黄曲霉毒素G_1对体外原代培养的大鼠肺泡Ⅱ型上皮细胞的影响

Effects of aflatoxin G_1 on primarily cultured rat lung alveolar typeⅡ cells in vitro

目的进一步研究黄曲霉毒素G1(AFG1)对可能作用的靶细胞SD大鼠肺泡Ⅱ型上皮细胞(AT-Ⅱ)损伤的生物学作用。方法以酶消化法原代培养SD大鼠AT-Ⅱ,纯化后培养36h,分别给予不同浓度(0.5,1.0和2.0mg.L-1)的AFG1处理。AFG1作用24h后,收集细胞和培养基上清液及细胞爬片,采用噻唑蓝(MTT)比色法检测细胞存活率、生物化学方法检测细胞培养上清液乳酸脱氢酶(LDH)和碱性磷酸酶(AKP)活力、透射电镜方法观察AT-Ⅱ超微结构的改变、激光扫描共聚焦显微镜(CLSM)检测Fluo-3/AM负载细胞内钙离子浓度([Ca2+]i)、免疫细胞化学、CLSM及流式细胞术(FCM)方法检测AT-Ⅱ特异分化标志物肺表面分泌蛋白C(SP-C)表达情况。结果给予不同浓度AFG1处理后,体外培养AT-Ⅱ细胞存活率分别为(88±3)%,(80±9)%和(72±8)%,明显低于溶剂对照组(101±2)%(n=6,P<0.01);培养上清液中LDH和AKP活力明显增高;透射电镜观察可见上皮细胞板层小体出现空化,线粒体肿胀、空泡化等损伤性变化。CLSM结果显示,AFG10.5,1.0和2.0mg.L-1处理组,AT-Ⅱ细胞内平均钙离子荧光强度分别为200±21,225±14和229±12,明显高于溶剂对照组161±28(n=6,P<0.01),有明显浓度依赖关系(r=0.849);免疫细胞化学、CLSM及FCM检测结果显示AFG1处理组AT-ⅡSP-C蛋白表达明显低于溶剂对照组。结论AFG1对体外培养的大鼠AT-Ⅱ具有明显致损伤作用,同时增加细胞[Ca2+]i、降低SP-C蛋白的表达。

AIM To further explore the effect of aflatoxin G1 （AFG1 ） on the possible target cell, primarily cultured lung alveolar type Ⅱ cell （ AT- Ⅱ ） from SD rats. METHODS The primarily cultured AT-Ⅱ cells isolated from the lung of SD rat by enzyme digestion method were treated with AFG1 at the concentration of 0.5, 1.0 and 2.0 mg· L-1, respectively for 24 h after purification culture for 36 h. The survival rates of AT-Ⅱ in vitro were evaluated by MTY assay. The activities of lactate dehydrogenase （LDH） and alkaline phosphotase （AKP） in culture medium of AT-Ⅱ cells were determined by biochemical method, while the uhrastructural changes were studied by transmission electron microscopy （TEM） observation. The concentration of intracellular free Ca2＋ （ [Ca^2＋ ]i） loaded with Fluo-3/AM was observed under confocal laser scanning microscopy （CLSM）. The immunocytochemical expression of SP-C of AT-Ⅱ was determined by both CLSM and flow cytometic （FCM） analysis. RESULTS The survival rates in all AFG1 treated 0.5, 1.0 and 2.0 mg· L^-1 groups were （ 88 ± 3 ） %, （ 80 ± 9） % and （ 72 ± 8 ） %, respectively, significantly lower than that in DMSO group （101 ± 2）% （n =6, P 〈0.01）. The activities of LDH and AKP in culture medium were significantly increased after AFG1 treatment. Injury changes at ultrastructural level, such as, turgid and evacuated lamellar bodies, vacuolar degeneration of mitochondria, etc were observed in AFG1-treated AT- Ⅱ under TEM. [ Ca^2＋ ] i in 0.5, 1.0 and 2.0 mg· L^ - 1 AFGl-treated groups were 200 ± 21,225 ± 14 and 229 ± 12, respectively, and significantly higher than that in solvent control group 161 ± 28 （n = 6, P 〈 0.01 ）. A significant concentration-depended response correlation could be found between [ Ca^2＋ ]i and AFG1 concentrations （r = 0. 849 ）. CLSM and FCM results showed the expression of SP-C protein was significantly decreased after AFG1 treatment. CONCLUSION AFG1 causes AT- Ⅱ injuries, increases the [ Ca^2＋ ]i and decreases the expression of SP-C in vitro.