[1]鲍庆东,刘太祥,罗鑫,等.腺相关病毒介导的增强型绿色荧光蛋白基因转染豚鼠巩膜成纤维细胞的实验研究[J].眼科新进展,2020,40(11):1029-1032.[doi:10.13389/j.cnki.rao.2020.0230]
 BAO Qingdong,LIU Taixiang,LUO Xin,et al.Expression of enhanced green fluorescent protein gene in guinea pig fibroblasts mediated by adeno-associated virus[J].Recent Advances in Ophthalmology,2020,40(11):1029-1032.[doi:10.13389/j.cnki.rao.2020.0230]
点击复制

腺相关病毒介导的增强型绿色荧光蛋白基因转染豚鼠巩膜成纤维细胞的实验研究/HTML
分享到:

《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
40卷
期数:
2020年11期
页码:
1029-1032
栏目:
实验研究
出版日期:
2020-11-05

文章信息/Info

Title:
Expression of enhanced green fluorescent protein gene in guinea pig fibroblasts mediated by adeno-associated virus
作者:
鲍庆东刘太祥罗鑫田祥
563000 贵州省遵义市,遵义医科大学附属医院眼科
Author(s):
BAO QingdongLIU TaixiangLUO XinTIAN Xiang
Department of Ophthalmology,Affiliated Hospital of Zunyi Medical University,Zunyi 563000,Guizhou Province,China
关键词:
腺相关病毒 增强型绿色荧光蛋白 豚鼠巩膜成纤维细胞 基因转染
Keywords:
adeno-associated virus enhanced green fluorescent proteinguinea pig scleral fibroblastgene transfection
分类号:
R778.1
DOI:
10.13389/j.cnki.rao.2020.0230
文献标志码:
A
摘要:
目的 比较四种不同血清型腺相关病毒(adeno-associatedvirus,AAV)介导增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因转染豚鼠巩膜成纤维细胞(guinea pig scleral fibroblast,GSF)的转染效率及安全性,筛选合适的基因转染载体。方法 原代分离培养GSF并鉴定,利用AAV2、AAV5、AAV8、AAV9介导EGFP基因并按不同感染复数(multiplicity of infection,MOI)10、100、1000、10 000转染GSF,空白对照组加入空白培养基,转染后2 d、3 d、4 d、5 d荧光显微镜下观察GFP表达强度及细胞生长状态,流式细胞术检测细胞转染率及凋亡率,CCK-8法检测细胞活力。结果 AAV感染GSF后2 d各组均可见弱荧光,荧光强度随时间延长及MOI值增大而增强,而AAV9-EGFP组全程弱荧光,空白对照组全程无荧光。转染后5 d,MOI=10 000时各组荧光最强,基因转染率组间整体比较,差异具有统计学意义(P<0.05);组间两两比较,AAV8-EGFP组均高于其他各组,差异均有统计学意义(均为P<0.05)。各组Annexin V阳性凋亡率与空白对照组比较,差异均无统计学意义(均为P>0.05)。各组在450 nm波长处光密度值与空白对照组比较,差异均无统计学意义(均为P>0.05)。结论 AAV8能有效地转染GSF,且对细胞凋亡、细胞活力无明显影响,是安全可行的。
Abstract:
Objective Tocompare the transfection efficiency and safety of enhanced green fluorescent protein(EGFP) gene in guinea pig scleral fibroblasts(GSF)mediated by four different adeno-associated virus(AAV)to screen the suitable gene-transfection vectors.Methods GSF was isolated and cultured in the primary generation and identified. The EGFP gene was mediated by AAV2, AAV5, AAV8, and AAV9, and the GSF was transfected at different multiplicity of infection (MOI) (MOI: 10, 100, 1000, 10 000), and blank transfected mediumwas added in blank control group. After staining 2 day, 3 day, 4 day, 5 day, we observed the GFP expression intensity and cell growth status under a fluorescence microscope, flow cytometry to detect the cell transfection rate and apoptosis rate, and CCK-8 method to detect cell viability.Results Weak fluorescence was seen in each group 2 days after AAV infection with GSF, and the fluorescence intensity increased as time went by and the MOI value increased, while the AAV9-EGFP group had weak fluorescence throughout the whole process, and the blank control group had no fluorescence during the whole process.Five days after transfection, each group had the strongest fluorescence when MOI=10 000, and the overall comparison of gene transfection rate between the groups showed statistically significant differences (P<0.05), and pairwise comparisons between the groups showed that the AAV8-EGFP group was higher than the other groups, and the differences were statistically significant (all P<0.05). Compared with the blank control group, the positive apoptosis rate of AnnexinV in each group was not statistically different (all P>0.05). There was no statistically significant difference in the optical density value of each group at 450 nm wavelength and the blank control group (all P>0.05).Conclusion The AAV8 can be steady and efficient to transfect GSF.It has no obvious effect on cell apoptosis and cell viability, which is safe and feasible.

参考文献/References:

[1] WU P C,HUANG H M,YU H J,FANG P C,CHEN C T.Epidemiology of myopia[J].Asia Pac J Ophthalmol,2016,5(6):386-393.
[2] ZADNIK K.The Glenn A,Fry Award Lecture (1995).Myopia development in childhood[J].Optom Vis Sci,1997,74(8):603-608.
[3] HARPER A R S J A.The dynamic sclera extracellular matrix remodeling in normal ocular growth and myopia development[J].Exp Eye Res,2015,133(4):100-111.
[4] RADA J A,SHELTON S,NORTON T T.The sclera and myopia[J].Exp Eye Res,2006,82(2):185-200.
[5] WOESSNER J F.The family of matrix metalloproteinases[J].Ann N Y Acad Sci,1994,732(3):11-21.
[6] YAMAOKA A,MATSUO T,SHIRAGA F,OHTSUKI H.TIMP-1 production by human scleral fibroblast decreases in response to cyclic mechanical stretching[J].Ophthalmic Res,2001,33(2):98-101.
[7] PANG J J,CHANG B,KUMAR A,NUSINOWITZ S,NOORWEZ S M,LI J.Gene therapy restores vision-dependent behavior as well as retinal structure and function in a mouse model of RPE65 Leber congenital amaurosis[J].Mol Ther,2006,13(3):565-572.
[8] HAN Z,CONLEY S M,MAKKIA R S,COOPER M J,NAASH M I.DNA nanoparticle-mediated ABCA4 delivery rescues Stargardt dystrophy in mice[J].J Clin Invest,2012,122(9):3221-3226.
[9] YU W,MOOKHERJEE S,CHAITANKAR V,HIRIYANNA S,KIM J W,BROOKS M.Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice[J].Nat Commun,2017,8:14716.
[10] BUCK THILO M,WIJNHOLDS J A N.Recombinant adeno-associated viral vectors (rAAV)-vector elements in ocular gene therapy clinical trials and transgene expression and bioactivity assays[J].Int J Mol Sci,2020,21(12):4197.
[11] CANTORE A,NALDINI L.WFH State-of-the-art paper 2020:In vivo lentiviral vector gene therapy for haemophilia[J].Haemophilia,2020,6:1-4.
[12] BYRNE B J,CORTI M A,SHUTTERLY A,CHAN Y K I.Management of neuroinflammatory responses to AAV-Mediated gene therapies for neurodegenerative diseases[J].Brain Sci,2020,10(2):119.
[13] 张兰兰,于健,吴炳义,刘琼,汤明芳,谭青,等.慢病毒介导绿色荧光蛋白基因转染豚鼠巩膜成纤维细胞的实验研究[J].眼科新进展,2011,31(12):1107-1110.
ZHANG L L,YU J,WU B Y,LIU Q,TANG M F,TAN Q,et al.Expression of green fluorescent protein in guinea pig fibroblasts mediated by lentiviral vector[J].Rec Adv Ophthalmol,2011,31(12):1107-1110.
[14] XIAO P J,SAMULSKI R J.Cytoplasmic trafficking endosomal escape and perinuclear accumulation of adeno-associated virus type 2 particles are facilitated by microtubule network[J].J Virol,2012,86(19):10462-10473.
[15] NICOLSON S C,SAMULSKI R J.Recombinant adeno-associated virus utilizes host cell nuclear import machinery to enter the nucleus[J].J Virol,2014,88(8):4132-4144.
[16] KELICH J M,MA J,DONG B,WANG Q,CHIN M,MAGURA C M,et al.Super-resolution imaging of nuclear import of adeno-associated virus in live cells[J].Mol Ther Methods Clin Dev,2015,2(1):15047.
[17] 汤明芳,于健,白浪,刘琼.8型重组腺相关病毒介导绿色荧光蛋白基因在角膜基质细胞中的表达及影响[J].国际眼科杂志,2011,11(5):769-771.
TANG M F,YU J,BAI L,LIU Q.Expression and effect of recombinat adeno-associated virus vector-8 transduced green fluorescent protein gene into rat keratocytes[J].Int Eye Sci,2011,11(5):769-771.
[18] 李连华,翁习生,邱贵兴,吴志宏,林进,金今,等.2型重组腺相关病毒转染新西兰兔关节软骨细胞的体外研究[J].中华医学杂志,2006(21):1489-1492.
LI L H,WENG X S,QIU G X,WU Z H,LIN J,JIN J,et al.In vitro gene transfection into rabbit articular chondrocytes mediated by recombinant adeno-associated virus vector[J].Natl Med J Chin,2006(21):1489-1492.

相似文献/References:

[1]丁芝祥 彭燕一 邱梅园 张玉明 曾新生.腺相关病毒介导HSV-tk/GCV 系统对晶状体上皮细胞的旁观者效应及机制[J].眼科新进展,2012,32(8):000.

备注/Memo

备注/Memo:
国家自然科学基金资助(编号:81660169)
更新日期/Last Update: 2020-11-05