乙型肝炎病毒前S2蛋白下调胰岛素受体启动子的研究

Down-regulating effect of hepatitis B virus pre-S2 protein upon insulin receptor gene promoter

目的探讨乙型肝炎病毒（HBV）前s2蛋白对胰岛素受体（INSR）启动子转录的下调作用。方法HBV前s2蛋白真核表达质粒pcDNA3．1（-）-preS2及INSR启动子真核报告质粒pCAT3-INSRp使用常规分子生物学方法进行构建，并经酶切、测序证实。接下来，将构建好的pcDNA3．1（-）-preS2质粒以1．0、1．5、2．0μg转染HepG2细胞，提取总RNA后进行相对实时荧光定量PCR检测以明确前S2蛋白mRNA的表达量。然后，分别将pCAT3-INSRp质粒以0．2μg递增量（0～2．0μg）转染HepG2、Huh7细胞系，CAT-ELISA法检测转染细胞的氯霉素乙酰转移酶（CAT）表达量以制作pCAT3-INSRp的量-效曲线。最后，根据量一效曲线选择合适的pCAT3-INSRp转染剂量（1．0μg）与不同剂量的pcDNA3．1（-）-preS2（1．0、1．5、2．0、2．5、3．0μg）共转染HepG2及Huh7细胞，同时使用抗前S2蛋白单克隆抗体以阻断前S2蛋白的作用，使用CAT-ELISA方法检测转染细胞的CAT表达量，以观察前S2蛋白对于INSR启动子的调节作用。结果转染了pcDNA3．1（-）-preS2的细胞可以正确表达HBV前s2蛋白的mRNA。pCAT3-INSRp质粒转染细胞后CAT的表达随着转染剂量的增加而增加，量-效曲线为S形。转染不同剂量pcDNA3．1（-）-preS2后，HepG2细胞内CAT基因的表达水平分别为对照的69．8％、60．1％、19．7％、10．3％、5．6％（转染后36h）和68．6％、56．O％、10．3％、8．6％、3．2％（转染后72h）。同时，在转染2．0txgpcDNA3．1（-）-preS2的细胞培养上清中加入前S2单抗后，其CAT的表达量恢复为对照的55．4％（36h）和69．7％（72h）。Huh7细胞的结果同HepG2细胞结果相似。结论克隆的INSR启动子有顺式激活下游基因的活性；HBV的前S2蛋白对INSR启动子转录具有下调作用，可以在分子水平解释肝源性糖尿病发病机制的一部分。

Objective To investigate the down-regulating effect of HBV pre-S2 protein upon insulin receptor （INSR） gene promoter. Methods Eukaryotic expression plasmid pcDNA3.1 （ -）-preS2 and report plasmid pCAT3-INSRp containing INSR gene promoter upstream of CAT gene were constructed with routine molecular biological methods and were confirmed by endonuclease digestion analysis and sequencing. Then HepG2 cells were transfected with pcDNA3. 1 （ - ） -preS2 plasmid and the total RNAs were examined with relative quantitative real-time PCR to confirm the expression of HBV pre-S2 protein. Later HepG2 and Huh7 cells were transfected with pCAT3-INSRp at various doses （0-2. 0 μg ）. The choloraphenical acetyhransferase （CAT） activity of transfected cells was detected with CAT-ELISA to generate the dose-effect curve of pCAT3-INSRp. Lastly pCAT3-INSRp （ 1.0 μg） and pcDNA3.1 （ - ）- preS2 （ 1.0, 1.5, 2. 0, 2. 5, 3.0 μg） were co-transfected into HepG2 and Huh7 cells respectively to study the regulation effect of pre-S2 protein upon INSR promoter. Meanwhile pre-S2 monoelonal antibody was added into additional cells transfected with 2. 0 μg of pcDNA3.1 （ - ） -preS2 plasmid to evaluate the effect when pre-S2 protein was blocked. Results The mRNA of pre-S2 protein could be detected with realtime PCR indicating that pre-S2 protein was properly expressed in pcDNA3. 1 （ - ） -preS2-transfected ceils. The expression of CAT increased proportionally with the incremental doses of pCAT3-INSRp. It suggested that the INSR gene promoter had its transcription activity. After eo-transfection of pCAT3-INSRp and pcDNA3.1 （ - ）-preS2, the CAT expression in HepG2 cells, comparing with that of controls, were 69. 8%, 60. 1%, 19.7%, 10.3%, 5.6% （36 h） and 68.6%, 56.0%, 10.3%, 8.6%, 3.2% （72 h） respectively. When pre-S2 monoclonal antibody was added into the supernatant of HepG2 cells transfected with 2. 0 μg of pcDNA3.1（ - ）-preS2, the CAT expression was partly restored to 55.4% （36 h）and 69.7% （72 h）of controls. The similar results were observed in Huh7 cells. Conclusion The pre-S2 protein down-regulates the activity of INSR gene promoter so as to reduce the expression of INSR. It may partly elucidate the pathogenesis of hepatogenous diabetes at the molecular level.