慢病毒介导可溶性肿瘤坏死因子受体1在小鼠骨髓未成熟树突状细胞中的表达

doi:10.3969/j.issn.1673-8225.2010.05.044

摘要/Abstract

摘要：

背景：肿瘤坏死因子α是介导树突状细胞成熟的重要细胞因子之一，可溶性肿瘤坏死因子受体1 与其结合可阻断肿瘤坏死因子α的作用，维持树突状细胞于不成熟状态，诱导免疫耐受。
目的：构建含有人sTNFR1的慢病毒表达载体，观察其在未成熟树突状细胞中的表达。
方法：以人外周血单个核细胞总RNA为模板，RT-PCR扩增出sTNFR1基因片段，亚克隆至慢病毒转移质粒pXZ208，通过IRES连接eGFP报告基因，建立双顺反子慢病毒转移质粒，命名为pXZ9-sTNFR1，DNA测序鉴定。采用脂质体转染293 FT细胞，根据报告基因eGFP测定病毒滴度。采用小剂量粒-巨噬细胞集落刺激因子+ 白细胞介素4体外培养扩增C57BL/6小鼠骨髓来源树突状细胞。培养第5天，以pXZ9-sTNFR1重组慢病毒上清感染未成熟树突状细胞， RT-PCR检测感染后sTNFR1转录，Western blot法检测sTNFR1蛋白表达，观察sTNFR1基因修饰及脂多糖刺激后树突状细胞的表型特征。
结果与结论：成功构建重组质粒 pXZ9-sTNFR1，转染293 FT细胞24 h后观察到eGFP表达，病毒滴度在106 U/L以上。RT-PCR显示pXZ9-sTNFR1感染的未成熟树突状细胞sTNFR1呈阳性表达，Western blot检测到sTNFR1蛋白存在于感染后未成熟树突状细胞和培养上清中。培养第5 天的树突状细胞低表达CD40、CD86、CD80和MHCⅡ类分子，脂多糖刺激后，高表达MHCⅡ类分子和CD40、CD80、CD86分子，显示出成熟型树突状细胞表型特征，sTNFR修饰的树突状细胞MHCⅡ类分子和CD40、CD80、CD86 分子表达水平无变化。提示：①成功构建了负载sTNFR1基因片段及含eGFP报告基因的慢病毒载体，获得了高滴度的重组慢病毒颗粒。②经慢病毒高效转导的未成熟树突状细胞sTNFR1 mRNA及蛋白稳定地表达，可以保护未成熟树突状细胞不被外源性脂多糖刺激活化，维持树突状细胞于非成熟状态。

关键词: 可溶性肿瘤坏死因子受体, 基因修饰, 慢病毒载体, 未成熟树突状细胞, 免疫耐受

Abstract:

BACKGROUND: Tumor necrosis factor-α (TNF-α) is one of important cytokines to promote the maturation of dendritic cells. Blockage of TNF-α action by binding with soluble tumor necrosis factor receptor 1 (sTNFR1) may arrest dendritic cells in an immature state and induce stable, long-term tolerance.
OBJECTIVE: To construct the lentiviral vectors carrying sTNFR1 gene and investigate sTNFR1 expression in immature dendritic cells.
METHODS: Total RNA of human peripheral blood mononuclear cells was taken as a template. The sTNFR1 gene fragment was amplified by RT-PCR, subcloned to the lentiviral vectors pXZ208, and ligated to the enhanced green fluorescent protein (eGFP) reporter gene to establish lentiviral vector, called pXZ9-sTNFR1. DNA sequencing was performed for lentiviral vector identification. Lentivirus was prepared by transfection of 293 FT cells with pXZ9-sTNFR1. Viral titer was determined by eGFP expression. C57BL/6 mouse bone marrow-derived dendritic cells were in vitro cultured with low-dose granulocyte-macrophage colony stimulating factors and interleukin 4. On day 5 of culture, immature dendritic cells were transfected with pXZ9-sTNFR1 recombinant lentiviral supernatant. sTNFR1 transcription was detected by RT-PCR, sTNFR1 protein expression by Western blot analysis. Following sTNFR1 gene modification and lipopolysaccharide stimulation, the phenotype characteristics of dendritic cells were observed.
RESULTS AND CONCLUSION: Recombinant plasmid pXZ9-sTNFR1 was successfully constructed. Twenty-four hours after 293 FT cell transfection, eGFP expression was observed and viral titer was over 106 U/L. RT-PCR demonstrated that pXZ9-sTNFR1-transfected immature dendritic cells showed sTNFR1 positive expression. Western blot analysis revealed that sTNFR1 protein appeared in the immature dendritic cells and supernatant following 293 FT cell transfection. On day 5 of culture, dendritic cells expressed low level of class Ⅱ major histocompatibility complex (MHC Ⅱ), as well as CD40, CD86, CD80, molecules. However, following lipopolysaccharide stimulation, dendritic cells expressed high level of MHC II, as well as CD40, CD80, and CD86, molecules, exhibiting the phenotype characteristics of mature dendritic cells. But after sTNFR modification, the expression level of MHC II, as well as CD40, CD80, and CD86, molecules was not altered obviously. Lentiviral vectors carrying sTNFR1 gene and eGFP reporter gene were successfully constructed, and recombinant lentiviral plasmids with high titer were acquired. Following high efficacy of lentiviral gene transfection, immature dendritic cells stably express sTNFR1 mRNA and protein, which prevents immature dendritic cells from activation by exogenous lipopolysaccharide and maintains the immature state.

中图分类号:

R318

黄一虹，晁亚丽，汤仁仙，王淑华，曾令宇，陈翀，潘秀英，徐开林. 慢病毒介导可溶性肿瘤坏死因子受体1在小鼠骨髓未成熟树突状细胞中的表达[J]. 中国组织工程研究, 2010, 14(5): 841-846.

Huang Yi-hong1, Chao Ya-li1, Tang Ren-xian2, Wang Shu-hua1, Zeng Ling-yu1, Chen Chong1, Pan Xiu-ying1, Xu Kai-lin1. Lentivirus-mediated soluble tumor necrosis factor receptor 1 expression in mouse bone marrow-derived immature dendritic cells　　[J]. Chinese Journal of Tissue Engineering Research, 2010, 14(5): 841-846.

参考文献

引言

With the research progress in immunobiology, dendritic cells have been shown to play an important role in inducing and maintaining immunological tolerance. Dendritic cells are presently known the most efficient antigen presenting cells and are a kind of heterogeneic cell population with different subsets and different functions. Dendritic cells can cause the activation, differentiation, or tolerance of initial T cells. These different roles correlate with the maturating statuses of dendritic cells[1-3]. If the interaction of initial T cells and specific antigen peptide is accomplished by immature dendritic cells, then T cell inexcitability will take place, but under the condition of inflammatory cytokines, the inexcitability will turn into immunogenicity over the maturation of dendritic cells. Therefore, a key to induce immunological tolerance is to block the maturation of dendritic cells or to maintain the immature state[4-6].
The regulation of dendritic cell maturation involves tumor necrosis factor receptor or related signal transduction pathways. Tumor necrosis factor α (TNF-α) has been shown to exert a critical role in the maturation of dendritic cells sourced from bone marrow precursor cells and mononuclear precursor cells[7-8]. TNF-α biological function is accomplished by binding with cell surface TNF-α receptor. TNF-α receptors can be categorized into and membrane-bound and soluble TNF-α receptor (mTNFR, sTNFR, respectively). Under the activation of TNF-α converting enzyme, mTNFR extracellular region liberates from cell membrane, dissociates in the blood, and forms sTNFR compound. But sTNFR can not mediate TNF-α signal transduction[9-10], and thereby indirectly inhibit the physiological role of TNF-α. Therefore, a promising method to induce long-lasting immunological tolerance is to utilize TNF-α to resist sTNFR1 gene-modified dendritic cells and maintain their immature status[11-13].
The present study constructed recombinant lentiviral vectors carrying human sTNFR1 gene and used them to transfect mouse bone marrow-derived immature dendritic cells, so as to detect the phenotype and maturity status of immature dendritic cells.

材料方法

讨论

Mature dendritic cells prone to immune activation, and immature dendritic cells prone to immune tolearance[15-17]. For mature dendritic cells, the ingested antigen binds to MHC Ⅱ molecules, and under the effects of synergic stimulation molecules, antigen peptide-MHC Ⅱ molecule complex binds to T cell surface TCR, and thereby to activate T cells. For immature dendritic cells, during the process of angtigen presentation, T cells can not be activated, and antigenic specific T cells inability is induced, due to lack of B7 molecules, thereby leading to antigen tolearance[18-19]. Great progress has been made to obtain tolerant dendritic cells. These methods include initial low-dose GM-CSF induction, classic GM-CSF plus interleukin 4 induction, and gene modification (transfecting transforming growth factor β, interleukin 10, sTNFR gene into mouse dendritic cells). These tolerant dendritic cells exhibit some advantages in inhibiting organ graft rejection and graft versus host disease[20-22].

Transformation of dendritic cells from immature precursor cells into mature cells is influenced by various factors, during which, changes in dendritic cell surface marker and function take place. Cytokine is an important factor that regulates the maturation of dendritic cells. An extremely small number of exogenous TNF-α added during in vitro culture can promote immature dendritic cells to develop into mature dendritic cells[7, 23-27]. Blocking TNF-α can inhibit the maturation and activation of dendritic cells. sTNFR gene, an antagonic molecule of TNF-α, can be used to modify tolerant dendritic cells and maintain their immature status, which is likely to be a more effective means to induce long-lasting immune tolerance[21]. At present, exogenous genes are transfected into dendritic cells through the use of viral vector systems, including adenoviral vector, adeno-associated viral vector, retroviral vector, and lentiviral vector. Our laboratory first successfully reconstruct a novel high titre inactivated lentiviral vector system in China, optimized internal promoters, and screened CMV promoter with highest expression efficiency[26]. The present study confirmed pCR2.1-sTNFR1 recombinant gene-engineered bacteria using enzyme digestion and nucleotide sequencing, compared sequencing factors with Genbank sequence, and obtained consistent results, without mutation. Then the pCR2.1-sTNFR1 was cloned into lentiviral transfer plasmid pXZ208, named pXZ9-sTNFR1. At 24 hours after transfecting 293FT cells, eGFP expression was observed under fluorescence microscope. Results demonstrated that viral titer exceeded 106 U/L, demonstrating that recombinant lentiviral expression vector carrying sTNFR1 gene was successfully reconstructed, and high viral titer was obtained.

(60.37±5.48) % of immature dendritic cells could be transfected by viral supernatant, and sTNFR1 mRNA and protein expression was stable, providing experimental evidence for further studying the immune tolerance of sTNFR1 gene modified immature dendritic cells during transplantation.

Some advancement has been presently made as to in vitro culture of dendritic cells. The amount of GM-CSF markedly correlates with maturation of dendritic cells cultured. Generally, high-dose GM-CSF primarily induce mature dendritic cells, while low-dose GM-CSF main induce immature dendritic cells[28-29]. 500-1 000 U/mL interleukin 4 can inhibit macrophage formation, and produce a synergistic effect with low-dose GM-CSF to enhance the amount and maturation degree of harvested cells[30]. The present study selected mouse bone marrow cells as the precursor cells of dendritic cells and used low-dose GM-CSF plus interleukin 4 to obtain higher purity of dendritic cells. This occurs possibly because GM-CSF produces proliferation-stimulating effects only on granulocytes, macrophages, and dendritic cells, while other cells, such as B cells and T cells, would gradually die during culture; granulocytes, the primarily contaminated cells, can not survive more than 10 days under the condition of 200 U/mL GM-CSF or lower concentration, and macrophages and other adherent cells would be separated in final cell harvesting. The present study also observed the development of dendritic cells. Results revealed that sTNFR1 gene could protect immature dendritic cells from activation by exogenous lipopolysaccharide, and compared with undigested immature dendritic cells, the phenotype of sTNFR1 gene-transfected immature cells did not change obviously. Exogenous lipopolysaccharide has been reported to promote dendritic cells to release TNF-α in the manner of autocrine and further promote the rapid differentiation and maturation of dendritic cells[31-32]. It is presumed that sTNFR1 gene modified immature dendritic cells maintain their immature status by local secretion of sTNFR1 to block the TNF-α autocrine by exogenous lipopolysaccharide-stimulated immature dendritic cells.

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