编码人白细胞介素-1β的重组痘苗病毒在肿瘤细胞中的构建与表达。

Construction and expression in tumor cells of a recombinant vaccinia virus encoding human interleukin-1 beta.

作者信息

Peplinski G R, Tsung K, Whitman E D, Meko J B, Norton J A

机构信息

Department of Surgery, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

出版信息

Ann Surg Oncol. 1995 Mar;2(2):151-9. doi: 10.1007/BF02303631.

DOI:10.1007/BF02303631

PMID:7728569

Abstract

BACKGROUND

Human interleukin-1 beta (hIL-1 beta) injected intratumorally has demonstrated growth inhibition of transplanted subcutaneous tumors in mice, regression of metastatic lesions, resistance to tumor rechallenge, and increased survival. Vaccinia virus (VV) can be genetically engineered to produce cytokines and may be an effective vector for gene therapy of cancer. This study was designed to (a) construct a VV expressing hIL-1 beta, (b) assess tumor cell infection in vitro with this construct, (c) measure hIL-1 beta production, and (d) assess the bioactivity of the secreted cytokine.

METHODS

The hIL-1 beta gene was amplified from a plasmid clone using polymerase chain reaction (PCR) and then cloned into a homologous recombination (HR) and expression vector, which was used to insert the hIL-1 beta gene into the VV genome. Selection of the recombinant VV (vMJ601hIL-1 beta) was based on inactivation of viral TK and expression of beta-galactosidase. vMJ601hIL-1 beta infectivity and cytokine production was assessed by infecting tumor cell lines and analyzing culture supernatants for hIL-1 beta. Bioactivity of the hIL-1 beta produced was demonstrated using an IL-1 dependent T helper cell line.

RESULTS

The hIL-1 beta gene was successfully cloned into the VV genome by HR, which was confirmed by PCR. vMJ601hIL-1 beta efficiently infected tumor cells, as shown by increased hIL-1 beta secretion (0 to > 500 ng/ml) and morphologic evidence of viral cytopathic effect. vMJ601hIL-1 beta-infected cells secreted large amounts of hIL-1 beta (mean 772 ng/10(6) cells/24 h). The secreted hIL-1 beta was bioactive (mean bioactivity 6.8 x 10(8) U/mg of hIL-1 beta).

CONCLUSIONS

(a) hIL-1 beta can be cloned into VV, (b) vMJ601hIL-1 beta retains its infectivity, (c) a large amount of hIL-1 beta is secreted, and (d) the secreted hIL-1 beta is bioactive. Recombinant VV may allow in situ cytokine gene delivery and expression in established tumors.

摘要

背景

瘤内注射人白细胞介素-1β（hIL-1β）已显示出对小鼠移植皮下肿瘤的生长抑制、转移灶消退、对肿瘤再次攻击的抗性以及生存期延长。痘苗病毒（VV）可通过基因工程改造来产生细胞因子，可能是癌症基因治疗的有效载体。本研究旨在：（a）构建表达hIL-1β的VV，（b）评估该构建体在体外对肿瘤细胞的感染情况，（c）检测hIL-1β的产生，以及（d）评估分泌的细胞因子的生物活性。

方法

使用聚合酶链反应（PCR）从质粒克隆中扩增hIL-1β基因，然后将其克隆到同源重组（HR）和表达载体中，该载体用于将hIL-1β基因插入VV基因组。重组VV（vMJ601hIL-1β）的筛选基于病毒TK的失活和β-半乳糖苷酶的表达。通过感染肿瘤细胞系并分析培养上清液中的hIL-1β来评估vMJ601hIL-1β的感染性和细胞因子产生。使用依赖IL-1的T辅助细胞系证明所产生的hIL-1β的生物活性。

结果

通过HR成功将hIL-1β基因克隆到VV基因组中，这通过PCR得到证实。vMJ601hIL-1β有效地感染肿瘤细胞，表现为hIL-1β分泌增加（从0至>500 ng/ml）以及病毒细胞病变效应的形态学证据。vMJ601hIL-1β感染的细胞分泌大量hIL-1β（平均772 ng/10⁶细胞/24小时）。分泌的hIL-1β具有生物活性（平均生物活性6.8×10⁸ U/mg hIL-1β）。