【作者】 张祥宏； 申海涛； 黄向华； 邢凌霄； 李月红； 王俊灵； 严霞； 王凤荣；

【Author】 Zhang Xiang-Hong Shen Hai-Tao Huang Xiang-Hua et al Lab of Pathology, Hebei Medical University, Shuiazhuang 050017, China

【机构】 河北医科大学基础所病理室；

【摘要】 目的:探讨AFG1对II型肺肺泡上皮细胞的致癌作用及其可能机理。方法:1以动物实验方法探讨了经口给予 AFG1对NIH小鼠的致癌作用,实验组分别灌喂不同剂量的 AFG1,对照组灌喂同等容量的生理盐水,3次／周,共24周。实验第58和74周处死实验动物,观察各器官组织病变。以免疫组化方法探讨AFG1诱发NIH小鼠肺腺癌组织学来源及其可能致癌机理。2以酶消化法原代培养大鼠肺泡Ⅱ型上皮细胞,纯化后培养36h,给予不同浓度AFG1处理24h, 黄曲霉毒素G1对体外培养的肺泡II型上皮细胞的影响。3按 30μg／kg剂量经支气管一次性给予雄性SD大鼠AFG1处理。以透射和扫描电镜观察大鼠肺组织Ⅱ型肺泡上皮细胞超微结构变化。原位杂交,免疫组化方法和FCM检测Ⅱ型肺泡上皮细胞特异分化标志物SP—C蛋白的表达情况。结果:1 AFG1诱发NIH小鼠肺癌的实验研究:结果发现对照组动物各器官组织均未见明显病理改变,而AFG1各处理组均有部分动物发生肺泡上皮增生和肺腺癌。AFG1 3 μ g／kg组和 AFG1 30 μg／kg组肺泡上皮增生,肺腺癌率分别为10．0 ％,30．0％和35．7％,42．9％。免疫组化结果发现,全部肺腺癌癌细胞均可见肺Ⅱ型肺泡上皮细胞特异分化标志物SP—C 蛋白的阳性表达,阳性表达率为100％,而Clara细胞特定分化标志物CC—10均为阴性表达(P<0．01)。肺腺癌细胞PCNA 平均阳性标记指数为73．32％,明显高于对照组22．43％(P<0． 01),但肺腺癌细胞未见突变型抑癌基因P53和癌基因Ras P21在蛋白水平上的表达。2 AFG1对体外培养的大鼠肺泡 II型上皮细胞的影响:MTT比色法检测显示不同浓度AFG1 处理组AT-Ⅱ细胞存活率明显低于对照组(p<0．01),随着 AFG1处理浓度的增加,细胞内钙离子浓度明显增加(r=0． 849,n=6,p<0．01,),透射电镜观察可见Ⅱ型肺泡上皮细胞明显损伤变化。FCM和激光扫描共聚焦显微镜检测结果显示不同浓度AFG1处理组SP—C蛋白表达明显低干对照组(p<0．05)。 3经支气管给予AFG1对大鼠肺组织Ⅱ型肺泡上皮细胞的影响:AFG1处理后Ⅱ型肺泡上皮细胞出现明显损伤性超微结构变化,TNF-α mRNA的表达明显增高。AFG1处理7d 时SP—C蛋白标记指数(9．10±1．28)％明显低于相应对照组 (13．19±3．30％,P<0．05)。结论:本研究结果提示,经口给予 AFG 1可诱发NIH小鼠肺癌前增生性病变和肺腺癌, 肺腺癌来源于II型肺泡细胞。AFG1对大鼠肺组织有一定的损伤作用,降低肺组织抗活性氧能力,上调肺泡细胞TNF-α mRNA的表达。AFG1对体外培养的AT-Ⅱ具有致损伤作用,可增加细胞内钙离子浓度,同时降低Ⅱ型肺泡上皮细胞特异分化标志物SP—C蛋白的表达。一次性支气管给予 A FG 1对大鼠肺组织Ⅱ型肺泡上皮细胞有一定的损伤作用, 作用7天时可降低Ⅱ型肺泡上皮细胞特异分化标志物SP—C 蛋白的表达。更多还原

【Abstract】 Objective: Aim To evaluate the carcinogenic effects of Aflatoxin G1 (AFG1) on alveolar type II cells and to explore the putative mechanisms. Methods: 1 The carcinogenic study in NIH mice: NIH mice were randomly divided into three groups. Two experimental groups were treated intragastrically by gavage with AFG1 3 μg/ kg and AFG1 30 μg/ kg respectively, 3 times a weekfor 24 weeks. The control group was treated with normal saline. All mice were fed with food free of AFGs as confirmed by HPLC analysis. The mice were sacrificed and examined pathologically at the 58th and 74th weeks respectively. The phenotype of the lung adenocarcinomas was determined by immunohistochemical expression of SP-C and CC-10 at protein level. The expression of P53, Ras P21 and PCNA was studied with immunohistochemical staining. 2 The effects of AFG1 on AT-II cell in vitro and in vivo: 1 Wistar rat AT-II cells were isolated with enzyme digest method and primarily cultured for 36h. The effects of AFG1 on AT-II cells in vitro were studied with ultrastructural observation, Confocal microscopic analysis, etc. 2 Male SD rats were intratracheally administrated with AFG1 (30 μg/kg body weight), and the animals were respectively sacrificed 1, 3, 7 and 14day (s) after AFG1 treatment The ultrastructural changes of lung alveolar type II cells were studied with transmission and scaning electron microscopy. The expression of SP-C was determined by immunohistochemical method and FCM analysis respectively. Results: The carcinogenic study in NIH mice: Compared with control mice receiving no AFG1, alveolar hyperplasia and adenocarcinoma of lung were observed in mice receiving AFG1 treatment The incidences of alveolar hyperplasia and adenocarcinoma of lung were 10.10 % and 30.10 % for mice receiving 3 μg/ kg AFG1 and 35.17 %, 42.19 %for mice receiving 30 μg/ kg of the toxin, respectively. Immunohistochemical staining showed that The positive expression of SP-C was found in all the lung adenocarcinomas, while no expression of CC-10 could be seen. The labelling index of PCNA in the adenocarcinomas were significantly higher that that of control (P<0. 01 ). No positive expression of mutant P53 and Ras at protein level could be found. The effects of AFGl on AT-II cell in vitro: MTT Results showed that the survival rates in AFGl treated AT-II cells were all significantly lower than that in control( p<0.01 ). As the AFGl concentration increased, the intracellular calcium ion concentration increased ( r=0.849,n=6,p<0.01 ). Significant ultrastructural injury changes could be found with transmiting electron microscopic observation. FCM and Confocal microscopic analysis showed significant decreases of SP-C expression in AFGl treated cells as compared with that of control (p <0.05). The Results of in vivo experiment by intratracheally administrated: Injury changes of AT- II cells after AFG1 treatment were found under SEM and TEM observation. Though no significant differences were found in更多还原