脂质体介导TGF-β_1质粒DNA转染家兔角膜上皮细胞的实验研究

Experimental observation of plasmid TGF-β_1 DNA gene transfer with lipofectamine to rabbit corneal epithelial cells in vitro

目的 :确定转化生长因子 β1(TGF β1)质粒DNA能否由脂质体携带进入家兔角膜上皮细胞 ,为角膜上皮细胞的研究提供方法。方法 :体外培养家兔角膜上皮细胞 ,用多聚阳离子脂质体携带重组的人的TGF β1质粒 ,向家兔角膜上皮细胞转染 ,在转染 12h后 ,表达 2d时 ,用免疫组化方法(SABC)检测TGF β1质粒DNA在家兔角膜上皮细胞的表达情况。结果 :外源TGF β1质粒DNA在家兔角膜上皮细胞中可以获得表达 ,在转染 12h后 ,表达 2d时的基因转染率为2 3%。结论 :稳定的外源基因可由脂质体介导转入生长中的收稿日期 :2 0 0 2 -0 3 -0 4;修回日期 :2 0 0 2 -0 4-10基金项目 :湖北省自然科学基金资助项目 (NO .98J0 70 )。作者简介 :黄琼 (1977-) ,女 ,湖北人 ,在读博士研究生 ,研究方向 :角膜病。通信作者 :黄琼 (E -mail:joanhuang77@2 1cn .com)。家兔角膜上皮细胞内 ,借此方法 ,可从外源基因入手 ,对角膜上皮细胞的生理、病理活动的机制进行研究 ;作为角膜上皮细胞基因类药物介入的基础研究和应用研究的介导体 。

Objective:To investigate whether the TGF β 1 plasmid DNA carried by lipofectamine can be introduced into cultured corneal epithelial rabbit cells.Methods:Eukaryotic expression plasmid pMAMTGF β 1 was prepared and purified. Then primary rabbit corneal epithelial cell culture was performed and transfection of pMAMTGF β 1 mediated by Lipofectamine was introduced into the second passage of corneal epithelial cells and allowed to transfect for 12 hours. After 2 days,specific expression of the plasmid pMAMTGF β 1 in the corneal epithelial cells was studied using immunohistochemical techniques. The positive rate of gene transfer of the corneal epithelial cells was measured.Results:A pure Plasmid DNA was obtained. Immunohistochemical staining showed that there was foreign plasmid TGF β 1 DNA product in the corneal epithelial cells. In the expression of 2 days,the positive transfer rate of expression was 23% after 12 hours of transfection.Conclusion:Efficient transfer of the functional gene can be achieved by introducing lipofectamine into the corneal epithelial cells. Lipofectamine is an available and promising vehicle for delivering a targeted gene to study the physiological and pathological mechanisms of corneal epithelial cells.