【作者】 朱进国；

【导师】 吴庆琛；

【作者基本信息】 重庆医科大学 ， 外科学， 2004， 硕士

【摘要】 目的: 研究体外人支气管上皮细胞经NNK诱发恶性转化过程中形态学及p53蛋白的表达，并探讨其在吸烟高危人群罹患肺癌预警及普查方面的意义。方法:用浓度为500(g/ml 烟草特异性亚硝胺NNK诱发BEAS-2B细胞恶性转化，并在此过程中利用光镜、电镜、图像分析及免疫组织化学法，动态观察细胞形态学、细胞形态参数（周长、最大直径、最小直径、核浆比和形状因子）变化和p53蛋白表达。结果: 1. NNK诱发BEAS-2B细胞恶性转化模型的建立 ① 实验组BEAS-2BNNK第5代细胞血清抗性显著增强。② 第15代BEAS-2BNNK细胞具有锚着独立性生长特性（软琼脂克隆形成）。③ 第25代BEAS-2BNNK细胞在裸鼠体内成瘤,病理为鳞形细胞癌(I-II级)。⒉ 形态学变化 ①显微结构改变：BEAS-2B细胞各代间形态无明显变化。BEAS-2BNNK细胞在第1、2、5代时细胞克隆形态及生长方式、细胞形态、细胞核形态及数目与相应代BEAS-2B无明显差异；第10、15、20、25代时细胞及胞核逐渐变圆,出现多个核仁，细胞由放射状生长逐渐变为膜状生长。② 超微结构改变： BEAS-2B细胞各代间无明显差别。与BEAS-2B相比较BEAS-2BNNK第1、2、5代细胞及胞核形态、细胞器<WP=7>形态及数量无明显差别；第10、15代细胞出现肿胀，细胞核逐渐变形，出现核畸形,核仁明显增大，边集，细胞器肿胀，数量明显增多，功能活跃具明显的转化细胞特征；第20、25代细胞及胞核明显畸变，出现明显的核碎裂及多个核仁，细胞器明显减少，具明显的肿瘤细胞特征。③ 形态学参数：BEAS-2B细胞各代间形态学参数无明显统计学意义。与BEAS-2B细胞比较：第1、2、5代BEAS-2BNNK细胞的形态学参数无明显差别（p>0.05）；第10、15代各形态学参数逐渐增大，差别有显著性(p<0.05)；第20、25代时各参数进一步增大，差别有极显著性(p<0.01)。⒊　p53蛋白表达 各代BEAS-2B（对照组）细胞表达均为阴性。各代BEAS-2BNNK细胞均有不同程度的表达，随着传代次数增加表达呈明显上升趋势，各代间存在极显著性差异。（p<0.001）。结论:⒈浓度为500(g/ml NNK可成功诱发BEAS-2B细胞恶性转化，建立体外细胞转化模型。⒉ 细胞显微结构、超微结构及形态学参数对转化细胞中晚期生物学特性可以做出较准确、客观、量化的判断，但对转化细胞早期生物学特性难以做出判断。⒊p53蛋白在细胞恶性转化的早期即有明显表达，并随恶性转化的程度增加而呈现逐渐增强的趋势。⒋ 细胞恶性转化过程中形态学特征性改变比p53蛋白表达相对较晚。⒌ p53蛋白在细胞转化成癌细胞以前已有较明显表达的特点，可能有助于吸烟高危人群罹患肺癌的预警及普查。更多还原

【Abstract】 Objective: To investigate morphology and p53 protein expression during the malignant transformation of immortalized human bronchial epithelial cell （BEAS-2B） induced by tobacco-specific nitrosamine （NNK）, and explore the significance of early-warning and mass screening of lung cancer in high-risk smoking population. Methods: Immortalized human bronchial epithelial cells （BEAS-2B）were induced to malignant transformation cells (BEAS-2BNNK) by NNK, and p53 protein expression was detected in the different passages of BEAS-2BNNK and BEAS-2B cells by Sp immunocytochemistry. The cell morphology and parameters (perimeter, maximum diameter, minimum <WP=9>diameter, ratio of karyoplasm and form factor) were observed under microscope and electron microscope in each group. Results: 1.The model of malignant transformation of BEAS-2B cells induced by NNK was created. ① The serum resistance was significantly increased in the 5th passage BEAS-2BNNK cells. ② The anchorage independent growth（soft agar colony formation）was appeared in the 15th passage. ③ The 25th passage BEAS-2BNNK cells formed tumors in nude mice, and tumors were squamous cell carcinoma(I-II grade) confirmed by histopathological examination. 2. Morphology change ① Microstructure: The microstructure of BEAS-2B cells was not significantly changed in different passages. Compared with the corresponding BEAS-2B cells, the shape of cell clone, the way of cell growth, the shape and quantity of cell and nuclear were not significantly changed in the 1st, 2nd, 5th passage BEAS-2BNNK cells. The shapes of cells and its nuclears of the 10th, 15th, 20th, 25th passage BEAS-2BNNK cells became round gradually, and the way of cell growth became from radio-form into membraniform and more than one nucleolus appeared. ②Ultrastructure change: The ultrastructure of BEAS-2B cells was not significantly changed in different passages. Compared with the corresponding BEAS-2B cells, the shapes of cells and nuclears, the shapes and quantity of cell organs were not significantly changed in the 1st, 2nd, 5th passage BEAS-2BNNK cells. The 10th, 15th <WP=10>passage BEAS-2BNNK cells, which had swelled cell and cell organs and abnormal nuclear and enlarged the nucleoli gradually and increased the quantity of the cell organs and activated their function, showed the characteristic of transformation cells. The 20th, 25th passage BEAS- 2BNNKcells, which had obvious aberration of the cell and nucleoli and had cataclasm of nucleoli and decreased cell organs, showed the characteristic of tumor cells. ③Morphological parameters: Morphological parameters of different passages of BEAS-2B cells were no difference in statistics. And the morphological parameters were also no difference between BEAS-2B cells and BEAS-2BNNKcells in 1st, 2nd, 5th passage（p>0.05）. Compared with BEAS-2B cells, the morphological parameters of BEAS-2BNNK cells became large gradually in 10th, 15th passage (p<0.05), especially in 20th, 25th passage (p<0.01). 3. p53 protein expressions: p53 protein expression of BEAS-2B cells was negative, but positive in the different passage BEAS-2BNNKcells. With the passage of BEAS-2BNNK cells increased gradually, p53 protein expression rised more and more obviously（p<0.001）. Conclusion: 1. The model of malignant transformation of BEAS-2B cells induced by NNK(500μɡ/ml) could be created successfully. 2. Biology characteristic of transformation cells in middle and late passages could be accurately, objectively and quantitatively judged by microstructure, <WP=11>ultrastructure and morphological parameters, but was difficult to judge in earlier passages. 3. There were obvious p53 protein expressions in earlier passages. With the passage of BEAS-2BNNKcells increased gradually, p53 protein expression rised more and more obviously. 4. During the malignant transformation, the change of morphological characteristic of BEAS- 2BNNKcells was later than that of p53 protein expression. 5. The characteristic of p53 being expressed before BEAS-2BNNK cells changed into cancer cells wa更多还原