构建消除糖皮质激素抵抗新靶点的BAG-1质粒表达载体

BAG-1 plasmid expression vector:A new target can reverse glucocorticoid resistance

目的:利用RNA干扰技术,建立脂多糖和地塞米松作用大鼠肺泡巨噬细胞BAG-1基因阻断模型,并阐明BAG-1表达对糖皮质激素受体抗炎活性的影响。方法:本实验于2006-02/09在南京军区南京总医院呼吸病实验室进行。选用健康SPF级Wistar大鼠10只,鼠龄四五周,雌雄不拘。采用支气管肺泡灌洗获取大鼠肺泡巨噬细胞,将分离培养得到的大鼠肺泡巨噬细胞随机分为4组:正常对照组、阴性对照组、SiBAG-1α转染组及SiBAG-1β转染组。通过在线寻找针对BAG-1基因的RNA干扰靶序列并设计对应的寡核苷酸序列,将退火的合成寡核苷酸序列插入pSilencer3.1H1-hygro质粒载体,构建RNA干扰质粒SiBAG-1α和SiBAG-1β,经测序确认后,转染大鼠肺泡巨噬细胞,阴性对照组是试剂盒提供的阴性对照质粒pSilencer3.1H1-negetive。运用细胞免疫组织化学法检测各组细胞核中BAG-1表达变化,以胞核中积分吸光度平均值表示BAG-1表达的相对强度;运用电泳迁移率改变分析法检测各组糖皮质激素受体活性变化,以扫描条带的灰度值与滞后带面积之积表示糖皮质激素受体的活性。结果:①合成的寡核苷酸片段正确连入pSilencer3.1H1-hygro质粒载体,成功构建针对BAG-1基因的RNA干扰重组质粒SiBAG-1α和SiBAG-1β(上海博亚公司测序号分别为1524-036和1524-037)。②质粒SiBAG-1α转染组胞核内BAG-1蛋白表达较正常对照组明显降低(3.46±0.75,13.58±0.47,P<0.05),而质粒SiBAG-1β转染组胞核内BAG-1蛋白表达与正常对照组比较差异无显著性意义(15.27±0.89,13.58±0.47,P>0.05)。③质粒SiBAG-1α转染组细胞糖皮质激素受体活性与正常对照组明显增强(9.62±0.47,2.65±0.22,P<0.05),质粒SiBAG-1β转染组细胞糖皮质激素受体活性与正常对照组比较差异无显著性意义(2.79±0.57,2.65±0.22,P>0.05)。结论:成功地构建并筛选出一个特异而高效地阻断BAG-1表达的质粒表达载体SiBAG-1α,抑制BAG-1表达可恢复糖皮质激素受体抗炎活性,逆转糖皮质激素受体活性下降所致的糖皮质激素抵抗。BAG-1是消除糖皮质激素抵抗的新靶点。

AIM： To establish a BAG-1 gene knockdown model of rat alveolar macrophage treated by lipopolysaccharide （LPS） and Dexamethasone （Dex） with RNA interference （RNAi） technique, and elucidate the effects of BAG-1 expression on anti-inflammatory activity of glucocorticoid receptor （GR）. METHODS： The experiment was carried out in the Laboratory of Respiration Diseases of Nanjing General Hospital of Nanjing Military Area Command of Chinese PLA from February to September in 2006. Ten Wistar rats of SPF grade were adopted, aged 4-5 weeks and either gender. All cultured alveolar macrophages were isolated with bronchoalveolar lavage, and randomly divided into normal control group, negative control group, SiBAG-1β trensfection group and SiBAG-1β transfection group. Targeted sequences of BAG-1 gene by RNAi were screened on line and corresponding nucleotide sequences were inserted into pSilencer3.1Hl-hygro vector. Two recombinant RNAi expression plasmids targeting to BAG-1 gene （named SiBAG-1α and SiBAG-1β） were constructed, and confirmed by sequencing prior to trensfecting rat alveolar macrophages. The expression changes of BAG-1 protein in nucleu were evaluated with immunocytochemistry technique, and integral absorptances in nucleus region indicated relative intensity of BAG-1 expression; Simultaneously GR activity was evaluated through method of electrophoretic mobility shift assay （EMSA）, and was demonstrated by product of multiplication of gray scale and areas of scanned band. RESULTS： ①Plasmids SiBAG-1α and SiBAG-1β （sequence of Shanghai Boya Company were 1524-036 and 1524-037, respectively） were successfully constructed and identified by inserting the synthesized oligonucleotide fragment to psilencer3.1Hl-hygro.②Compared with normal control group, BAG-1 protein expression in nuclear protein of SiBAG-1α trensfection group were significantly decreased （03.46±0.75, 13.58±0.47, P 〈 0.05）, but no difference was found in SiBAG-1β trensfection group （15.27±0.89, 13.58±0.47, P 〉 0.05）. ③Similar results were the increased GR activity in SiBAG-1α trensfection group compared with normal control group （9.62±0.47, 2.65±0.22, P 〈 0.05）, but no difference to SiBAG-1β trensfection group （2.79±0.57, 2.65±0.22, P 〉 0.05）. CONCLUSION： A recombinant RNAi expression plasmids SiBAG-1α, which can knockdown BAG-1 gene specially and efficiently, is successfully constructed. Repressing BAG-1 expression can bring the recovery of anti-inflammatory activity of GR, which can reverse glucocorticoid resistance induced by the decrease of GR activity. BAG-1 is a new target of reversing glucocorticoid resistance.