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目的建立本实验云锡矿粉(无氡)诱导永生化大鼠肺泡Ⅱ型上皮细胞RLE-6TN恶性转化模型,观察在诱导过程中不同阶段的细胞形态、超微结构和某些相关蛋白改变。方法高、低2个剂量(200和50 mg/L)矿粉对RLE-6TN细胞进行72 h染毒,隔代染毒至第10代,常规传代直至软琼脂克隆形成实验阳性;通过透射电镜、流式细胞术、免疫印迹技术及免疫细胞化学观察实验细胞的改变。结果染毒24 h细胞内见大量矿粉,板层小体变性减少,出现细胞凋亡;第25代高剂量组及第30代高低剂量组的细胞均可在软琼脂上形成细胞集落,空白对照组克隆形成实验阴性(P<0.01);第30代高剂量组细胞增殖指数(PI)高于空白对照组;高剂量组转化细胞p53(+)、MDM2(+)、FHIT(-);空白对照组p53(-)、MDM2(+)、FHIT(+)。结论云锡矿粉可引起离体培养的RLE-6TN细胞发生恶性转化。p53阳性表达及FHIT失表达可作为RLE-6TN细胞转化的参考指标。
Objective To establish a malignant transformation model of alveolar epithelial type Ⅱ epithelial cells induced by tin powder (no radon) in this experiment and observe the changes of cell morphology, ultrastructure and some related proteins during different stages of induction. METHODS: RLE-6TN cells were exposed to high and low dose (200 and 50 mg / L) of mineral powder for 72 h. The cells were passaged to the tenth passage from generation to generation, and were passaged routinely until soft agar colonies were formed. Transmission electron microscopy , Flow cytometry, immunoblotting and immunocytochemistry to observe the changes of experimental cells. Results A large amount of mineral powder was observed in the cells exposed to 24 h and the degeneration of lamellar bodies was reduced. Apoptosis was observed. Cells in the 25th generation and 30th generation of high and low dose groups could form colony on soft agar and blank (P <0.01). The proliferation index (PI) of the 30th generation high dose group was higher than that of the blank control group. The expression of p53 (+), MDM2 (+) and FHIT The blank control group p53 (-), MDM2 (+), FHIT (+). Conclusion Cloud tin ore powder can cause malignant transformation of RLE-6TN cells cultured in vitro. p53 positive expression and loss of expression of FHIT RLE-6TN cells can be used as a reference indicator of transformation.