조선병독재체병전입배양적대서신상선기락세포류세포주(PC12),장배양적PC12세포분성PC12조、PC12+ Ad5-GFP조화PC12+ Ad5-ELOVL4조,근거분조정황분별장Ad-GFP화Ad-ELOVL4병독질립안1×104～2×104서반형성단위(pfu/ml)적적도가입함질량분수2％태우혈청(FBS)적세포배양액전도24 h,채용실시정량PCR법(qRT-PCR)정량분석각조세포중ELOVL4 mRNA적상대표체량;채용Western blot법검측세포중ELOVL4단백적표체.분별재각조배양기중가입화생사희산(AA혹20:4n6)、이십탄오희산(EPA혹20:5n3)화이십이탄륙희산(DHA혹22:6n3),부육48 h후진행지방산제취,통과기상색보-질보련용의(GC-MS)분석각조세포중초장련지방산산물. 결과 PC12+Ad-ELOVL4조、PC 12+ Ad-GFP조화PC12조간세포중ELOVL4 mRNA표체수평(ELOVL4 mRNA/RPL19 mRNA)분별위0.833±0.138、0.027±0.002、0.024±0.002,3개조적총체차이유통계학의의(F=102.700,P=0.000),기중PC 12+ Ad5-ELOVL4조PC12세포중ELOVL4 mRNA표체수평시PC12조화PC12+Ad5-GFP조적30 ～ 40배;Western blot법검측결과증실,PC12+Ad5-ELOVL4조PC12세포중ELOVL4단백정양성표체.GC-MS법검측발현PC 12+ Ad5-ELOVL4조PC12세포중유C28-C38다불포화지방산적합성,기중균이C34화C36다불포화지방산합성량최다.결론 ELOVL4단백매가최화C28-C38다불포화지방산적합성,저개결론위STGD3환자적치료제공료신적사로.
Background Researches determined that ELOVL4 gene is a disease-causing gene of Stargard tmacular dystrophy and is a elongation enzyme of very long chain fatty acids.Stargardt type Ⅲ(STGD3) may be associated with the metabolism of extensive enzyme of very long chain fatty acids.To explore the effect of ELOVL4 in STGD3 and its relationship with the metabolism of extensive enzyme of very long chain fatty acids is of important clinical significance.Objective This study was to determine the role of ELOVL4 gene for the pathogenesis of STGD3.Methods Recombinant adenovirus type 5 carrying mouse ELOVL4 gene and green fluorescent protein (GFP) was transfected into pheochromocytoma cells (PC12 cells),and then the cells were divided into PC12 group,PC12+Ad5-GFP group and PC12+AdS-ELOVL4 group.Ad-GFP or Ad-ELOVL4 was added into the culture medium for 24 hours with the virus plasmid 1 × 104-2× 104 pfu/ml.Expression of ELOVL4 mRNA in the PC12 was quantified by quantitative real time PCR(qRT-PCR) and was described as relative value to the expression of RPL19.ELOVL4 protein was assayed by Western blot.The transfected cells were treated with arachidonic acid (AA,20:4n6),eicosapentaenoic acid (EPA,20:5n3) and docosahexaenoic acid (DHA,22:6n3) individually for 48 hours.The cells were collected,and total lipids were extracted,and fatty acid methyl esters were prepared and analyzed by gas chromatography-mass spectrometry (GC-MS).Results The relative expression levels of ELOVL4 mRNA in PC12 cells in the PC12+Ad5-ELOVL4 group,PC12+Ad5-GFP group and PC12 group were 0.833± O.138,0.027t±0.002 and 0.024 ±0.002,with a significant difference among the 3 groups (F =102.700,P =0.000),and relative expression levels of PC12+Ad-ELOVL4 were 30-40 folds more than those in the PC12 group and the PC12+Ad-GFP group.Western blot assay showed a stronger response band for ELOVL4 protein in the PC12+Ad-ELOVL4 group.GC-MS found that abundant polyunsaturated fatty acids (C28-C38) were synthesized by PC12 cells in the PC12+Ad-ELOVL4 group,with the more levels in C34 and C36.Conclusions ELOVL4 can promote the synthesis of C28-C38 polyunsatured fatty acid in PC12 cells,which offers a novel clue for the treatment of STGD3.
