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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 an in vitro model that mimics the exposure of alveolar type Ⅱ epithelial cells (AT Ⅱ) to the soot environment of Yunxi refinery. Methods Immortalized ATⅡ (RLE-6TN cell line) was divided into high and low dose exposure groups and blank control group. The first two groups were used in the first, third, fifth, seventh and ninth generations of the cells respectively with the final concentrations of 200 and 50 mg / L of soot suspension, and the control groups were cultured synchronously without dust suspension. From the tenth generation onwards, high and low dose groups were routinely passaged until soft agar colony formation assay was positive. The ultrastructure of the cells was observed by electron microscope. The cell cycle was observed by flow cytometry. The expressions of SP-B, SP-C and MDM2 were detected by immunoblotting and immunohistochemistry. Protein, FHIT Protein Changes. Results The ultrastructure of RLE-6TN cells was observed to be damaged by smoke and dust for 24 h after exposure to tobacco smoke. The changes were mainly attributed to reduced degeneration of lamellar body and decreased number of apoptotic cells, as well as cell apoptosis and necrosis. The transformed cells consisted of round or similar Round paved stone-like growth into a long fusiform. In the high and low dose groups, the colony formation assay of soft agar was positive at 25 passages, and the clonogenic rates were 3.33 ‰ and 1.67 ‰, respectively. After 35 passages, the clonogenic rates of high and low dose groups were observed Respectively, 12.50 ‰ and 10.00 ‰, the blank control group has never been cloned. There was a significant correlation between the rate of colony formation and exposure to culture algebra in the high and low dose groups (P <0.01), but no significant correlation with the dose of smoke and dust exposure (P> 0.05). Flow cytometry showed that the proportion of G2 / M phase + S phase in high and low dose groups was higher than that in blank control group. The MDM2 protein positive, SP-B and FHIT protein in the 25th and 30th generation cells in the high and low dose groups were negative, while in the control group, the MDM2 protein was negative in the same generation, SP-B and FHIT protein were positive. High and low dose exposure group 25,30,35 cells showed SP-C loss of expression, blank control group SP-C expression. Conclusion AT Ⅱ long-term exposure to the smoke tin refinery can lead to its malignant transformation. AT Ⅱ-specific protein SP-B, SP-C, FHIT loss of expression, MDM2 positive expression can be used as a reference for RLE-6TN cell transformation.