ANF基因转染人血管平滑肌肉瘤细胞及其表达的实验研究

The Study on Transfected ANF Gene Expression of Human Leiomyosarcoma Cell in Vitro

通过对体外传代培养的人血管起源平滑肌肉瘤细胞进行直接和脂质体Ｔｆｘ－５０包裹的人心钠素（ＡＮＦ）基因表达质粒ｐｃＤＮＡ３／ＡＮＦ转染，并以非同位素地高辛标记的ＲＮＡ分子探针对转染细胞中的ＡＮＦｍＲＮＡ水平进行ＲＮＡＤｏｔＢｌｏｔ分子杂交检测，同时利用放射免疫方法测定转染细胞培养上清中的ＡＮＦ含量。结果显示，与未经脂质体Ｔｆｘ－５０包裹的ｐｃＤＮＡ３／ＡＮＦ直接进行细胞转染及低浓度脂质体Ｔｆｘ－５０包裹的ｐｃＤＮＡ３／ＡＮＦ进行细胞转染相比，脂质体Ｔｆｘ－５０与ｐｃＤＮＡ３／ＡＮＦ以３∶１（荷质比）进行包裹，并当ｐｃＤＮＡ３／ＡＮＦ在细胞数为１０５达到２．０μｇ时，细胞中ＡＮＦｍＲＮＡ表达阳性，转染细胞培养上清中ＡＮＦ含量达到５８．０ｎｇ／Ｌ。表明通过脂质体Ｔｆｘ－５０介导可以提高ｐｃＤＮＡ３／ＡＮＦ的转染效率，并使细胞表达ＡＮＦ显著增加。

Both Tfx 50 liposome coated and uncoated pcDNA3/ANF containing the human ANF gene were transfected into leiomyosarcoma (LMS) cells in vitro. The ANF mRNA of the LMS cells was detected by Dot Bloting method using the Diglabeled RNA probe. The ANF peptide levels in the LMS cell culture media were determined by RIA. The results showed that on day 3 after the gene transfection the ANF mRNA expression was positive and the ANF concentration of the cell culture media was up to 58 ng/L when pcDNA3/ANF DNA was coated with Tfx 50 under the charge ratio of 3∶1 (Tfx 50 liposome: pcDNA3/ANF DNA) and the quantity of the DNA was 2.0 μg/10 5 cell. These indicate that Tfx 50 reagent can be used to enhance pcDNA3/ANF transfection efficiency and gene expression, and it may be used for the study of ANF gene therapy on cardiovascular obstructive disease.