中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (23): 4186-4190.doi: 10.3969/j.issn.1673-8225.2010.23.002

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

人白细胞介素12腺病毒载体构建及在人骨髓间充质干细胞中的表达

陈媛媛1,2，谭晓华1，马晶莹1   

  1. 1解放军北京军区总医院生物治疗中心，北京市  100700；
    2山西医科大学，山西省太原市  030001
  • 出版日期:2010-06-04 发布日期:2010-06-04
  • 通讯作者: 谭晓华，博士后，主任医师，硕士生导师，解放军北京军区总医院血液科，北京市 100700 xiaohua_t@hotmail.com
  • 作者简介:陈媛媛★，女，1984年生，山西省太原市人，汉族，山西医科大学在读硕士。 yuanyuan840725@126.com
  • 基金资助:

    国家自然科学基金(30972804)

Construction and expression of an adenoviral vector encoding human interleukin-12 in human bone marrow mesenchymal stem cells

Chen Yuan-yuan1,2, Tan Xiao-hua1, Ma Jing-ying1   

  1. 1Biotherapy Center, General Hospital of Beijing Military Area Command of Chinese PLA, Beijing  100700, China;
    2Shanxi Medical University, Taiyuan  030001, Shanxi Province, China
  • Online:2010-06-04 Published:2010-06-04
  • Contact: Tan Xiao-hua, Doctor, Chief physician, Master’s supervisor, Biotherapy Center, General Hospital of Beijing Military Area Command of Chinese PLA, Beijing 100700, China xiaohua_t@hotmail. com
  • About author:Chen Yuan-yuan, Studying for master’s degree, Biotherapy Center, General Hospital of Beijing Military Area Command of Chinese PLA, Beijing 100700, China; Shanxi Medical University, Taiyuan 030001, Shanxi Province, China yuanyuan840725@126.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30972804*

PDF

437

被引次数

8

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12 5型腺病毒载体(Ad hIL-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-12 p35和p40基因cDNA，p35和p40 cDNA通过脑心肌炎病毒内部核酸进入位点连接，构建pDC515 hIL-12 p35/IRES/p40腺病毒穿梭质粒。采用Ad MaxTM腺病毒载体体系，pDC515 hIL-12 p35/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Ad hIL-12。Ad hIL-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-12p70 ELISA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-12 p35和p40基因cDNA序列与GenBank NM_000882(762 bp)和NM_002187(987 bp)提供的序列完全一致。用Ad MaxTM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；Ad hIL-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。

关键词: 白细胞介素12, 骨髓间充质干细胞, 腺病毒载体, 基因治疗, 干细胞

Abstract:

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.

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