摘要
背景:研究表明,白细胞介素12是一种强有力的抗肿瘤细胞因子,骨髓间充质干细胞易于外源基因的导入和表达,且具有较弱的免疫抗原性和免疫调节功能,因此,利用携带白细胞介素12基因的间充质干细胞对于肿瘤的治疗具有极大的发展前景。目的:构建人白细胞介素125型腺病毒载体(AdhIL-12),感染人骨髓间充质干细胞,检测白细胞介素12的表达。方法:提取成熟的树突状细胞的总RNA,用RT-PCR方法获得hIL-12p35和p40基因cDNA,p35和p40cDNA通过脑心肌炎病毒内部核酸进入位点连接,构建pDC515hIL-12p35/IRES/p40腺病毒穿梭质粒。采用AdMaxTM腺病毒载体体系,pDC515hIL-12p35/IRES/p40穿梭质粒与pBHGfrt△E1,3FLP骨架质粒共转染至293细胞,通过重组酶位点特异性重组获得AdhIL-12。AdhIL-12感染人骨髓间充质干细胞后经γ射线照射使细胞失去增殖能力,用hIL-12p70ELISA试剂盒检测细胞上清中白细胞介素12的水平。结果与结论:经测序证实,RT-PCR所获得的hIL-12p35和p40基因cDNA序列与GenBankNM_000882(762bp)和NM_002187(987bp)提供的序列完全一致。用AdMaxTM腺病毒载体体系可高效、快捷地获得所要包装的腺病毒载体,通过IRES连接hIL-12的两个亚基p40和p35可有效地表达IL-12p70蛋白;AdhIL-12腺病毒载体感染人骨髓间充质干细胞后可持续高水平分泌白细胞介素12,在以人骨髓间充质干细胞为载体细胞的基因治疗中有潜在的应用前景。
BACKGROUND：Previous studies have suggested that interleukin-12 （IL-12） is a powerfull anti-tumor cell factor.Bone marrow mesenchymal stem cells （BMSCs） are prone to introduction and expression of exogenous gene,and have weak immunizing antigen and immunological regulation functions.Therefore,mesenchymal stem cells （MSCs） carrying IL-12 gene possess great prospects for tumor treatment.OBJECTIVE：To construct an adenoviral （Ad） vector encoding human IL-12 （hIL-12） gene,infect human BMSCs,and detect the expression of IL-12.METHODS：The total RNA of human dendritic cells （DCs） was obtained.hIL-12 p35 and p40 cDNA were amplified from the total RNA by RT-PCR,and p35 and p40 fragments were linked by internal ribosomal entry sites （IRES）,to construct the shuttle vector pDC515 p35/IRES/p40.Using Ad MaxTM adenovirus vector system,pDC515 p35/IRES/p40 and pBHGfrt△E1,3FLP were cotransfected into 293 cells,and Ad hIL-12 was generated by FLP recombinase-mediated site-specific recombination.After Ad hIL-12 infection,hBMSCs were irradiated with γ-ray to lose proliferative activity.IL-12 levels were determined in cell supernatants utilizing hIL12p70 enzyme linked immunosorbent assay.RESULTS AND CONCLUSION：The sequences of p35 and p40 fragments were identical with those provided by GenBank NM_000882 （762 bp） and NM_002187 （987 bp）,respectively.Ad MaxTM was an efficiently and quickly packaging system of adenoviral vectors.hIL-12 gene in which the two subunits p35 and p40 were linked by IRES can efficiently express the protein of IL-12p70.The high expression of IL-12 is consecutively found after the infection of human BMSCs with Ad hIL-12,suggesting a potential application to the gene therapy of human BMSCs as a carrier.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2010年第23期4186-4190,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金(30972804)~~
