小鼠膀胱癌气囊模型的构建与效果评估

Establishment of air-pouch bladder cancer model and investigation of its feasibility for evaluating the effect of intravesical therapy

目的构建一类肿瘤可视化的小鼠膀胱癌气囊(APBCa)模型.并评估药物灌注疗效。方法本研究于2019年6-12月构建人源性肿瘤APBCa(H-APBCa)模型和具有免疫功能的APBCa(I-APBCa)模型。在BALB/c裸鼠背部皮下注射无菌空气建立大小为2.5 cm×3.5 cm的气囊,24 h后在气囊内壁通过种植入膀胱癌细胞EJ建立H-APBCa模型。对比灌注吉西他滨与生理盐水20d后H-APBCa小鼠模型肿瘤的体积,并利用Tunel染色法比较灌注吉西他滨与生理盐水20d后肿瘤细胞凋亡情况。在C57BL/6小鼠背部皮下注射无菌空气建立大小为2.5 cm×3.5 cm气囊,24 h后在气囊内壁通过种植小鼠膀胱癌细胞MB49建立I-APBCa模型。比较灌注卡介苗与生理盐水20d后1-APBCa小鼠模型肿瘤的体积,并用免疫荧光染色法观察灌注卡介苗与生理盐水20d后两组肿瘤组织CD80^+巨噬细胞及CD3^+T细胞浸润情况。结果H-APBCa和I-APBCa两种小鼠模型均成功建立,肉眼能直视观察且可通过游标卡尺测量肿瘤大小。H-APBCa小鼠模型灌注治疗20d,灌注吉西他滨组肿瘤体积小于灌注生理盐水组[(781.02±241.02)mm^3与(1213.88±214.02)mm^3,P<0.05]。灌注吉西他滨组小鼠肿瘤细胞凋亡多于灌注生理盐水组(77.33±4.63与14.67±2.60,P<0.05)。H-APBCa模型灌注治疗20d,灌注卡介苗组肿瘤体积小于灌注生理盐水组[(645.02±156.63)mm^3与(948.84±221.76)mm^3,P<0.05];灌注卡介苗组肿瘤组织浸润的CD80±巨噬细胞多于灌注生理盐水组(49.67±7.57与16.33±5.69,P<0.05),肿瘤组织浸润的CD3^+T细胞也多于灌注生理盐水组(18.00±3.46与4.67±1.45,P<0.05)。结论H-APBCa和I-APBCa两种小鼠模型均成功建立。APBCa模型化疗以及卡介苗灌注治疗后肿瘤均明显缩小,与临床中膀胱癌灌注治疗效果相似,可作为评价药物灌注疗效的新型动物模型。

Objective To establish an air-pouch bladder cancer(APBCa)model and investigate whether it could be a new animal model to evaluate the efficacy of intravesical therapy through chemotherapeutics and BCG instillation.Methods Filtered sterile air was injected subcutaneously into the backs of BALB/c-Nude mice to create a 2.5 cm×3.5 cm air pouch.After 24 hours,human bladder cancer cells EJ were seeded on the inner face of the pouch wall to establish APBCa of human cancer(H-APBCa).Gemcitabine instillation was used to investigate whether chemotherapy could inhibit tumor growth in the H-APBCa model,and Tunel staining was used to verify the apoptosis of tumor cells 20-day treatment.Filtered sterile air was injected sulicutaneously into the backs of C57BL/6 mice to create a 2.5 cm×3.5 cm air pouch.After 24 hours,mice bladder cancer cells MB49 were seeded on the inner face of the pouc h wall to establish APBCa with intact immunity(I-APBCa).BCG instillation was used to investigate whether BCG could inhibit tumor growth in the APBCa model.Immunofluorescence was used to verify the infiltration of immune cells after 20-day treatment.Results H-APBCa and I-APBCa mice models could be established by immune deficiency and intact mice.At day 20,chemotherapeutic instillation therapy could inhibit tumor growth(781.02±241.02 vs.1213.88±214.02 mm^3,P<0.05)by inducing tumor cell apoptosis with statistically significant differences(77.33±4.63 vs.14.67±2.60,P<0.05).BCG instillation was able to inhibit tumor growth(645.02±156.63 vs.948.84±221.76,P<0.05)hy increasing CD80^+macrophage(49.67±7.57 vs.16.33±5.69,P<0.05)and T cells in the tumor with statistically significant differences(18.00±3.46 vs.4.67±1.45,P<0.05).Conclusions APBCa model could evaluate the efficacy of ding instillation and was expected to be a new animal model for studying drug for intravesical therapy.