脂多糖刺激下的人肺微血管内皮细胞血管紧张素Ⅱ受体1放射受体分析法的建立

Establishment of radioligand binding assay for angiotensin Ⅱ type 1 receptor of human pulmonary microvascular endothelial cell after lipopolysaccharide stimulation

目的建立一种脂多糖（LPS）刺激下人肺微血管内皮细胞（HPMEC）血管紧张素Ⅱ（AngⅡ）1型受体（ATR1）与其配体AngⅡ结合的放射配体结合分析法（RLBA）。方法体外培养的HPMEC接种于24孔培养板,当细胞达到80%-90%融合,饥饿24 h后用浓度为100 ng/ml的LPS刺激8 h,用0.025、0.05、0.1、0.2、0.4、0.8、1.62、.0 fmol 8个不同剂量的放射性配体125I-AngⅡ（TL表示其放射性）在板孔中与ATR1结合（存在或不存在5μg AngⅡ）,然后γ计数仪计数,测定ATR1的总结合（TB）与非特异性结合（NSB）,TL减去TB得游离放射配体（F）,TB减去NSB得ATR1的特异性结合（B）。由TB与TL作图得ATR1饱和曲线;由B/F与B作图得出Scatchart曲线。结果随放射性配体125I-AngⅡ加入量的增加,NSB也相应逐渐增加,且由直线相关与回归分析得到NSB与加入125I-AngⅡ剂量呈直线关系（r2=0.9929）;Scatchart作图得B/F与B也呈直线关系,其相关系数r2=0.9514。HPMEC上仅存在单一亲和力的AngⅡ受体,反应亲和力的Kd值为109 pmol/5×10^5细胞,从Scatchart曲线与X轴截距得到ATR1的最大结合位点Bmax为29 pmol/5×10^5细胞;ATR1饱和曲线分为两段,前段斜率较大,随125I-AngⅡ加入量的增加TB增加明显;后端曲线较为平坦,TB随125I-AngⅡ的增加变化不明显,125I-AngⅡ在1600 pmol/5×10^5细胞与2000 pmol/5×10^5细胞之间TB的增加与NBS增加基本相当,能使ATR1饱和的125I-AngⅡ剂量为1600 pmol/5×10^5细胞。结论本实验建立的RLBA方法能得到ATR1的Kd与Bmax两个基本参数,可用于LPS刺激下HPMEC的ATR1功能研究。

Objective To establish radioligand binding assay（RLBA） for angiotensinⅡ（AngⅡ） type 1 receptor（ATR1） of human pulmonary microvascular endothelial cell（HPMEC） after lipopolysaccharide（LPS） stimulation.Methods HPMEC cultured in vitro was seeded and culture in 24-well plate.After 80%-90% cell confluency,cell was cultured in the BSA free medium for 24 hours.Then LPS（100 ng/ml） stimulated the cell for eight hours.Subsequently 125I-AngⅡ was added to each well at the dose of 0.025,0.05,0.1,0.2,0.4,0.8,1.6,2.0 fmol/well（TL）.Cell was incubated with 0.5 ml reaction buffer in the absence or presence of unlabeled AngⅡ to determine the total（TB） and nonspecific（NSB） ATR1 binding through γ-counter.Free 125I-AngⅡ（F） was obtained from TL by subtracting TB and specific binding（B） was obtained from TB by subtracting NSB.At last saturation curve and Scatchart graph was drawn.ResultsLinear correlation and regression analysis confirmed that NSB and TL had linear correlation（r2=0.9929）.NSB was increased following the increasing dose of 125I-AngⅡ each well.Scatchart graph,linear correlation coefficient r2 was 0.9514,showed that ATR1 maximum binding（Bmax） values was 29 pmol/5×105 cell and dissociation constants（Kd） was 109 pmol/5×10^5 cell.From the linear correlation,there was just solo affinity AngⅡ receptor.Saturation curve showed that the dose of 1600 pmol/5×10^5 cell 125I-AngⅡ can bring the ATR1 saturation,because the increasing 125I-AngⅡ from 1600 pmol/5×10^5 cell to 2000 pmol/5×10^5 cell caused the increase of TL about equal degree as the NSB increased and the B increase was weak.Conclusions The establishment of ATR1 RLBA method meets the request of experiment and is qualified for the function research of HPMEC ATR1 after the LPS stimulation.