烟尘慢性染毒致肺泡Ⅱ型上皮细胞恶性转化离体实验研究  

Experimental study on malignant transformation of lung alveolar epithelial type Ⅱ cells chronically exposed to Yunnan tin mine smelting dust in vitro

作　　者：唐莹[1] 边莉[2] 孟庆印[3] 金克炜[4] 高倩[4] 阮永华[4] 

机构地区：[1]昆明医科大学电子显微镜室,云南昆明650500 [2]昆明医科大学第一附属医院病理科,650031 [3]昆明医科大学附属肿瘤医院病理科,650032 [4]昆明医科大学基础医学院,650500

出　　处：《中国职业医学》2014年第1期20-25,共6页China Occupational Medicine

基　　金：国家自然科学基金项目(81060189);云南省应用基础研究基金项目(2010ZC107)

摘　　要：目的建立模仿肺泡Ⅱ型上皮细胞(ATⅡ)暴露于云锡炼厂烟尘环境的离体研究模型。方法以永生化ATⅡ(RLE-6TN细胞株)为实验对象,分为高、低剂量染毒组和空白对照组。前2组分别使用终浓度为200、50 mg/L的烟尘悬浊液在第1、3、5、7、9代对细胞染毒,同代对照组不加烟尘悬浊液同步培养。从第10代起,高、低剂量染毒组常规传代直至软琼脂克隆形成检测实验呈阳性。采用透视电镜观察细胞超微结构,流式细胞术观察细胞周期,免疫印迹法及免疫组织化学观察各组细胞肺表面活性蛋白(SP)-B、SP-C、鼠双微体2(MDM2)蛋白、脆性组氨酸三联体(FHIT)蛋白的改变。结果染毒24 h后即可观察到烟尘对RLE-6TN细胞超微结构造成损伤,主要表现为板层小体变性且数量减少,以及细胞凋亡和坏死;转化后的细胞由圆形或类圆形铺路石样生长变为长梭形。高、低剂量染毒组细胞培养至25代时软琼脂克隆形成检测实验均呈阳性,克隆形成率分别为3.33‰和1.67‰;继续培养至35代观察高、低剂量染毒组克隆形成率分别为12.50‰和10.00‰,空白对照组始终没有克隆形成。高、低剂量染毒组克隆形成率与染毒培养代数相关性有统计学意义(P<0.01),但与烟尘染毒剂量相关性无统计学意义(P>0.05)。流式细胞仪检测示高、低剂量染毒组G2/M期+S期的比例较空白对照组增高。高、低剂量染毒组第25、30代细胞MDM2蛋白阳性、SP-B及FHIT蛋白阴性,而空白对照组同代细胞MDM2蛋白阴性、SP-B及FHIT蛋白阳性。高、低剂量染毒组第25、30、35代细胞示SP-C失表达,空白对照组SP-C有表达。结论 ATⅡ长期暴露于云锡炼厂烟尘中可导致其恶性转化。ATⅡ特异性蛋白SP-B、SP-C、FHIT失表达,MDM2阳性表达可作为RLE-6TN细胞转化的参考指标。Objective To establish a model in vitro that mimics the chronic exposure of alveolar epithelial type Ⅱ cells to Yunnan tin mine smelting dust. Methods Immortalized alveolar epithelial type Ⅱ cells （ RLE-6TN） were set into three groups. The high-and low- concentration groups were respectively exposed to Yunnan Tin mine smelting dust at mass con- centrations of 200 mg/L and 50 mg/L for 72 hours per cycle during cycle 1, 3, 5, 7 and 9. The control group was not treated with the dust. From the cycle 10, all cells were transmitted routinely until anchorage independent growth was deter-mined. The uhrastructure of RLE-6TN cells were observed by electron microscope. Cell cycle was tested by flow cytome-try. Immunocytochemistry （ICC） and western blot （WB） were used to test the expressions of surfactant protein （SP）-B, SP-C,murine double minute 2 （MDM2） and fragile histidine triad （FHIT）. Results After exposure to tin mine smelting dust for 24 hours, the uhrastructure of RLE-6TN was injured. The number of osmiophilic lamellar body decreased signifi-cantly and the structure of osmiophilic lamellar body degenerated. Apoptosis could also be found. In cycle 25, anchorage independent growth test showed positive results, and respective cloning efficiencies for high and low concentration groups were 3.33%0 and 1.67%0; in cycle 35, the cloning efficiencies reached to 12. 50%0 and 10. 00%0 respectively. There was no cell clone in the control group in this test. After statistics analysis, statistical significant difference could be found be-tween cloning efficiency and cell cycles （P 〈 0. 01 ） , while there was no statistical significant difference between cloning ef-ficiency and dust concentration （ P 〉 0. 05 ）. Flow cytometry test showed the percentage of S-phrase cell increased in high and low concentration groups. ICC and WB results were MDM2（ ＋ ）, SP-B（ - ）, FHIT（ - ）, SP-C（ - ） in both two concentration groups while MDM2（ - ）, SP-B（ ＋ ）, FHIT（ ＋ ） SP-C�

关 键 词：肺泡Ⅱ型上皮细胞 RLE-6TN 云锡烟尘 恶性转化 肺表面活性蛋白 鼠双微体2 脆性组氨酸三联体 软琼脂克隆 

分 类 号：R135.2[医药卫生—劳动卫生] R734.2[医药卫生—公共卫生与预防医学]