Huang Fei, Zhao Feng, Liang Li-Ping, Zhou Mei, Qu Zhi-Ling, Cao Yan-Zhen, Lin Chen
Department of Pathology, The Affiliated Tumor Hospital Of Xinjiang Medical University, Urumqi, China E-mail :
Asian Pac J Cancer Prev. 2015;16(17):7749-54. doi: 10.7314/apjcp.2015.16.17.7749.
Currently, cationic liposome has become the commonly used vehicles for gene transfection. Furthermore, one of the most significant steps in microRNAs expression studies is transferring microRNAs into cell cultures successfully. In this study we aim to approach the feasibility of transfection of cervical cancer cell lines mediated by liposome and to obtain the optimized transfection condition for cervical cancer cell lines.
Lipofectamine(TM)2000 as the carrier, miR-101 mimic was transfected into Hela cells and Siha cells. Using green fluorescent protein as reporter gene, to set different groups according to cell seeding density, the amount of miRNA , miRNA and the proportion of Liposomes, Whether to add serum into medium to study their impact on the liposomal transfection efficiency. Finally, MTT assay was used to analyze the relative minimal cell toxicity of liposome reagents.
The seeding density of Hela cell line and Siha are 1.5 x 10(4) (per well of 24 well plates), miRNA amount is 1ul of both, the ratio of miRNA and liposome is 1:0.5 of Hela cell line; 1:0.7 of Siha cell line respectively, after 24 hours we can get the highest transfection efficiency. Compared with serum medium, only Siha cells cultured with serum-free medium obtained higher transfection efficiency before transfection (P<0.01).MTT assay showed that according to the above conditions which has the lowest cytotoxicity.
The method of Liposome to transfected is a suitable way and it can be an efficient reagent for miRNA delivery for Hela cells and Siha cells in vitro. It may serve as a reference for the further research or application.
目前，阳离子脂质体已成为基因转染常用的载体。此外，在微小RNA表达研究中最重要的步骤之一是将微小RNA成功导入细胞培养物中。在本研究中，我们旨在探讨脂质体介导的宫颈癌细胞系转染的可行性，并获得宫颈癌细胞系的优化转染条件。
以Lipofectamine™2000为载体，将miR-101模拟物转染至Hela细胞和Siha细胞。以绿色荧光蛋白作为报告基因，根据细胞接种密度、miRNA用量、miRNA与脂质体的比例以及培养基中是否添加血清设置不同组，研究它们对脂质体转染效率的影响。最后，采用MTT法分析脂质体试剂的相对最小细胞毒性。
Hela细胞系和Siha细胞的接种密度均为1.5×10⁴（24孔板每孔），miRNA用量均为1μl，Hela细胞系中miRNA与脂质体的比例为1:0.5；Siha细胞系中分别为1:0.7，24小时后可获得最高转染效率。与血清培养基相比，仅无血清培养基培养的Siha细胞在转染前获得了更高的转染效率（P<0.01）。MTT法显示，按照上述条件细胞毒性最低。
脂质体转染方法是一种合适的方法，它可以成为体外向Hela细胞和Siha细胞递送miRNA的有效试剂。它可为进一步的研究或应用提供参考。
