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小鼠体内RU486诱导的及肝脏特异性的IL-12基因表达 被引量:1

RU486-inducible and liver-specific expression of IL-12 gene in mice
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摘要 目的研究含有RU486调控系统质粒的可诱导能力。方法通过水流动力学注射的方法,将含有RU486调控系统、肝脏特异性启动子以及转基因IL-12的质粒pRS22注射到小鼠体内,质粒注射后不同时间点腹腔注射RU486。通过ELISA试剂盒检测血清中IL-12表达水平,通过PCR、RT-PCR以及免疫组织化学染色的方法,在DNA、RNA及蛋白质水平测试质粒pRS22在小鼠体内的可诱导性表达。结果为了确定质粒pRS22活性的持续时间,在水流动力学注射10μg质粒后,每7天给小鼠注射1次RU486(250μg/kg),发现尽管每次诱导后血清中IL-12的水平逐渐下降,但直到质粒注射后第15周,仍然可以被诱导表达。为了测试不同诱导方式对IL-12表达趋势的影响,5μg pRS22被注射到小鼠体内后,在6d内分别每天或每两天给小鼠注射1次RU486。结果每两天诱导1次后IL-12表达呈波浪形,而每天诱导1次则可获得持续的IL-12表达。PCR和RT-PCR结果显示,无论有无RU486诱导,都能在肝脏检测到pRS22质粒及GLp65调节子mRNA的表达,但是仅能够在接受RU486的小鼠肝脏中观察到IL-12p35亚基mRNA表达。免疫组织化学染色结果提示,IL-12蛋白只有在RU486作用下才会在肝细胞中表达。结论在肝脏特异性启动子TTR控制下,RU486调控系统能够在时间和空间上精确控制转基因IL-12的表达。 Objective To investigate the inducible ability of plasmid DNA carrying a RU486 regu-latory system. Methods Plasmid pRS22 containing RU486 regulatory system, liver specific promoter and transgene IL-12 was injected into mice by hydrodynamic injection. RU486 was injected intraperitoneally into mice at different time points after plasmid administration. The IL-12 level in serum was tested by an ELISA kit. The distribution and inducible expression of pRS22 in mice were assayed by measuring DNA, RNA and protein levels by PCR, RT-PCR and immunohistochemical staining. Results To determine the duration of the activity of plasmid pRS22, mice injected with 10μg of pRS22 were treated repeatedly with 250μg/kg of RU486 per 7 days after hydrodynamic injection of plasmid. IL-12 expression in serum abruptly increased to peak was detected at 10 h after induction and declined to baseline on day 6. Though peak values of IL-12 decreased gradually after each induction, IL-12 in serum could be induced until 15 weeks after plasmid administration. A total of 5μg of pRS22 was injected into mice to detect the effect of different induction manners on the IL-12 expression. The mice were treated with RU486 per day or per 2 days in 6 days, respectively. The induction per 2 days resulted in a wavelike pattern of serum IL-12 expression with peak levels on the day of induction alternating with lower values on the following day. In contrast, sustained levels of IL-12 could be achieved by administering RU486 per day. Plasmid DNA and GLp65 mRNA were detected in liver of mice with or without RU486 until at least day 28 after plasmid administration. However, IL-12 p35 mRNA was detected only in the liver of mice with RU486 induction. IL-12 immunohistochemical staining in liver demonstrated that IL-12 expressed predominantly in the hepatocytes near the surface of liver or between the central vein and portal area after induction with RU486. In contrast, no IL-12 expression was observed in the hepatocytes after induction with sesame oil. Conclusion Tight temporal and spatial control of transgene IL-12 expression could be achieved by RU486 regulatory system driven by liver specific promoter.
出处 《中华微生物学和免疫学杂志》 CAS CSCD 北大核心 2008年第4期294-298,共5页 Chinese Journal of Microbiology and Immunology
基金 基金项目:国家自然科学基金资助项目(30370547) 教育部新世纪优秀人才支持计划资助(2004) 教育部优秀青年教师资助计划项目(2003)
关键词 RU486调控系统 基因表达 白细胞介素12 水流动力学注射 RU486 regulatory system Gene expression Interleukin-12 Hydrodynamic injection
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参考文献17

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二级参考文献36

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