构建人白细胞介素 10 和增强型绿色荧光蛋白双顺反子重组腺病毒载体在骨髓间充质干细胞中的表达。

Construction of a bicistronic recombinant adenoviral vector for human interleukin-10 and enhanced green fluorescent protein expression in bone marrow mesenchymal stem cells.

机构信息

Department of Anesthesiology, First Affiliated Hospital of Fujian Medical University, Institute of Technology and Engineering, Fujian Medical University, Fuzhou, Fujian 350005, China.

出版信息

Chin Med J (Engl). 2012 Jan;125(1):102-8.

PMID:22340474

Abstract

BACKGROUND

Human interleukin-10 (hIL-10) is a cytokine synthesis inhibitory factor, which is involved in various immune responses. The purpose of this study was to construct an adenoviral vector carrying the hIL-10 gene for expression of biologically active hIL-10 in rat bone marrow mesenchymal stem cells (rMSCs).

METHODS

A pSNAV2.0-hIL10 plasmid was used as a template to obtain a hIL-10 cDNA fragment that was subcloned by restriction enzyme digestion and ligation into a pDC316-IRES-EGFP-lacZ alpha plasmid carrying an enhanced green fluorescent protein (EGFP) marker gene. The pDC316-hIL-10-IRES-EGFP plasmid was linearized by PmeI digestion and used to transfect HEK293 packaging cells using the adenovirus packaging system AdMax. Virus particles were amplified by repeatedly infecting HEK293 cells with the seed virus and then purified by ion exchange. After the number of virus particles and titer was determined, rMSCs were infected with the adenoviral vector. The infection rate was determined by fluorescence microscopy and flow cytometry, and hIL-10 protein expression in rMSCs was measured by Western blotting.

RESULTS

The virus particle concentration, OD260/280 value and virus titer of the amplified and purified recombinant adenovirus were 3.2 × 10(11) VP/ml, approximately 2.0, and 1.1 × 10(10) TCID50/ml, respectively. Bright green fluorescence was observed by fluorescence microscopy and flow cytometry in the recombinant adenovirus-infected rMSCs. GFP expression was considered the multiplicity of infection (MOI) and was time-dependent. The infection rate was 92.9% at 100 MOI.

CONCLUSIONS

A bicistronic recombinant adenoviral vector for hIL-10 and EGFP gene expression were successfully constructed. The infection rate of rMSCs by the adenovirus was high (92.9% at 100 MOI) and the target gene hIL-10 was highly expressed in cells. The present study provides an experimental basis for further research of immunosuppressive therapy using hIL-10. The expression level of hIL-10 protein as detected by Western blotting was also MOI- and time-dependent.

摘要

背景

人白细胞介素-10（hIL-10）是一种细胞因子合成抑制因子，参与各种免疫反应。本研究的目的是构建携带 hIL-10 基因的腺病毒载体，在大鼠骨髓间充质干细胞（rMSCs）中表达具有生物活性的 hIL-10。

方法

以 pSNAV2.0-hIL10 质粒为模板，通过限制性内切酶消化和连接亚克隆获得 hIL-10 cDNA 片段，该片段克隆到携带增强型绿色荧光蛋白（EGFP）标记基因的 pDC316-IRES-EGFP-lacZα质粒中。用 PmeI 消化线性化 pDC316-hIL-10-IRES-EGFP 质粒，使用腺病毒包装系统 AdMax 转染 HEK293 包装细胞。通过反复用种子病毒感染 HEK293 细胞来扩增病毒颗粒，然后通过离子交换进行纯化。确定病毒颗粒数和滴度后，用腺病毒载体感染 rMSCs。通过荧光显微镜和流式细胞术确定感染率，并通过 Western 印迹法测定 rMSCs 中 hIL-10 蛋白的表达。

结果

扩增和纯化的重组腺病毒的病毒颗粒浓度、OD260/280 值和病毒滴度分别为 3.2×10(11)VP/ml、约 2.0 和 1.1×10(10)TCID50/ml。重组腺病毒感染的 rMSCs 可见亮绿色荧光，通过荧光显微镜和流式细胞术观察到 GFP 表达，被认为是感染复数（MOI），并具有时间依赖性。在 MOI 为 100 时，感染率为 92.9%。

结论

成功构建了携带 hIL-10 和 EGFP 基因表达的双顺反子重组腺病毒载体。rMSCs 被腺病毒感染的效率很高（MOI 为 100 时为 92.9%），细胞内目的基因 hIL-10 表达水平较高。本研究为进一步研究 hIL-10 免疫抑制治疗提供了实验基础。Western 印迹法检测 hIL-10 蛋白的表达水平也与 MOI 和时间有关。