串珠素反义cDNA质粒转染对喉癌细胞Hep-2增殖能力的影响

目的：研究串珠素反义cDNA质粒转染对喉癌细胞Hep-2增殖能力的影响。方法：利用阳离子脂质体作为载体，把重组真核表达载体串珠素反义cDNA质粒pAP转染喉癌细胞Hep-2。通过RT—PCR、Westernblot及MTT法检测转染后Hep-2细胞串珠素mRNA、蛋白质表达及细胞增殖水平，并观察转染后Hep-2细胞对碱性成纤维细胞生长因子（bFGF）的反应性。将Hep2细胞分3组：①未转染的Hep-2细胞组（WT组）；②空载体ph8Apr—neol转染组（neo组）；③串珠素反义cDNA质粒pAP转染组（pAP组）。结果：将质粒pAP成功地转入Hep2细胞，获得了稳定表达串珠素反义cDNA基因的细胞系。串珠素mRNA和蛋白质水平在pAP组低表达，在WT组和neo组高表达，均差异有统计学意义（均P〈O．01）。在0．1％FCS的RPMI1640培养基培养下，WT组、neo组和pAP组细胞的增殖率均降低，但pAP组细胞的增殖与WT组和neo组细胞相比明显减低；在0．1％FCS加1μg／L bFGF的RPMI1640培养液培养下．WT组和neo组细胞增殖接近正常，但pAP组细胞的增殖能力较弱。结论：串珠素反义cDNA质粒转染可以有效抑制喉癌细胞Hep2的增殖能力。

Suppression of proliferation of Hep-2 cells by stable expression of anti-sense perlecan cDNA

OBJECTIVE: To study the suppression of proliferation of Hep-2 cells by stable expression of anti-sense perlecan cDNA. METHOD: In this study, the plasmids of recombination eukaryotic expression vector perlecan anti-sense cDNA (pAP) were transfected into Hep-2 cells by using cationic liposome (lipofectamine 2000) and divided into three groups: non-transfected group, WT group; transfection with no load carrier, neo group; and transfection with the pAP plasmid, pAP group. Semi quantify RT-PCR, western blot assay and MTT assay were used to detected the expression of perlecan mRNA and protein in the three groups; the level of cell proliferation; and the responsivity of basic fibroblast growth factor (bFGF). RESULT: It was showed that the expression of perlecan mRNA and protein were significantly reduced in the pAP group compared with WT group and ph beta Apr-neol transfected group ( P < 0.01). In the presence of 1 microg/L of bFGF in low serum (0.1% FCS), the pAP transfected cells showed a reduced proliferation rate (MTT assay) while the wild type cells and ph beta Apr-neol transfected cells grew rapidly. CONCLUSION: The growth of Hep-2 cells could be inhibited significantly by perlecan anti-sense cDNA plasmids transfection.