敲减人MCHR2基因抑制CHO细胞的放射性配体受体结合能力及Ca^2＋释放

Down-regulate Human MCHR2 Expression Inhibites the Receptor Binding to Its Radioligands and Reduces Calcium Release in CHO Cells

研究人黑色素浓集激素受体2（MCHR2）基因特异的小发夹RNA（shRNA）真核表达载体pGenesil-1-MCHR2-shRNA对MCHR2表达及特征的影响．将pGenesil-1-MCHR2-shRNA转染到稳定表达人MCHR2基因的CHO细胞中，通过RT-PCR和Western印迹检测MCHR2表达的变化；放射性配体结合实验（RBA）检测受体最大结合容量Bmax及平衡解离常数Kd值的变化；钙流检测实验观察配体MCH刺激后单个细胞Ca^2＋释放及MCH半数有效浓度EC50的变化,与转染pGenesil-1空载体组比较，pGenesil-l-MCHR2-shRNA能使MCHR2基因mRNA表达减少45．8％～66．4％；蛋白表达减少44．2％～81．0％；Bmax减少39．4％～78．7％，Kd值升高40．9％～81．9％；EC50升高114．8％～822．4％．MCHR2基因shRNA真核表达载体能有效抑制MCHR2基因的表达，减少Bmax、升高虬值及EC50，从而对MCHR2生物活性等特征产生影响。

To investigate and characterize the effects of the transfection of shRNA eukaryotic expression vectors specifically targeted to human melanin-concentrating hormone receptor 2 （MCHR2）, we introduced pGenesil- 1-MCHR2-shRNAs into a CHO cell line that stably expresses human MCHR2. The levels of MCHR2 expression were determined by RT-PCR and Western blotting, the maximum binding （ Bmax ） and dissociation constant（ Kd ）of MCHR2 were determined by radioligand binding assays（RBA）, and the EC50 of MCH and the intracellular Ca^2＋ stimulated by MCH were determined by calcium influx assays in single cells. Compared with the pGenesil-1 blank vector transfected group, the MCHR2 transcripts were reduced by 45.8 % -66.4% and the MCHR2 proteins were reduced by 44.2% - 81.0% using four different transfectants. The observed Bmax values of MCHR2 were decreased by 39.4% ～ 78.7%, whereas the Ka and EC50 values were increased by 40.9% - 81.9% and 114.8% - 822.4% respectively.