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鱼精蛋白硫酸盐对DNA纳米结构入胞能力及胞内溶酶体逃逸的影响

葛奕辰 崔伟同 蔡潇潇

葛奕辰, 崔伟同, 蔡潇潇. 鱼精蛋白硫酸盐对DNA纳米结构入胞能力及胞内溶酶体逃逸的影响[J]. 四川大学学报(医学版), 2020, 51(6): 783-789. doi: 10.12182/20201160202
引用本文: 葛奕辰, 崔伟同, 蔡潇潇. 鱼精蛋白硫酸盐对DNA纳米结构入胞能力及胞内溶酶体逃逸的影响[J]. 四川大学学报(医学版), 2020, 51(6): 783-789. doi: 10.12182/20201160202
GE Yi-chen, CUI Wei-tong, CAI Xiao-xiao. Influence of the Protamine Sulfate on Endocytosis and Intracellular Lysosome Escape of DNA Nanostructure[J]. JOURNAL OF SICHUAN UNIVERSITY (MEDICAL SCIENCE EDITION), 2020, 51(6): 783-789. doi: 10.12182/20201160202
Citation: GE Yi-chen, CUI Wei-tong, CAI Xiao-xiao. Influence of the Protamine Sulfate on Endocytosis and Intracellular Lysosome Escape of DNA Nanostructure[J]. JOURNAL OF SICHUAN UNIVERSITY (MEDICAL SCIENCE EDITION), 2020, 51(6): 783-789. doi: 10.12182/20201160202

栏目: 论 著

鱼精蛋白硫酸盐对DNA纳米结构入胞能力及胞内溶酶体逃逸的影响

doi: 10.12182/20201160202
基金项目: 国家重点研发计划(No. 2019YFA0110600)和国家自然科学基金(No. 81970986)资助
详细信息
    通讯作者:

    E-mail:xcai@scu.edu.cn

Influence of the Protamine Sulfate on Endocytosis and Intracellular Lysosome Escape of DNA Nanostructure

  • 摘要:   目的  探究鱼精蛋白硫酸盐对四面体框架核酸入胞能力及胞内稳定性的影响。  方法  取3日龄C57BL小鼠肢端软骨进行软骨细胞培养,并收集第1~2代细胞用于实验。利用4条DNA单链S1(标记Cy5荧光)、S2、S3、S4,经过退火程序合成四面体框架核酸并超滤提纯。用高通量毛细电泳验证四面体框架核酸合成并拍摄透射电镜图进行表征。向新合成四面体框架核酸中缓慢滴入1 mg/mL鱼精蛋白硫酸盐溶液(以原子数N/P=5/1混合),检测Zeta电位。将细胞分为3组:细胞中加入100 nmol/L经过鱼精蛋白硫酸盐孵育的四面体框架核酸作为实验组1;细胞中加入100 nmol/L未经孵育处理的四面体框架核酸为实验组2;对照组细胞不进行加药处理。在加药后6 h及12 h,用流式细胞术定量检测胞内Cy5荧光,并取部分12 h组细胞,进行免疫荧光染色,于激光共聚焦显微镜下定性观察分析胞内Cy5荧光。加药5.5 h及11.5 h后,加入溶酶体探针对活细胞溶酶体进行染色,30 min后(6 h及12 h)观察Cy5荧光与溶酶体位置关系。  结果  经过鱼精蛋白硫酸盐孵育处理后,溶液Zeta电位由负转正,即由(-1.567±0.163) mV转为(4.700±0.484) mV;在6 h及12 h两个时间点,流式细胞仪检测到实验组1胞内四面体框架核酸荧光强度均高于实验组2,差异有统计学意义(P<0.05)。12 h的免疫荧光染色观察结果与流式细胞术检测结果一致。溶酶体染色结果显示,加药6 h及12 h后,实验组1的Cy5荧光与溶酶体位置重叠现象较实验组2更少;且12 h后,实验组1依然可见大量Cy5荧光,而实验组2的Cy5荧光较少较弱。  结论  鱼精蛋白硫酸盐孵育处理可有效提升四面体框架核酸入胞能力,并在一定程度上实现四面体框架核酸胞内溶酶体的逃逸。
  • 图  1  四面体框架核酸合成及鱼精蛋白硫酸盐孵育模式图

    Figure  1.  Schematic diagram of synthesizing tFNAs with protamine sulfate incubation

    图  2  tFNAs的合成、形貌与鱼精蛋白硫酸盐处理前后的Zeta电位

    Figure  2.  Synthesis, morphological characteristics and Zeta potential of tFNAs

    A: High-performance capillary electrophoresis (the unit of values in the figure is bp); B: Transmission electron microscope (TEM); C: Detection of Zeta potential before/after protamine incubation (n=3).

    图  3  tFNAs入胞能力定量分析(n=3)

    Figure  3.  Quantitative analysis of internalized tFNAs (n=3)

    A: Cy5 fluorescence after 6 h; B: Cy5 fluorescence after 12 h; C: Cell count of fluorescence after 6 h; D: Cell count of fluorescence after 12 h. *P<0.05, **P<0.01, ***P<0.001.

    图  4  鱼精蛋白硫酸盐孵育tFNAs 12 h后入胞能力增强现象定性观察

    Figure  4.  Protamine incubation enhanced endocytosis of tFNAs after 12 h

    图  5  加药6 h后tFNAs与溶酶体荧光共定位

    Figure  5.  Fluorescence colocalization between tFNAs and lysosomes after 6 h

    图  6  加药12 h后tFNAs与溶酶体荧光共定位

    Figure  6.  Fluorescence colocalization between tFNAs and lysosomes after 12 h

    表  1  DNA单链碱基序列

    Table  1.   Base sequences of DNA single strands

    Single strandBase sequence (5'→3')
    S1 ATTTATCACCCGCCATAGTAGACGTATCACCAGGCAGTTGAGACGAACATTCCTAAGTCTGAA
    S2 ACATGCGAGGGTCCAATACCGACGATTACAGCTTGCTACACGATTCAGACTTAGGAATGTTCG
    S3 ACTACTATGGCGGGTGATAAAACGTGTAGCAAGCTGTAATCGACGGGAAGAGCATGCCCATCC
    S4 ACGGTATTGGACCCTCGCATGACTCAACTGCCTGGTGATACGAGGATGGGCATGCTCTTCCCG
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出版历程
  • 收稿日期:  2020-05-03
  • 修回日期:  2020-08-25
  • 刊出日期:  2020-11-20

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