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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 Sciences), 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 Sciences), 2020, 51(6): 783-789. DOI: 10.12182/20201160202

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

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。

     

    Abstract:
      Objective  To investigate the influence of the protamine sulfate on endocytosis and intracellular stability of tetrahedral framework nucleic acid (tFNA).
      Methods  Articular cartilage cells were collected from 3-day-old C57BL mice. Cells at passage 1-2 were used in the experiments. 4 single-strand DNAs (S1 was marked by Cy5) were utilized to synthesize tFNAs via annealing process and ultrafiltration for purification. High-performance capillary electrophoresis (HPCE) was used to verify synthesis of tFNAs and transmission electron microscope was used to photo morphological characteristics. The 1 mg/mL protamine sulfate solution was slowly dropped into newly synthesized tFNAs (N/P=5/1). Then, Zeta potential was detected. Cells were treated with 100 nmol/L tFNAs with protamine sulfate in Dulbecco’s Modified Eagle’s medium (DMEM) (Exp.1), 100 nmol/L tFNAs in DMEM (Exp.2), and DMEM (Control), respectively. Flow cytometry was used to quantitatively detect intracellular Cy5 fluorescence after 6 h and 12 h treatments. Immunofluorescence staining was used to qualitatively observe internalized Cy5 fluorescence after 12 h treatment by laser confocal microscope. Lysosome of living cells were stained with lysosome probe. Colocalization between lysosome and tFNAs was observed by laser confocal microscope.
      Results  After incubating protamine sulfate, negative potential was transformed into positive one ( (−1.567±0.163) mV to (4.700±0.484) mV). The fluorescence intensity of tFNAs in the Exp.1 group was higher than that of the Exp.2 group in 6 h and 12 h (P<0.05). This was consistent with the results of immunofluorescence staining after 12 h. Colocalization of Cy5 fluorescence and lysosome in the Exp.1 group was more rare than that in the Exp.2 group at 6 h and 12 h. Furthermore, a large amount of Cy5 fluorescence was still seen in the Exp.1 group at 12 h, while Cy5 fluorescence of the Exp.2 group was less.
      Conclusion  Protamine sulfate can effectively enhance endocytosis, and to some extent it can achieve lysosome escape of tFNAs.

     

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