Ebadi Padideh, Karimi Mohammad Hossein, Pourfathollah Ali Akbar, Saheb Ghadam Lotfi Abbas, Soheili Zahra Soheila, Samiee Shahram, Hajati Smerdis, Nadali Fatemeh, Geramizadeh Bita, Moazzeni Seyyed Mohammad
Department of Biology, Science and Research Branch, Islamic Azad University.
Iran J Immunol. 2009 Mar;6(1):1-11.
Dendritic cells (DCs) are ideal accessory cells in the field of gene therapy. Delivery of DNA and siRNA into mammalian cells is a useful technique in treating various diseases caused by single gene defects. Selective gene silencing by small interfering RNAs (siRNAs) and antisense oligodeoxynucleotides (ODN)s is an efficient method for the manipulation of cellular functions. An efficient, functional delivery system with no toxicity problems would be attractive.
We compared two commercially available cationic lipids, Lipofectamine and FuGENE6, in the delivery of both siRNA and antisense ODNs into mice spleen-derived DCs.
Cellular uptake was measured by the means of fluorescein-labelled non-silencing siRNA and antisense ODNs as a model system using flow cytometry. Cytotoxicity of the two delivery systems was compared with propidium iodide and annexin-V staining, and quantified with flow cytometry. The efficiency of our oligonucleotide delivery systems was compared by measuring CD40 expression by flow cytometry.
CD40 expression in DCs was 38%. After siRNA transfection by Lipofectamine, CD40 expression decreased to 13%, and after transfection by FuGENE6, it decreased to 18%. The difference was statistically significant. CD40 down regulation in DCs transfected with the two different antisense sequences by Lipofectamine was 21% and 23%, and down regulation after transfection by FuGENE6 was 19% and 18%, respectively. The differences were not statistically significant. The effects of siRNA and antisense ODNs were specific.
Lipofectamine was a more potent delivery system in siRNA effect, followed by FuGENE6. There was no significant difference between Lipofectamine and FuGENE6 as a delivery system of antisense ODNs.
树突状细胞（DCs）是基因治疗领域理想的辅助细胞。将DNA和小干扰RNA（siRNA）导入哺乳动物细胞是治疗由单基因缺陷引起的各种疾病的有用技术。小干扰RNA（siRNAs）和反义寡脱氧核苷酸（ODN）介导的选择性基因沉默是操纵细胞功能的有效方法。一种高效、无毒性问题的功能性递送系统将具有吸引力。
我们比较了两种市售阳离子脂质Lipofectamine和FuGENE6在将siRNA和反义ODN递送至小鼠脾脏来源的DCs中的效果。
以荧光素标记的非沉默siRNA和反义ODN作为模型系统，采用流式细胞术测量细胞摄取。通过碘化丙啶和膜联蛋白-V染色比较两种递送系统的细胞毒性，并用流式细胞术进行定量。通过流式细胞术测量CD40表达，比较我们的寡核苷酸递送系统的效率。
DCs中CD40表达为38%。用Lipofectamine转染siRNA后，CD40表达降至13%，用FuGENE6转染后降至18%。差异具有统计学意义。用Lipofectamine转染两种不同反义序列的DCs中CD40下调分别为21%和23%，用FuGENE6转染后下调分别为19%和18%。差异无统计学意义。siRNA和反义ODN的作用具有特异性。
在siRNA作用方面，Lipofectamine是一种更有效的递送系统，其次是FuGENE6。作为反义ODN的递送系统，Lipofectamine和FuGENE6之间没有显著差异。
