材料与方法   Material and Method

1. 材料Experimental material

1.1 实验动物 Experimental animals

健康成年雌性清洁级SD大鼠（10-12w，体重260±20g，共24只），购自实验动物中心，分笼饲养，自由饮水。

Healthy and apinoid female SD large rat(10-12W, weight 260±20g,total 24), purchased from experimental animal center, divided cage rearing and free water.

1.2 主要试剂  Main Reagent

LPS（Sigma，美国），HID-AB-PAS染色试剂盒（Baso，珠海），抗大鼠COX-2（1:200，Santa Cruz，美国），抗大鼠ERK1 （1:200，Santa Cruz，美国），抗大鼠Stat3（1:200，Santa Cruz，美国），抗大鼠TNF-α（1:200，Abcam，英国），生物素化山羊抗兔IgG （1:200，中杉金桥）， DAB显色试剂盒（博士德，武汉），TIANScript RT试剂盒（TIANGEN，北京），总RNA提取试剂盒（TIANGEN，北京），SYBR 溶液（TIANGEN，北京）。

LPS（Sigma，US）, HID-AB-PAS Staining Kit(Baso, Zhuhai), In rats COX-2（1:200, Santa Cruz, US）, In  rats ERK1 （1:200, Santa Cruz, US）, In  rats Stat3 （1:200, Santa Cruz, US）,In Rats TNF-α（1:200，Abcam，UK, Biotinylated  Goat anti rabbit IGG （1:200，Zhongshanjinqiao）, DAB Chromogenic reagent kit(Boshide, Wuhan), TIANScript RT Reagent kit （TIANGEN，Beijing）, General RNA extraction kit （TIANGEN，Beijing）, SYBR solution（TIANGEN，Beijing）.

1.3 主要仪器 Main Equipment

PCR仪（Life Express，BIOER，杭州），电泳仪（DYY-7C，六一，北京），荧光定量PCR仪（Exicycler 96，BIONEER，韩国）。

PCR apparatus(Life Express,Bioer, Hangzhou), Electrophoresis apparatus( DYY-7C , Liuyi, Beijing), Fluorescence quantitative apparatus （Exicycler 96，BIONEER，Korea）.

2. 方法  Method

2.1 动物模型的建立 Animal Model Creating.

取健康清洁系SD雌性大鼠24只，适应性饲养后，随机分为3组（正常对照组4只，生理盐水对照组10只，脂多糖组10只）。各组大鼠每隔2d导尿后，脂多糖组膀胱内灌注脂多糖0.2ml(浓度为1g/L)，生理盐水对照组膀胱内灌注同等剂量生理盐水，正常对照组不做处理。膀胱内灌注方法:用10%水合氯醛麻醉，将大鼠仰卧位固定，常规消毒会阴部。将成人麻醉用硬膜外导管用石蜡油润滑后，经大鼠尿道外口向膀胱内推进4～5cm，当有尿液滴出后导尿，再用1ml注射器注入LPS或无菌生理盐水，闭合导管防止液体流出。3组分别常规饲养8w。

Choose 24 healthy and clean female rats, divide them into 3 groups randomly (4 for normal control group, saline control group and lipopolysaccharide group)after adaptive feeding. After catheterization per 2d, inject lipopolysaccharide into rats’ bladder of lipopolysaccharide group0.2ml(density is 1g/L), same dosage of saline injected to the saline control group, keep the normal control group same. Method for injecting into bladder: 10% water mixed with chloral for anesthesia, make rat to lie face up and fix them, conventional disinfect rats’ pudendum. Pick an anesthesia catheter for audit and lubricate the catheter with Paraffin oil, propel it into bladder 4-5cm through rats’ urethra, carry catheterization after urine dropped, use 1ml syringe to inject LPS or sterile saline water, close the catheter to avoid liquid running out.

2.2 膀胱标本处理及染色 Bladder Specimen Dealing and Dyeing

每组大鼠达到饲养时间后，将大鼠处死，取出膀胱，观察膀胱三角区，中性甲醛固定12h，连续切片后常规制片，行HE染色。

Put rats into death after get to feeding time, take bladder out, watch what’s going on of bladder triangular area, put it into neutral formaldehyde for 12 hours, make into pieces after serial sections, dye it with HE.