乙酰肝素酶反义寡核苷酸对Hep-2细胞侵袭力抑制的研究

Inhibition of invasiveness of human Hep-2 cell lines by heparanase antisense oligodeoxynucleotide

目的探讨乙酰肝素酶(heparitinase,HPSE)反义寡核苷酸(antisense oligodexyribonucle-otides,AS-ODN)对Hep-2细胞HPSE mRNA、HPSE蛋白的表达及其侵袭力的活性抑制。方法设计合成互补于HPSE mRNA起始密码区的HPSEAS-ODN,以阳离子脂质体Lipofectin包埋后转染人喉癌Hep-2细胞进行培养,采用流式细胞分析技术和逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)方法检测Hep-2细胞HPSE蛋白及其mRNA表达的变化;以Matrigel细胞侵袭试验分别对HPSEAS-ODN对Hep-2细胞侵袭力的抑制进行检测。结果AS-ODN各组与正常对照组和脂质体组比较及AS-ODN各组间比较HPSEmRNA和HPSE蛋白的表达及细胞侵袭力均有明显抑制作用,差异有统计学意义(P<0.01);AS-ODN在终浓度为100、200、400nmol/L时对Hep-2细胞侵袭力的抑制率分别为60.1%、80.8% and 94.0%。结论HPSEASON通过下调HPSE mRNA和HPSE蛋白的表达可显著抑制Hep-2细胞的侵袭力并呈剂量依赖性。

OBJECTIVE To evaluate the inhibitory effect of HPSE AS-ODN on the invasiveness of human Hep-2 cell lines. METHODS HPSE AS- ODN which was complementary with initiation codon region of HPSE mRNA was designed and synthesized. After embedded by cation lipofectin, it was transfected into Hep-2 cells of human laryngocarcinoma. The expression of HPSE protein and HPSE mRNA in Hep-2 cell lines were detected by flow cytometry and RT- PCR. Meanwhile Matrigel invasive assay was used to measure the inhibitory effect of HPSE AS-ODN on the invasiveness of human Hep-2 cell lines. RESULTS The HPSE protein and HPSE mRNA expression and invasiveness of human Hep-2 cells treated with AS- ODN of different concentrations were significantly decreased as the AS-ODN concentration increasing. There was a significantly difference between control group and each group of AS-ODN respectively （P 〈0.01）, between lipofectin group and each group of AS-ODN respectively （P 〈0.01）, and among the groups of AS-ODN （P 〈0.01）. Besides, the difference of inhibitory effect was significantly among the cells treated with different AS-ODN concentrations （P 〈0.01）. The inhibition rates of Hep-2 cell invasiveness were 60.1%, 80.8 % and 94.0 % treated by AS-ODN at final concentration of 100 nmol/L, 200 nmol/L and 400 nmol/L respectively. CONCLUSION By down-regulating the expression of HPSE mRNAand HPSE protein, HPSE AS- ODN has significant inhibitory effect on the invasiveness of human Hep-2 cell line in a dose-dependent manner.