Culture Of Single Gene Of β₃-AR From Human Detrusor Cell And Intervention Experiment

ObjectiveHuman urinary bladder detrusor muscle hyperactivity is one of the common causes of inferior urinary tract syndromes, which rnanisfested urinary frequency, urgency and uroclepsia in clinical. Recent reports showed thatβ-adrenoceptor subtype revealed a relaxant effect on the human urinary bladder detrusor and played a key role in urine storage in the human urinary bladder. However, researchers still disputed which sudtype acted. To lay the foundation of further research of the function ofβ-adrenoceptor subtype, we applied RNA interference technology to knockdown two subtypes ofβ-AR to obtain a clone expressing single subtype ofβ-AR, based on which we will do further research to confirm the effect of activeβ-AR on the relaxation and proliferation of the human detrusor muscle cells. The result may help us to find new treatment of the human urinary bladder detrusor muscle hyperactivity.To study the signal transduction pathways of isoprenaline on primary cultured human detrusor cells.Materials and MethodsRNAi nterference pGCsi system was used to constructβ_1-AR andβ_2-AR small interfering RNAs (siRNAs) sxpressing vector: 1.oliigo insert design and synthesis. 2. anneal oligos. 3. linearize the vector. 4. ligation into pGCsi vector. 5. transformation In bacteria. Transfaction of mammalian cells We used lipided-mediated transfection. identification of a stably expressing clone Reverse transcription-polymerase chain reaction and western blot analysis were used to detectβ-AR expression of transfected cells.After the isoprenaline which both can increase the cAMP level of cells,the level of cAMP were measured by immunoassay KIT. Four group of isoprenaline (10^-10-10(-7)mol/L) were added to the primary cultured human detrusor cells with fluo3 in order to determine the changes of fluorescence.RESULTS1. The sequence of plasmid was conformity to that of our predesigned by sequencing, and the DNA oligos of dsRNA was inserted into the pGCsi vector.2. The result of plasmid tranfection analysed by RT-PCR assay: Analysis by RT-PCR assay, the expression of densitometric scan were both lower in dsRNA plasmid (containingβ₁-AR orβ₂-AR dsRNA dsRNA) tranfected cells than that of pGCsi vector transfected cells, whereas the expression of GAPDH mRNA was equally. Analysised by densitometric scan, the inhibition rate of expression of pGC-β₁-AR or pGC-β₂-AR plasmid transfected cells were 70% and 75% respectively, compared with pGCsi vector transfected cells.3. The result ofβ₁-AR or -β₂-AR gene translation after plasmid transfection determined by westwern blot assay: The expression of PC21 protein was analysised by western blot. We found that the expression of PC21 was significantly inhibited in pGC-β₁-ARi plasmid transfected cells compare with compared with pGCsi vector transfected cells, whereas the expression ofβ₂-actin was no difference. That indicated that RNAi specificly inhibited expression of PC21 protein. Analysised by densitometric scan, we found that the inhibitor rate of PC21 protein by pGC-β₁-ARi or pGC-β₂-ARi were 81%å’Œand 85%.4. various concentrations of isoprenaline (10^-10～10^-7mol/L) increased the cAMP production in a dose dependent manner in mesenteric. The effects of adrenomedullin for increasing cAMP on the smooth muscle cell showed concent ration independence. The four kinds of isoprenaline all could reduce the concent ration of fluorescence compared with controls. The concent ration of fluorescence was decreased obviously with L-arginine to the minimal value. Adrenomedullin affected signal transduction of vascular endothelial cells by influcing cAMP of mobilization. Singal gene clone ofβ₃-AR from human detrusor product the more increasing of cAMP in contrast withβ₁-AR and -β₂-AR.ConcluionWith the above actions isoprenaline can exert many bioiogical actions on primary cultured human detrusor cells,β₃-AR may play a more important role on relaxation of human detrusor among threeβ-adrenergic acceptor.