重组hIFN-β腺病毒对食管癌细胞增殖及细胞周期的影响

Effects of hIFN-β recombinant adenovirus on proliferation and cell cycle of esophageal carcinoma cells in vitro

背景与目的:Ⅰ型干扰素包括IFN-α和IFN-β,是多功能的细胞因子,具有抗病毒,抗增殖,抗血管生成及免疫调节作用。许多研究已经证明,IFN-β能明显抑制多种肿瘤的生长。然而,IFN-β蛋白极短的半衰期是临床应用中面临的主要问题。此外,临床实验表明最大剂量给药后机体IFN-β血清浓度远远低于抗肿瘤效应所需要的有效药物浓度。由载体介导的基因治疗解决了这一难题。许多研究已经表明,腺病毒载体介导的IFN-β基因能在肿瘤组织局部高效表达,抑制这些肿瘤细胞和组织的生长,并能诱导其凋亡。所以,基因治疗是一种具有应用前景的治疗手段,本实验用重组腺病毒载体转染食管癌细胞,观察腺病毒(Ad)介导的hIFN-β基因对人食管癌细胞体外增殖及细胞周期的影响。方法:重组腺病毒AdhIFN-β转染食管癌细胞系KYSE150,应用逆转录聚合酶链反应(RT-PCR)从RNA水平检测hIFN-β基因在KYSE150细胞中的表达,细胞生长抑制实验、集落形成能力实验检测hIFN-β基因对KYSE150细胞的体外增殖的影响;流式细胞术(FCM)检测细胞周期改变。结果:重组腺病毒经HEK293细胞扩增、纯化后滴度可达2×1011pfu/ml,且30 MOI(multiplicity of infection)的病毒可使95%以上的KYSE150细胞感染;RT-PCR检测到外源性hIFN-β基因在KYSE150细胞中的表达;MTT实验和集落形成能力实验表明,与对照组相比,AdhIFN-β转染后细胞生长受到明显抑制;流式细胞仪检测显示细胞阻滞于S期。结论:腺病毒介导的hIFN-β基因能抑制食管癌细胞的增殖及诱导S期阻滞,为该基因应用于食管癌的治疗提供了理论基础。

Background and purpose： Type Ⅰ IFNs including the IFN-α family and IFN-β are multifunctional cytokines with antiviral, antiproliferative, antiangiogenic activity and immune cell stimulation. Many investigators have demonstrated that IFN-β could inhibit significantly the growth of many tumors. However, the problem in the use of IFN-β protein for clinical treatment is its short half-life. Moreover, clinical studies have shown that the serum concentrations of IFN-β after systemic administration of the maximally tolerated dose is far below the level required to achieve the anti-proliferative effect documented in vitro studies. Gene therapy may overcome this limit because of continuous intracellular secretion of cytokine with IFN-β gene transduction. Many studies have demonstrated that the systemic delivery of an adenovirus vector secreting IFN-β may generate high local concentrations of IFN-β and achieve cytostatic and cytotoxic levels. So, gene therapy provides a promising approach for patients. This study investigated the effect of replication-deficient recombinant adenovirus vector expressing hIFN-β gene on the proliferation and cell cycle of esophageal carcinoma cell line KYSE150 in vitro. Methods： hIFN-β gene was transduced into KYSE150 cells mediated by recombinant adenovirus and then the hIFN-β expression was detected by RT-PCR and agarose gel electrophoresis. Cell growth assay and colony formation test were used to observed the anti-proliferation of AdhIFN-β on the KYSE150 cells. FACS was used to analysis the cell cycle of KYSE150 cells. Results： The titers of AdhIFN-β reached 2×10^11 pfu/ml and more than 95% KYSE150 cells could be infected by 30 MOI（multiplicity of infection） AdhIFN-β. The results of RT-PCR showed obvious hIFN-β mRNA in KYSE150 cells after transfection. The proliferation of KYSE150 cells was obviously inhibited after transfected with AdhIFN-β observed in the growth curve and cloning efficiency test compared with that of the control cells. The results of FACS test showed that AdhIFN-β could inhibit the cell cycle progression in S phage. Conclusions： AdhIFN-β adenovirus vector can inhibit the growth of KYSE150 cells and induces S phase arrest. This experiment provides an elementary basis for the gene therapy of esophageal carcinoma by using hIFN-β gene.