pEGFP-N1脂质体转染方法的优化及其应用

doi:10.13481/j.1671-587x.20160338

吉林大学学报(医学版) ›› 2016, Vol. 42 ›› Issue (03): 612-616.doi: 10.13481/j.1671-587x.20160338

pEGFP-N1脂质体转染方法的优化及其应用

李凤娥¹, 向国艳², 孔繁利³, 郝峰², 杨紫嫣⁴, 郑岩⁴, 方芳⁴

1. 北华大学附属医院神经二科, 吉林吉林 132013;
2. 北华大学基础医学院病理生理教研室, 吉林吉林132013;
3. 吉林医药学院生物化学检验教研室, 吉林吉林 132013;
4. 吉林医药学院病原教研室, 吉林吉林 132013

收稿日期:2016-01-17 发布日期:2016-06-17
通讯作者: 孔繁利,副教授,硕士研究生导师(Tel:0432-64608018,E-mail:kongfanli5@163.com);郝峰,副教授(Tel:0432-64560542,E-mail:haof863@126.com) E-mail:kongfanli5@163.com;haof863@126.com
作者简介:李凤娥(1975-),女,吉林省榆树市人,医学硕士,主要从事脑血管疾病基础和临床方面的研究。
基金资助:
国家自然科学基金资助课题(812022031);吉林省卫计委科研计划项目资助课题(2014Z103);吉林省教育厅科研基金资助课题(2013-162,2013-351)

Optimization of liposome-mediated transfection of pEGFP-N1 and its applification

LI Fenge¹, XIANG Guoyan², KONG Fanli³, HAO Feng², YANG Ziyan⁴, ZHENG Yan⁴, FANG Fang⁴

1. Second Department of Neurology, Affiliated Hospital, Beihua University, Jilin 132013, China;
2. Department of Pathophysiology, School of Basic Medical Sciences, Beihua University, Jilin 132013, China;
3. Department of Biochemistry Laboratory, Jilin Medical College, Jilin 132013, China;
4. Department of Pathogen, Jilin Medical College, Jilin 132013, China

Received:2016-01-17 Published:2016-06-17

摘要/Abstract

摘要：

目的: 应用脂质体介导的转染方法将pEGFP-N1转染入Fisher大鼠甲状腺滤泡上皮细胞中,并获得较佳的优化方案,阐明该优化条件在转染其他目的基因时的适用性。方法: 分别选取不同配比的质粒和脂质体,将pEGFP-N1转入Fisher大鼠甲状腺滤泡上皮细胞。转染后36h,倒置荧光显微镜和流式细胞术检测细胞中增强型绿色荧光蛋白(EGFP)的表达情况和转染效率;台盼蓝染色法检测细胞存活率,以此获得优化的转染条件。同时将pEGFP-Aquaporin2、pEGFP-Aquaporin4和pEGFP-Aquaporin5分别以上述条件转染,并获取相应优化的转染条件。结果: 在质粒质量固定的条件下,随着脂质体质量的增加,其转染效率和细胞存活率升高;但是当质粒:脂质体>1:4时,转染效率和细胞存活率下降。在质粒和脂质体比例固定而质量增加时,其转染效率和细胞存活率下降;当质粒:脂质体>3.2μg:12.8μL时,转染效率降低。应用脂质体介导的方法转染pEGFP-N1和转染pEGFP-Aquaporin2、pEGFP-Aquaporin4和pEGFP-Aquaporin5时,在质粒与脂质体的配比为1:4(3.2μg:12.8μL)时,转染效率达最高。结论: 在应用脂质体介导的目的基因转染时,pEGFP-N1优化的转染条件具有较为广泛的适用性,可用作后续目的基因高效转染优化条件。

关键词: 脂质体, pEGFP-N1, 转染, 甲状腺滤泡上皮细胞

Abstract:

Objective: To optimize the liposome-mediated transfection conditions based on transfecting the pEGFP-N1 into thyroid cells of Fischer rats,and to investigate its effectiveness on other target gene transfection. Methods: Based on the different ratios and quantities of plasmids and liposomes,the thyroid follicular epithelial cells of Fischer rats were transfected with pEGFP-N1.The expression of enhanced green fluorescence protein(EGFP) and transfection efficiency were observed under inverted fluorescence microscope and flow cytometry, respectively.Then the cell viability was detected by Trypan Blue stainning.Meanwhile the thyroid follicular epithelial cells of Fischer rats were transfected with pEGFP-Aquaporin2,pEGFP-Aquaporin4 and pEGFP-Aquaporin5 under the above conditions,and the optimized conditions of transfecion were obtained. Results: When the quantity of plasmid was certain,the transfection efficiency and cell viability were increased with the increasing of liposomes,but the transfection efficiency and cell viability were decreased when the ratio of plasmid to liposome was over 1:4.When the ratio of plasmid to liposome was certain and the quantities were increased gradully,the transfection effciency was increased,and the cell viability was decreased.With the ratio of plasmids to liposomes was 1:4 (3.2 μg:12.8 μL),the transfecion efficencies of pEGFP-N1,pEGFP-Aquaporin2,pEGFP-Aquaporin2, and pEGFP-Aquaporin2 reached the highest values,respectively. Conclusion: The optimized conditions of transfection pEGFP-N1 is the same as the other plasmids, which can be used as optimized conditions for efficient transfection of the other target gene.

Key words: liposome, pEGFP-N1, transfection, thyroid follicular epithelial cells

中图分类号:

R361

李凤娥, 向国艳, 孔繁利, 郝峰, 杨紫嫣, 郑岩, 方芳. pEGFP-N1脂质体转染方法的优化及其应用[J]. 吉林大学学报(医学版), 2016, 42(03): 612-616.

LI Fenge, XIANG Guoyan, KONG Fanli, HAO Feng, YANG Ziyan, ZHENG Yan, FANG Fang. Optimization of liposome-mediated transfection of pEGFP-N1 and its applification[J]. Journal of Jilin University Medicine Edition, 2016, 42(03): 612-616.

参考文献

[1] Montier T,Benvegnu T,Jaffrès PA,et al. Progress in cationic lipid-mediated gene transfection:a series of bioinspired lipids as an example[J].Curr Gene Ther,2008,8(5):296-312.

[2] Bochicchio S,Dalmoro A,Barba AA,et al.Liposomes as siRNA delivery vectors[J].Curr Drug Metab,2014,15(9):882-892.

[3] Haynes MT,Huang L.Lipid-coated calcium phosphate nanoparticles for nonviral gene therapy[J].Adv Genet,2014,88:205-229.

[4] Tsien RY.The green fluorescent protein[J].Annu Rev Biochem,1998,67:509-544.

[5] Verkhusha VV,Lukyanov KA.The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins[J].Nat Biotechnol,2004,22(3):289-296.

[6] Zheng K,Xu HJ,Zang YX,et al.Effect of enhanced green fluorescent protein fusion on Ano1 physiological feature[J].Sheng Li Xue Bao,2015,67(6):623-628.

[7] Verkman AS,GaLietta LJ.Chloride channels as drug targets [J].Nat Rev Drug Discov,2009,8 (2):153-171.

[8] 郝峰,白雪松, 鞠晓红.TMEM16A钙激活氯离子通道在Fisher大鼠甲状腺滤泡上皮细胞的表达及其电生理特性研究[J].中国病理生理杂志,2014,30(9):1633-1639.

[9] 李凤娥,郝峰,藏雨轩,等.重组EGFP-aquaporin-4融合蛋白真核表达载体的构建及其在FRT细胞中的表达和定位[J].吉林大学学报:医学版,2013,39(2):273-277,428.

[10] Piechowicz KA,Truong EC,Javed KM,et al.Synthesis and evaluation of 5,6-disubstituted thiopyrimidine aryl aminothiazoles asinhibitors of the calcium-activated chloride channel TMEM16A/Ano1[J].J Enzyme Inhib Med Chem,2016:1-7.

[11] Nath S,Dancourt J,Shteyn V,et al.Lipidation of the LC3/GABARAP family of autophagy proteins relies on a membrane-curvature-sensing domain in Atg3[J].Nat Cell Biol,2014,16(5):415-424.

[12] Xu H,Li Z,Si J.Nanocarriers in gene therapy:a review[J].J Biomed Nanotechnol,2014,10(12):3483-3507.

[13] Ramamoorth M,Narvekar A.Non viral vectors in gene therapy-an overview[J].J Clin Diagn Res,2015,9(1):GE01-6.

[14] Junquera E,Aicart E.Cationic lipids as transfecting agents of DNA in gene therapy[J].Curr Top Med Chem,2014,14(5):649-663.

[15] Yang J,Liu H,Zhang X.Design,preparation and application of nucleic acid delivery carriers[J].Biotechnol Adv,2014,32(4):804-817.

pEGFP-N1脂质体转染方法的优化及其应用

Optimization of liposome-mediated transfection of pEGFP-N1 and its applification

PDF (PC)

赞

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

Metrics

本文评价

推荐阅读 10

[1]	王明月, 王浩, 王冬梅, 杨泽斌, 崔梅英, 刘宁, 黄莉莉, 关新刚. SIRPα-GFP真核表达载体构建及其在HEK293T细胞中的表达[J]. 吉林大学学报(医学版), 2020, 46(05): 925-929.
[2]	孔维健, 方红娟, 潘肃, 杨利丽, 郑爽, 齐治平, 付川, 钟磊. 搭载紫杉醇脂质体的聚乳酸-羟基乙酸共聚物静电纺丝膜对神经干细胞增殖分化的影响[J]. 吉林大学学报(医学版), 2020, 46(03): 509-514.
[3]	华进, 程志彬, 林春霖, 戴起宝, 朱广伟. 一种高效稳定的磷酸钙转染293T细胞方法的建立及评价[J]. 吉林大学学报(医学版), 2019, 45(05): 1177-1181.
[4]	李里, 宫亮, 吴玉斌. 沉默WNT4基因对稳定转染PAX2基因大鼠肾小管上皮细胞迁移和侵袭能力的影响[J]. 吉林大学学报(医学版), 2018, 44(06): 1212-1217.
[5]	李浩渤, 杜勇, 陈勇, 李文静. Notch 1对舌麟状细胞癌Tca8113细胞表皮生长因子受体表达的影响及其意义[J]. 吉林大学学报(医学版), 2017, 43(04): 715-719.
[6]	陈志华, 林素勇, 韩宏景, 苏小宝, 陈绍勤, 戴起宝. 慢病毒转染KISS1基因对人结直肠癌HCT116细胞增殖、侵袭和迁移能力的影响[J]. 吉林大学学报(医学版), 2017, 43(03): 577-581.
[7]	于海帆, 郭传辉, 李当当, 张虹亮, 耿爽, 杨占清, 郭斌, 岳占碰. 多效生长因子对小鼠子宫基质细胞蜕膜化的影响[J]. 吉林大学学报(医学版), 2016, 42(05): 855-859.
[8]	韩春耀, 张斌, 马雪, 刘明媛, 薛中原, 郝丽静, 葛树卿. 沉默α-catulin基因的人舌鳞状细胞癌TSCCA细胞模型的建立[J]. 吉林大学学报(医学版), 2016, 42(03): 481-485.
[9]	李春梅, 张林, 侯艳红, 李楠. 潜在抑癌基因TXNIP对胃癌MKN-45细胞生物学特性的影响[J]. 吉林大学学报(医学版), 2015, 41(03): 588-592.
[10]	王子航, 张宇虹, 夏稻子, 礼广森, 武俊, 由悦, 黄冬梅, 薄华颖, 胡滨, 毛鑫. 超声靶向破坏微泡技术介导小鼠肝癌细胞株JNK1基因表达、细胞迁移和侵袭抑制[J]. 吉林大学学报(医学版), 2015, 41(01): 21-26.
[11]	齐亚灵，赵文杰，方艳秋，陈玉强，孟德欣，王伟群，李尧，李文 . Survivin-ASODN对肝癌SMMC-7721细胞凋亡的促进作用及其机制[J]. 吉林大学学报(医学版), 2014, 40(04): 757-762.
[12]	许会静，杜彤华，孙艳，李校堃，肖业臣. FGFR3真核表达载体的构建及其在人白血病细胞系K562中的表达[J]. 吉林大学学报(医学版), 2014, 40(03): 465-470.
[13]	王宇，陈葳，李旭，张红，张晓芹，史迎莉. UCA1a(CUDR) 基因真核表达载体的构建及其在膀胱癌UM-UC-2 细胞中的表达[J]. 吉林大学学报(医学版), 2014, 40(03): 504-507.
[14]	李里，南晓娟，吴玉斌 . 配对盒基因2对正常肾小管上皮细胞间充质转分化的诱导作用[J]. 吉林大学学报(医学版), 2013, 39(3): 565-569.
[15]	李凤娥，郝峰，藏雨轩，马睿泽，孔繁利，刘磊，李艳. 重组EGFP-aquaporin-4融合蛋白真核表达载体的构建及其在FRT细胞中的表达和定位[J]. 吉林大学学报(医学版), 2013, 39(2): 273-277.