Investigation Of Relationship Of β₃-AR Gene Trp64ArgSNP And CAMP In Detrusor Cells Existing OAB

ObjectiveNowdays,studies of overactive bladder(OAB) centered on abnormity of detrusor receptor.It is confirmed thatβ₃ adenoreceptor can play a role in relaxation of detrusor cells.By cultivating detrusor cells,Comparatively analyze Trp64ArgSNP mutation rate and genotype ofβ₃-AR between OAB patients and normal control group and compare cAMP expression in OAB patients withβ₃-AR mutation and other control groups after activated by different BRL37344 concentrations to make it clear thatβ₃-AR gene mutation is one of the reasons of incidence of OAB and drug resistance in therapy.Methods1.Cultivating detrusor cellsSelecting 22 patients existing OAB and 20 normal control persons,we cultivate their detrusor cellss respectively.(1)detachmenting initial detrusor cells(2)cultivating initial detrusor cells(3)detrusor cells transfer of culture(4)detrusor cells immunohistochemisty2.Comparatively analyze Trp64ArgSNP gene mutation rate in OAB and control group.(1) extracting DNA(2) sequencing DNA3.Comparatively analyze the relationship ofβ₃-AR Trp64ArgSNP and cAMP expression level.The cells were divided into 3 groups:OAB group withβ₃-AR Trp64ArgSNP,OAB group with wildβ₃-AR and control group with wildβ₃-AR.Add 100μl BRL37344(four concentration—10^-7,10^-8,10^-9,10^-10mol/L) into the 3 group,Disintegrate cells with ultrasonic cell disintegrator(30 seconds×5 times),12000r/min centrifuge at 4℃for 10 minutes,then suck the clear supernatant liquid to use.Detect cAMP according to the description of cAMP radioimmunity kit.Results1.Cultivating detrusor cells(1) the growing form of detachrnenting initial detrusor cells(2) detrusor cells immunohistochemisty2.Comparatively analyze Trp64ArgSNP gene mutation rate in OAB and control groupIn 22 patients of OAB group,12 patients hadβ₃-AR Trp64ArgSNP,the mutation rate was 55%.In 20 patients of control group,4 hadβ₃-AR Trp64ArgSNP,the mutation rate was 20%.The mutation rate difference of the two groups was prominently different(X²=5.30,P＜0.05).3.Relationship betweenβ₃-AR Trp64ArgSNP and cAMP expression level.cAMP levels in detrusor cellss of OAB group withβ₃-AR Trp64ArgSNP,OAB group with wildβ₃-AR and control group with wildβ₃-AR,were dosage depended ascending under the action of BRL37344(four concentration—10^-7,10^-8,10^-9,10^-10mol/L).The difference has statitically significance by q statistical test(P＜0.01). The maximal effect of BRL37344 was at 10^-7mol/L.At different concentration of BRL37344(10^-7,10^-8,10^-9,10^-10mol/L),we analyzed the cAMP levels produced by Trp64ArgSNPβ₃-AR detrusor cells in OAB group,wildβ₃-AR detrusor cells and wildβ₃-AR detrusor cells in control group,the results showed that the cAMP level was highest in wildβ₃-AR detrusor cells in control group,and the cAMP level was lowest in Trp64ArgSNPβ₃-AR detrusor cells in OAB group,by q statistical test(P＜0.01).Conclusion1.Trp64ArgSNP mutation rate in OAB group was higher,and in different concentration of BRL37344,cAMP expression levels inβ₃-AR and wild typeβ₃-AR detrusor cells of OAB group with Trp64ArgSNP were lower than wild typeβ₃-AR detrusor cells in control group.In OAB patients,functional impairment ofβ₃-AR in relaxation of detrusor muscles was related toβ₃-AR subtype Trp64ArgSNP.2.The tendency of BRL37344 concentration depended cAMP expression ascending inβ₃-AR detrusor cells with Trp64ArgSNP mutation was lower than in wild typeβ₃-AR detrusor cells in OAB group.Trp64ArgSNP mutation is the reason of poor efficacy when treated with adenorecepter agonist in some OAB patients.