Expression Of P2X₁ Purinergic Receptor In The Rat Detrusor Of Overactive Bladder Disease

IntroductionOveractive bladder(OAB ) is a new conception that is based on symptoms diagnosis.International Continence Society formally definites the conception of OAB as a syndrome symptoms that hint dysfunction of lower urinary tract.The main performance is urgency,with or without urge incontinence,usually with frequent micturition and nocturia.The most serious one may appear Hydronephrosis and dydroureter because of urine dripping wet,accordingly forming renal dysfunction,even endanger life.OAB may ascribe to uninhibited contraction of detrusor,its disease incidence is very high,and have serious influence on the patients' physical and psychological development,so that decline the quality of life.The cause of OAB is complex,and is not entirely clear at present.Manly related to the neurogenic anatomy and dysfunction of bladder and urethra.Although there are many treatments to cure OAB,at present,It can only alleviate the symptoms,and can't heal.So it's very necessary for a correct treatments and handling of OAB.P2X receptor which was discovered recently is a purinergic receptor gated by ATP ion channel.It is widely distributed in the various organizations of body.Mainly involved in the regulation of contraction and relaxation of smooth muscle.They play an important role in sensory of human bladder and contraction of detusor.However,its specific mechanism in the occurrence and development of OAB is not entirely clear.It's difficult to obtain the tissue samples of human OAB,so in this study,on the basis of establishing the rats model of overactive bladder successfully,through making urodynamic diagnosis,obtain rat bladder tissue of OAB.By RT-PCR and Immunohistochemistry detection methods,observing the expression of P2X₁ receptor in the detrusors of both normal rats and overactive bladder rats.To evaluate the function and role of the P2X₁ receptors in the OAB bladder,and discuss the relationship between P2X₁ receptor and bladder dysfunction.And provide a new theoretical basis for clinical treatment of OAB caused by bladder dysfunction.Experimental materials1.Experimental animals32 Female Wistar rat which is 3-4 month years old and each weight is about 250g, provided by Medical Animal Center in Shengjing Hospital of China Medical University.2.Experimental reagentsTotal RNA separating reagents were purchased from Chinese tiands genetic engineering corporation.RT-PCR kit,DNA polymeras were purchased from TAKARA biotechnology(DALIAN) CO.,LTD.Gene mRNA primers of P2X₁ receptor subtype was the United States National Library of Medline search gene pool purchased by Yingjun Biological Technology Co,Ltd.The specific antibody to P2X₁ receptor antibody which was 1st antibody was purchased from chemicon corporation.The S-P kits for immuhistochmistry were purchased from MaiXin biological limited company, FuJian Province.3.Main experimental instrumentsOphthalmic operating sets and ultraviolet spectrophotometer were purchased from Japan.PCR amplification apparalus was purchased from Germany.Desktop low temperature high speed centrifugal machine,level electrophoresis bath and electrophoresis apparalus were purchased from China.Automatic electrophoresis gelatum image analysis system was purchased from USA.Leica RM2025-based paraffin section machine was purchased from Germany.Nikon optical microscope was purchased from Japan.Galantz medical microwave ovens was purchased from China.Experimental methods1.All 32 rats were observed in experimental study after urodynamic testing,with the result is normal.And divided the experimental rats into normal control group(group A,10 rat)and OABgroup(group B,22 rat).Put all rats of group B into animal experiment.Ligating the junction of bladde rand urethra,and cause the outlets of bladder to obstuct partly.By urodynamic testing again,taking filling detrusor contraction occur without inhibition as standards for the diagnosis of OAB.and divided group B into OAB group(group B2) and model control group(group B1).2.By RT-PCR and Immunohistochemistry detection methods,Observe the expression of P2X₁ receptor in the detrusors of both normal rats and OAB rats. Evaluate the function and role of the P2X₁ receptors in the OAB bladder,and discuss the relationship between P2X₁ receptor and bladder dysfunction.3.All datum were expressed as the means±SD,the t-test was carried out using SPSS 13.0 statistical software,The difference that P＜0.05 has statistical significance.Results1.Results of the animal models and urodynamic testing10 rat in group A were all alive,without emergence of OAB.Its bladder wall was smooth,and the flexibility of its bladder was good.3 rat in 22 of group B died because of urinary tract infection,and the rest 19 rat were alive.By urodynamic testing,Group A shows a smooth curve at filling period,without detrusor contraction.The reflection of detrusor was normal at the voiding period(18.1±4.7cmH₂O ).The largest infusion of the bladder was 0.5 milliliter.52%rat(10 rat in 19) existed OAB(group B2),which appearred to thickening and poor flexibility for the bladder wall,and also appearred irregular uninhibited contracting at filling period of model group.And there was overflow urine in external orifice of urethra as detrusor contracting.Frequency of uninhibited contracting in unit time was 2.9±1.2 times per minute.At voiding period,There was no obvious detrusor reflection.47%rat(9 rat in 19)without OAB(group B1),which appearred smooth and good flexiblity for the bladder wall.and also appearred no obvious detrusor contraction and overflowing.2.Results of RT-PCRThe mRNA expression of P2X₁ receptor was detected in the normal control group, the model control group and also OAB group.The relative expression was as follows respectively:1.805±0.102,1.821±0.068 and 1.968±0.063.The mRNA expression of P2X₁ receptor in the rats detrusor of group B2 was higher than the group A and group B1,and with statistical significance(P＜0.01).There was no significant difference between group A and group B1 in the mRNA expression of P2X₁ receptor,and without statistical significance(P＞0.05).3.Results of ImmunohistochemistryThere was expression of P2X₁ receptor in cytoplasms of bladder detrusor cells in group A,compared group A and group B1,the bladder mucosa and muscle fiber of group B2 was thicken,and its myofilament was disordered.The color of immunopositive granules in the rats detrusor of group B2 was deeply stained,and the amount is much more compared group A and group B1.The brown immunopositive granules in the rats detrusor of group A and group B1 was fewer than group B2.And the color was lighter.There was no obvious difference between group A and group B1.Conclusion1.Lower urinary tract obstruction of rats may do harm to bladder which embraces neural and functional respects.And then induces OAB.Therefore,It can be confirmed that lower urinary tract obstruction is an important stimulus factor to detrusor instability.2.The expression of P2X₁ receptor in the detrusor of OAB rats is higher than normal rats.It tips that P2X₁ receptor may play an important role in the mechanism of OAB bladder systolic and diastolic dysfunction.This is likely related to bladder dysfunction closely.