【摘要】 目的 建立苯并[a]芘（Ba P）恶性转化人支气管上皮细胞T-16HBE-C1（THBEc1）的裸小鼠原位肺癌模型，评估血清人源神经纤维网蛋白2（NRP2）对该模型的诊断价值。方法 12只雄性ICR小鼠随机分为3组，右肺分别一次性注射无菌生理盐水20,30和40μL，观察14 d内小鼠死亡数，以确定小鼠肺内注射安全体积。16只雄性BALB/c-nu裸小鼠随机分为4组：对照组[皮下和肺内同时一次性接种人支气管上皮16HBE（HBE）细胞，皮下1×10⁶，肺内2×10⁵]、皮下荷瘤模型组（皮下一次性接种THBEc1细胞1×10⁶）、原位肺癌模型组（肺内一次性接种THBEc1细胞2×10⁵或5×10⁵）。接种后每7 d监测小鼠体重和皮下肿瘤体积；HE染色检测肺肿瘤和皮下肿瘤组织病理特征，计算成瘤率；酶联免疫吸附实验（ELISA）检测裸小鼠血清和胸水中人源NRP2水平；采用受试者工作特征（ROC）曲线确定血清人源NRP2对THBEc1细胞导致的裸小鼠原位肺癌模型的诊断价值。结果 肺内注射40μL无菌生理盐水可致2/4小鼠死亡，20和30μL组未见小鼠死亡，但整体表现欠佳，故后续实验注射体积选择20μL。对照组裸小鼠均未在接种部位检出肿瘤，小鼠体重持续增长；皮下荷瘤模型组祼小鼠全部成瘤，体重显著低于对照组（P<0.05）。接种细胞后第10天，原位肺癌模型5×10⁵细胞组裸小鼠全部成瘤（4/4）；第14天，原位肺癌模型2×10⁵细胞组裸小鼠3/4成瘤，且2个剂量组小鼠体重均低于对照组（P<0.01,P<0.05），肿瘤组织均呈鳞状细胞癌特征。ELISA结果显示，对照组裸小鼠血清人源NRP2水平均显著低于皮下荷瘤模型（4只）和原位肺癌模型（7只）裸小鼠（P<0.05）。原位肺癌模型组（4只）裸小鼠胸水中均可检出高水平的人源NRP2。ROC曲线显示，血清人源NRP2水平能有效区分THBEc1细胞在裸小鼠肺内是否成瘤，最佳截断值为123.00 ng·L^-1。结论 通过肺内注射接种THBEc1细胞建立了裸小鼠原位肺癌模型，血清人源NRP2可用于该模型诊断。更多还原

【Abstract】 OBJECTIVE To establish an orthotopic lung cancer model in BALB/c-nu nude mice caused by benzo[a]pyrene-transformed human bronchial epithelial cell T-16HBE-C1（THBEc1） and to assess the potential diagnostic value of serum human-derived neuropilin-2 （NRP2）.METHODS Twelve male ICR mice were randomly divided into three groups and the right lungs were injected with20,30 and 40μL of sterile normal saline in a single injection.The safe volume of intrapulmonary injection was determined by observing the death of animals within 14 d.Sixteen male BALB/c-nu nude mice were randomly divided into four groups:control group[subcutaneous and intrapulmonary simultaneous inoculation of human bronchial epithelial 16HBE （HBE） cells,subcutaneous （1×10⁶cells per mouse）,lung （2×10⁵cells per mouse）],subcutaneous tumor-bearing model group[subcutaneous inoculation of THBEc1 cells （1×10⁶cells per mouse）],and the in situ lung cancer model group[intrapulmonary inoculation of THBEc1 cells （2×10⁵cells per mouse or 5×10⁵cells per mouse）].All the mice were inoculated at one sitting.The animals were monitored for body weight and subcutaneous tumor volume every 7 d after cel inoculation.The histopathological characteristics of lungs and subcutaneous tumors were detected by HE staining,and the tumourigenic rate was calculated based on whether tumour cel s could be observed.The levels of human-derived NRP2 in the sera and pleural fluid of nude mice were determined by enzymelinked immunosorbent assay （ELISA）.The diagnostic value of serum human-derived NRP2 for the orthotopic lung cancer model caused by THBEc1 cells in nude mice was assessed via the receiver operating characteristic （ROC） curve.RESULTS A single intrapulmonary injection of 40μL of sterile saline resulted in the death of 2/4 of the animals,but no animal death was observed below 30μL.However,the overall performance was not good,so a 20μL injection volume was selected for the subsequent experiment.During the experiment,no tumours were detected at the inoculation site in the control group,and the animals continued to increase in body weight.All nude mice in the subcutaneous tumour-bearing group became tumori,and their body weight was significantly lower than that of the control group at the end of the experiment （P<0.05）.On the 10^thday after cell inoculation,all nude mice in the 5×10⁵group of the in situ lung cancer model developed tumors （4/4）.On the 14^thday,3/4 of the nude mice in the 2×10⁵group of the in situ lung cancer model developed tumors and the body weight of the mice in the two dosage groups was lower than in the control group （P<0.01,P<0.05）,and the tumors were characterized by squamous cell carcinoma.The ELISA results showed that the serum levels of human-derived NRP2 in the control group was significantly lower than that of the subcutaneous tumor-bearing model（4） and the nude mice in situ lung cancer model （7 mice,P<0.05）.High levels of human-derived NRP2were detected in the pleural fluid of nude mice in the model group of lung cancer in situ （4 mice）.The ROC curve showed that serum human-derived NRP2 levels were effective in differentiating whether THBEc1 cells were tumorigenic in the lungs of nude mice with an optimal cut-off value of 123.00 ng·L^-1.CONCLUSION An orthotopic lung cancer model in nude mice can be established by using intrapulmonary injection of THBEc1 cel s,and serum human-derived NRP2 can be used for the diagnosis of this model.更多还原