纳米基因载体介导RNA干扰沉默Survivin基因治疗大肠癌的实验研究
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摘要
目的：制备转染率高、安全无毒的新型壳聚糖-聚乙二醇纳米基因载体,研究其介导的靶向人survivin的RNAi基因纳米复合物对结肠癌细胞HT-29中survivin mRNA的表达,及其对HT-29细胞凋亡和增殖的影响和意义。
方法：通过两步接枝共聚法,优化制备工艺,制备粒径较小、粒度分布均匀的壳聚糖-聚乙二醇纳米颗粒。使用Malvem Zetasizer3000E对纳米粒的粒径及zeta电位进行检测；利用原子力显微镜对纳米颗粒的形态进行观察。
选取人survivin基因(NM_001168)序列中的相关位点序列构建靶向survivin的shRNA重组质粒,通过序列测定检测重组子的正确性,再从中筛选出最佳的survivin shRNA重组质粒载体。合成壳聚糖-聚乙二醇纳米粒介导的survivin RNAi基因纳米复合物,将其导入结肠癌细胞HT-29中(以携带相同shRNA的脂质体载体,survivin shRNA以及阴性对照shRNA为对照)。用RT-PCR和Western-blot分别检测转染后的HT-29细胞中Survivin mRNA和蛋白表达量的变化；通过MTT法检测细胞凋亡情况；
结果：Malvem Zetasizer 3000E激光粒度分析仪进行粒径及分布的检测结果显示,制备出的壳聚糖-聚乙二醇纳米颗粒的平均粒径值为61nm。粒径大小分布呈正态分布,分布均匀。原子力显微镜进行观测。纳米颗粒呈圆球形,表面平滑完整,分散良好,较少粘附团聚现象；且细胞毒性低、转染率高。序列测定重组质粒载体survivin-shRNA构建正确。转染壳聚糖-聚乙二醇介导的survivin shRNA基因纳米复合物后发现,该基因纳米复合物对结肠癌细胞HT-29的转染效率显著高于survivin shRNA和脂质体载体(P<0.01)；HT-29细胞中Survivin mRNA拷贝数及蛋白表达显著降低(P<0.01),且结肠癌细胞HT-29的细胞死亡率和凋亡率明显提高(P<0.01)。
结论：本研究成功制备出平均粒径为61nm,分布均匀的壳聚糖-聚乙二醇纳米颗粒；其细胞毒性低、转染率高。合成的壳聚糖-聚乙二醇介导survivin shRNA基因纳米复合物能够高效转染结肠癌细胞株HT-29并有效干扰Survivin,且较之携带相同shRNA的脂质体载体,survivin shRNA和阴性对照shRNA具有更高的干扰效率,能高效地诱导结肠癌细胞凋亡并抑制其增殖。其介导RNA干扰能长时间高效率地沉默结肠癌细胞HT-29中的Survivin基因表达。
Objective:To prepare the PEGylated chitosan (CS-PEG) nano-gene carrier with high transfection rate and low cytotoxicity by a novel preparing method; construct the CS-PEG induced Survivin shRNA gene nano complex, which can transfect to HT-29 cells, to investigate its effect on the expression of survivin mRNA.To investigate its effect on the propagation and apoptosis of colon carcinoma cell lines HT-29, and to discuss its treatment effect on colon carcinoma
Methods:CS-PEG nanoparticles with small particle size were prepared by an optimized graft copolymerization method. Malvem Zetasizer 3000E was used to measure the particle size and zeta potential; and atomic force microscope was used to observe the morphology.
Construct the survivin shRNA recombinant plasmid targeted at human survivin gene (NM_001168).The accuracy of the sequence was tested by gene sequencing analysis and the best survivin shRNA recombinant plasmid was selected. Synthesized the CS-PEG induced survivin RNAi gene nano complex and transfected to HT-29 (lipsome that carried the same shRNA was used as positive control and nonsense shRNA was used as negative control).RT-PCR and Western blot were used to check the change of survivin mRNA expression, respectively; and MTT was performed to see the apoptosis.
Results:Malvem Zetasizer 3000E was used to test the particle size and distribution, the results showed that the CS-PEG particles size was 61nm. Atomic force microscope testing results showed that the shape of the particles was spherical like, with smooth surface and less adsorption. The nano particles had low cytotoxicity and high transfection rate. Gene sequencing analysis showed that the survivin shRNA recombinant plasmid was accurately constructed. The transfection rate of the gene nano complex was much higher than the controls (P<0.01);the expression of survivin mRNA in gene nano complex group was lower than the lipsome group (P<0.01);besides, the death rate and apoptosis (P<0.01).
Conclusion:In this study the CS-PEG nanoparticles with a particle size of 61nm, and well distributed are prepared;its cytotoxicity is low and transfection rate is high. The gene nano complex can effectively transfect to colon carcinoma cell lines HT-29 and interfere survivin. Compared with that of the control groups, the gene nano complex group shows higher effect in inducing the apoptosis and suppressing the propagation. It can effectively silence the expression of survivin in HT-29 at a comparatively long time.
方法：通过两步接枝共聚法,优化制备工艺,制备粒径较小、粒度分布均匀的壳聚糖-聚乙二醇纳米颗粒。使用Malvem Zetasizer3000E对纳米粒的粒径及zeta电位进行检测；利用原子力显微镜对纳米颗粒的形态进行观察。
选取人survivin基因(NM_001168)序列中的相关位点序列构建靶向survivin的shRNA重组质粒,通过序列测定检测重组子的正确性,再从中筛选出最佳的survivin shRNA重组质粒载体。合成壳聚糖-聚乙二醇纳米粒介导的survivin RNAi基因纳米复合物,将其导入结肠癌细胞HT-29中(以携带相同shRNA的脂质体载体,survivin shRNA以及阴性对照shRNA为对照)。用RT-PCR和Western-blot分别检测转染后的HT-29细胞中Survivin mRNA和蛋白表达量的变化；通过MTT法检测细胞凋亡情况；
结果：Malvem Zetasizer 3000E激光粒度分析仪进行粒径及分布的检测结果显示,制备出的壳聚糖-聚乙二醇纳米颗粒的平均粒径值为61nm。粒径大小分布呈正态分布,分布均匀。原子力显微镜进行观测。纳米颗粒呈圆球形,表面平滑完整,分散良好,较少粘附团聚现象；且细胞毒性低、转染率高。序列测定重组质粒载体survivin-shRNA构建正确。转染壳聚糖-聚乙二醇介导的survivin shRNA基因纳米复合物后发现,该基因纳米复合物对结肠癌细胞HT-29的转染效率显著高于survivin shRNA和脂质体载体(P<0.01)；HT-29细胞中Survivin mRNA拷贝数及蛋白表达显著降低(P<0.01),且结肠癌细胞HT-29的细胞死亡率和凋亡率明显提高(P<0.01)。
结论：本研究成功制备出平均粒径为61nm,分布均匀的壳聚糖-聚乙二醇纳米颗粒；其细胞毒性低、转染率高。合成的壳聚糖-聚乙二醇介导survivin shRNA基因纳米复合物能够高效转染结肠癌细胞株HT-29并有效干扰Survivin,且较之携带相同shRNA的脂质体载体,survivin shRNA和阴性对照shRNA具有更高的干扰效率,能高效地诱导结肠癌细胞凋亡并抑制其增殖。其介导RNA干扰能长时间高效率地沉默结肠癌细胞HT-29中的Survivin基因表达。
Objective:To prepare the PEGylated chitosan (CS-PEG) nano-gene carrier with high transfection rate and low cytotoxicity by a novel preparing method; construct the CS-PEG induced Survivin shRNA gene nano complex, which can transfect to HT-29 cells, to investigate its effect on the expression of survivin mRNA.To investigate its effect on the propagation and apoptosis of colon carcinoma cell lines HT-29, and to discuss its treatment effect on colon carcinoma
Methods:CS-PEG nanoparticles with small particle size were prepared by an optimized graft copolymerization method. Malvem Zetasizer 3000E was used to measure the particle size and zeta potential; and atomic force microscope was used to observe the morphology.
Construct the survivin shRNA recombinant plasmid targeted at human survivin gene (NM_001168).The accuracy of the sequence was tested by gene sequencing analysis and the best survivin shRNA recombinant plasmid was selected. Synthesized the CS-PEG induced survivin RNAi gene nano complex and transfected to HT-29 (lipsome that carried the same shRNA was used as positive control and nonsense shRNA was used as negative control).RT-PCR and Western blot were used to check the change of survivin mRNA expression, respectively; and MTT was performed to see the apoptosis.
Results:Malvem Zetasizer 3000E was used to test the particle size and distribution, the results showed that the CS-PEG particles size was 61nm. Atomic force microscope testing results showed that the shape of the particles was spherical like, with smooth surface and less adsorption. The nano particles had low cytotoxicity and high transfection rate. Gene sequencing analysis showed that the survivin shRNA recombinant plasmid was accurately constructed. The transfection rate of the gene nano complex was much higher than the controls (P<0.01);the expression of survivin mRNA in gene nano complex group was lower than the lipsome group (P<0.01);besides, the death rate and apoptosis (P<0.01).
Conclusion:In this study the CS-PEG nanoparticles with a particle size of 61nm, and well distributed are prepared;its cytotoxicity is low and transfection rate is high. The gene nano complex can effectively transfect to colon carcinoma cell lines HT-29 and interfere survivin. Compared with that of the control groups, the gene nano complex group shows higher effect in inducing the apoptosis and suppressing the propagation. It can effectively silence the expression of survivin in HT-29 at a comparatively long time.
引文
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