建立小鼠膀胱肿瘤原位模型的优化方法

Optimization for establishment of mouse models of orthotopic bladder tumors

目的探讨硝酸银、盐酸、胰酶和乙醇预处理构建鼠膀胱肿瘤的成瘤机制。方法 24G静脉留置针插入膀胱,PBS冲洗后,将小鼠随机分为5组,每组6只:(1)乙醇作用组:22%乙醇0.1 mL保留20 min;(2)胰酶作用组:0.2%胰酶保留30 min;(3)酸碱作用组:0.1 mmol/L HCl 0.1 mL作用15 s后,PBS冲洗,0.1 mmol/L NaOH0.1 mL作用5 s,排空膀胱;(4)硝酸银作用组:0.15 mol/L硝酸银保留10 s;(5)对照组:0.1 mL生理盐水。术后1和24h随机处死每组3只小鼠,摘取膀胱,HE染色观察膀胱黏膜病理变化;戊二醛固定,电镜下观察膀胱黏膜细胞微结构变化;甲苯胺蓝染色,观察膀胱黏膜固有层肥大细胞数目变化;过碘酸-希夫(PAS)染色,观察膀胱黏膜GAG层变化。40只小鼠应用上述前四组预处理因素处理膀胱后,建立膀胱癌原位模型,计算各组成瘤率。结果胰酶和乙醇处理1h后,局部上皮伞状细胞脱落,黏膜下层暴露;酸碱和硝酸银处理组大部黏膜完整性破坏,黏膜下层暴露较多,连续性中断;对照组和实验组间炎症细胞浸润均不表现出统计学差异。24 h后,胰酶和乙醇组可见局部轻度水肿并充血,黏膜完整性恢复较好,细胞间见紧密连接;而酸碱和硝酸银组上皮黏膜薄厚不均一,仍可见部分脱落黏膜。结论利用硝酸银和酸碱预处理膀胱可作为鼠膀胱肿瘤原位模型构建的首选方法。

Objective To assess the efficacy of establishment of mouse models of orthotopic bladder tumor in- duced by pretreatment with silver nitrate, HCI, trypsin and ethanol. Methods Seventy 8- to 10-week old female C57BL/ 6 mice were anesthetized and catheterized with 24G i.v. catheter, then divided into 5 groups （n = 6 in each group） after washing the bladder with phosphate-buffered saline （PBS）. （ 1 ） Ethanol group ： incubation with 22% ethanol for 20 rain ; （2） Trypsin group： incubation with 0. 2% trypsin for 30 min; （3） HC1 group： pretreated with 0. 1 mmol/L HCI for 15 s and 0. 1 mmol/L NaOH for 5 s after washing the bladders with PBS; （4） Silver nitrate group： incubation with 0. 15 mol/ L silver nitrate for 10 s; （5） Vehicle group： normal saline. The mice were killed to obtain bladder tissue samples after 1 or 24 hours pretreatment. Pathological changes were observed using hematoxylin and eosin staining. The mieroenvironment modification was assessed by electron microscopy. Mast cells were counted with toluidine blue staining. The glycosamin- oglycan （GAG） layer was observed by periodic acid-Sehiff staining. Forty mice were intravesieally pretreated with the a- bove four reagents, respectively, then incubated with MB49 tumor cells for 2 hours, and the rate of tumor formation was de- termined. Results In the model mice, a part of umbrella cells were sloughed off, the submueosal layer was exposed and incontinuous in some areas at one hour after pretreatment with trypsin and ethanol. Mucosal damages were more serious in the HC1 and silver nitrate groups. However, there was no significant difference in the inflammatory cell infiltration between the experiment and control groups. Mild edema and congestion in the mucosa, tightly arranged epithelial cells, and contin- uous mucosa were observed in the mice after trypsin and ethanol pretreatment for 24 hours. By contrast, the thickness of mucosa was uneven and incomplete mueosa in some areas were still observed in the mice 24 hours after silver nitrate and HCL and NaOH pretreatment. The mice were sacrificed at 50 clays after tumor induction and the tumor formation rate was 100% in the mice pretreated with HC1, NaOH, and silver nitrate, respectively, 90% in the mice pretreated with tripsin, and 80% in the mice pretreated with ethanol （n = 10 in each group）. Conclusions Pretreatment with silver nitrate and HC1, NaOH cause more serious damage in the bladder mueosa and may be regarded as an optimal choice in establishing or- thotopic bladder tumor models in mice.