环磷酰胺诱发永生化人支气管上皮细胞的恶性转化

研究环磷酰胺 (CP)对人类支气管上皮细胞的致癌转化效应 .实验利用永生化人支气管上皮细胞(BEAS 2B)为靶细胞模型 ,研究CP诱导的BEAS 2B细胞 (BEAS CP)的恶性转化 ,并伴随研究了BEAS CP转化细胞的增殖动力学 ,细胞周期和非贴壁依赖性生长等转化表型特征 .结果显示 ,经传代和软琼脂培养基筛选 ,BEAS CP细胞已具有较为稳定的转化表型和细胞生物学特性 ,可能是具有裸小鼠致瘤性的恶性转化细胞 .结果表明CP对BEAS 2B细胞具有恶性转化效应     

凝血酶诱发人支气管上皮细胞恶性转化

目的探讨凝血酶对人类支气管上皮细胞的致癌性和转化涉及的相关机制。方法用75nmo·lL-1凝血酶持续刺激人类乳头状瘤-18永生化的人支气管上皮细胞(BEP2D)70d,观察不同代龄细胞的血清抗性、软琼脂克隆形成率,流式细胞术观察细胞周期改变,逆转录-聚合酶链反应检测凝血酶受体-蛋白酶活化受体(PAR1),c-myc和p21~(cip1)基因表达水平,蛋白质免疫印迹检测C-MYC和P21~(cip1)蛋白表达变化,鉴定细胞的恶性转化表型。结果经传代培养,凝血酶处理的第10代BEP2D细胞血清抗性增强,接种效率提高,第20代细胞呈非贴壁依赖性生长,软琼脂克隆形成率升高达(0.461±0.009)%。流式细胞术结果显示,第22代细胞处于S期比例为55.63%。凝血酶转化第25代、第27代细胞PAR1基因、c-myc基因及C-MYC蛋白表达水平上调。相反,p21~(cip1)基因和P21~(cip1)蛋白表达减弱。凝血酶处理的第20代细胞已具有较为稳定的转化表型和细胞生物学特性,可能是具有裸小鼠致瘤型的恶性转化细胞。每升2×104抗凝血酶单位水蛭素能够拮抗凝血酶对BEP2D细胞的恶性转化作用。结论凝血酶对人支气管上皮细胞具有恶性转化作用,c-myc基因转录和蛋白表达活化与p21~(cip1)失活在这一过程中发挥关键作用。     

塞替派诱发永生化人支气管上皮细胞的恶性转化（一）

目的 研究塞替派对人类支气管上皮细胞的致癌转化效应。方法 利用永生化人支气管上皮细胞（BEAS－2B）为靶细胞模型,以塞替派诱导BEAS－2B细胞的恶性转化,并伴随研究了塞替派转化细胞（EBAS－TE）的增殖动力学、细胞周期和锚着独立生长能力等转化表型特征。结果 经传代和软琼脂培养基筛选,BEAS－TE具有较为稳定的转化表型和细胞生物学特性。结论 塞替派对人支气管上皮细胞具有较强的恶性转化效应。     

α 粒子辐射诱发人支气管上皮细胞恶性转化

α粒子辐射诱发人支气管上皮细胞恶性转化周晓程书钧１吴德昌郭素萍张欣舒为群１（北京放射医学研究所毒理室，北京１００８５０；１中国医学科学院肿瘤所病因室，北京１０００２１）本研究采用２３８ＰｕＯ２α粒子沉积源照射永生化的人支气管上皮细胞，在国内首次建立了...     

塞替派、环磷酰胺诱发的人支气管上皮恶性转化细胞的致瘤性

目的　观察塞替派 (TE)及环磷酰胺 (CP)能否诱发永生化人支气管上皮恶性转化细胞成瘤。方法以永生化人支气管上皮细胞 (BEAS 2B)为对照 ,以TE诱发转化细胞 (BEAS TE)的琼脂糖克隆扩增细胞(BEAS STE)及CP诱发转化细胞 (BEAS CP)的琼脂糖克隆扩增细胞 (BEAS SCP)为靶细胞接种于 3～ 4周龄裸鼠 ,每组接种 6只 ,♂♀各半。结果　接种后2 6周 ,对照组无一长出肿瘤 ,BEAS STE组有 5只长出肿瘤 ,BEAS SCP组有 1只长出肿瘤 ,其中BEAS STE组有 3只裸鼠的肿瘤直径在 1cm以上。经病理组织形态和免疫组织化学检查证实肿瘤为低分化癌肉瘤组织。结论　TE、CP诱发恶性转化的人支气管上皮转化细胞均具有裸鼠致瘤性。     

塞替派诱发人支气管上皮恶性转化成瘤细胞的染色体畸变

目的　旨在了解转化细胞在成瘤过程中的细胞遗传学改变。方法　运用染色体G显带技术研究永生化人支气管上皮细胞 (BEAS 2B)恶性转化后的裸小鼠接种成瘤细胞 (BEAS TT)的染色体畸变。结果　瘤细胞在传代早期基本以近二倍体细胞为主 ,随着细胞代龄的增加 ,各肿瘤细胞系的细胞染色体数目变化趋势不同 ,其中BEAS TTa逐渐形成以多倍体细胞为主的细胞群 ,而BEAS TTb ,BEAS TTc则以近二倍体细胞为主份额细胞。核型分析表明 3个瘤细胞系的核型与BEAS TE不同 ,在其基础上有新的染色体 (14号染色体 )丢失和标记染色体 (M4 )的增加。结论　细胞染色体数目不稳定 ,14号染色体的丢失和M4染色体的增加 ,可能与BEAS TE的裸小鼠成瘤性有关     

GMA致人支气管上皮细胞恶性转化过程中全基因组甲基化水平的研究北大核心CSCD

目的分析甲基丙烯酸环氧丙酯(glycidyl methacrylate,GMA)致人支气管上皮细胞(16HBE细胞)恶性转化过程中不同时点全基因组甲基化水平的改变,探讨GMA诱导16HBE细胞全基因组甲基化水平改变的意义。方法通过细胞毒性试验,确定8μg/ml浓度GMA诱导16HBE细胞,收集GMA转化前期(第10代)、转化中期(第20代)、转化后期(第30代)细胞和同步溶剂对照(DMSO)细胞,以及去甲基化制剂(5-氮杂-2-脱氧胞苷,5-aza-cdr)处理的各时点转化细胞,应用DNA甲基化定量检测试剂盒,检测不同转化时期细胞全基因组甲基化水平。结果 DNA甲基化定量检测结果显示,不同时点GMA转化组较同代龄DMSO组细胞全基因组甲基化水平降低,转化前期GMA组、DMSO组、5-aza-cdr组细胞甲基化水平较转化中期及转化后期相对应各组的全基因组甲基化水平低。结论 GMA诱导16HBE细胞在转化前期已出现全基因组低甲基化现象,故可将其视为细胞恶性转化前期生物标志。     

塞替派诱发人支气管上皮细胞恶性转化

目的观察抗癌药物塞替派诱发永生化人支气管上皮细胞恶性转化过程中相关基因的突变并分析其意义.方法以塞替派作为致癌原诱导永生化人支气管上皮细胞(BEAS-2B),获得转化细胞(BEAS-TE)和克隆化多倍体癌前转化细胞(BEAS-STE).采用PCR-SSCP法检测上述3种细胞p53、p16和Ki-ras 3种基因是否出现点突变,进一步测序确定其突变情况.结果 SSCP结果阳性的有BEAS-TE细胞p53第7外显子,BEAS-STE细胞p53第8外显子以及这二种细胞的p16基因第1外显子;Ki-ras基因第1外显子的结果仅为可疑阳性.测序证明,p53、Ki-ras基因存在多位点的碱基突变,而p16基因仅为单位点的碱基突变.结论塞替派可诱发人支气管上皮细胞p53、Ki-ras多位点的碱基突变和p16的单位点突变.分析前者为塞替派诱导细胞转化过程中发生的重要分子事件,后者是次要分子事件.     

环磷酰胺及塞替派诱导人支气管上皮细胞恶性转化的超微结构

为了解抗癌化疗药环磷酰胺 (CP)及塞替派(TE)诱导的永生化人支气管上皮细胞恶性转化过程中的细胞超微结构的动态变化 ,采用透射电镜方法观察的细胞模型由对照永生化人支气管上皮细胞(BEAS- 2 B) ,受环磷酰胺 (BEAS- CP)和塞替派(BEAS- TE)诱导的恶性转化细胞组成 .发现经 CP和 TE暴露的细胞可出现细胞死亡 ,凋亡和转化 ,它们具有各自的超微结构特征 .说明逃逸细胞死亡 ,凋亡的支气管上皮基细胞呈增生性改变 ,这种增生性基细胞经过扩增后将成为转化细胞群主体并且具有肿瘤细胞的增生特性     

环磷酰胺及塞替派诱导人支气管上皮细胞恶性转化的超微结构

为了解抗癌化疗药环磷酰胺(CP)及塞替派(TE)诱导的永生化人支气管上皮细胞恶性转化过程中的细胞超微结构的动态变化,采用透射电镜方法观察的细胞模型由对照永生化人支气管上皮细胞（BEAS-2B）,受环磷酰胺（BEAS-CP）和塞替派（BEAS-TE）诱导的恶性转化细胞组成. 发现经CP和TE暴露的细胞可出现细胞死亡,凋亡和转化,它们具有各自的超微结构特征．说明逃逸细胞死亡,凋亡的支气管上皮基细胞呈增生性改变,这种增生性基细胞经过扩增后将成为转化细胞群主体并且具有肿瘤细胞的增生特性.     

Overexpression of SLURP-1 and -2 alleviates the tumorigenic action of tobacco-derived nitrosamine on immortalized oral epithelial cells

Recent research has demonstrated that mucocutaneous epithelial cells express functional nicotinic acetylcholine receptors (nAChRs) and that tobacco-derived carcinogenic nitrosamines, such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and SLURP (secreted mammalian Ly-6/urokinase plasminogen activator receptor-related protein)-1 and -2 can act as non-canonical ligands of these receptors. It was found that recombinant SLURP-1 and -2 can lessen tumorigenic activity of nitrosamines. The immortalized esophageal keratinocytes (Het-1A cells) exhibit low SLURP-1 and -2 mRNA levels that decrease further after treatment with NNK. Based on these observations, we hypothesized that overexpression of full length SLURP proteins may protect Het-1A cells from malignant transformation by NNK. The Het-1A cells transfected with either SLURP-1 or -2 vector produced the highest amounts of respective proteins between 24 and 48 h, at which point they were exposed to 1 microM NNK for 24 h and their tumorigenic activities were subsequently evaluated by plating in soft agar and injecting subcutaneously to Nu/Nu mice. Transfection with either SLURP-1 or -2 cDNA in both cases significantly (p<0.05) diminished the number of colonies produced by NNK exposed cells. SLURP-1 was more efficient than SLURP-2 in abolishing the tumorigenic effect in nude mice. Thus, the anti-tumorigenic activities of SLURP-1 and -2 were demonstrated both in vitro and in vivo. The obtained results suggest that SLURP-like proteins may become useful for developing novel anti-cancer therapies.     

NLRP3 inflammasome activation involved in LPS and coal tar pitch extract‐induced malignant transformation of human bronchial epithelial cells

Inflammatory microenvironment has been found as a new characteristic of cancer; however, the mechanisms of inflammation‐related lung cancer remain unclear. To explore the role of NLRP3 inflammsome activation in inflammation‐related lung carcinogenesis, a cell model was set up. Human bronchial epithelial cells (BEAS‐2B) were stimulated with 1 μg/mL lipopolysaccharide (LPS) for 24 hours, and then treated with 2.4 μg/mL coal tar pitch extract (CTPE) for 24 hours, after removal of LPS and CTPE, the cells were numbered passage 1 and were passaged and treated in this way until passage 30, which was called LPS + CTPE group. DMSO and Saline were used as vehicle controls. Malignant transformation of cells in passage 30 was evaluated by morphological change, platelet clone formation assay, and tumor formation in nude mice. The mRNA levels of NLRP3 and IL‐1β were detected by real time‐PCR. The combination of NLRP3 and caspase‐1 were determined using immunofluorescence and confocal. The protein expression of NLRP3, cleaved caspase‐1(p10), and cleaved IL‐1β was detected using Western blot. It was shown that CTPE, LPS + CTPE‐stimulated BEAS‐2B cells of passage 30 changed a lot morphologically. The clone formation rates, the rates of positive cells of NLRP3 and caspase‐1 combination, the mRNA levels of NLRP3 and IL‐1β, the protein expression of NLRP3, cleaved caspase‐1(p10) and cleaved IL‐1β of cells exposed with CTPE and LPS + CTPE at passage 30 were significantly increased compared to vehicle controls. Furthermore, the ability of tumor formation in nude mice, the rates of clone formation and positive cells, mRNA and protein levels of NLRP3 inflammasome activation‐related factors in LPS + CTPE‐induced cells were all higher than those in cells stimulated with CTPE alone. In conclusion, the cell model of inflammation‐related lung cancer is set up successfully, and NLRP3 inflammasome activation may be involved in the malignant transformation of BEAS‐2B cells which induced by CTPE alone or LPS combined with CTPE.     

塞替派诱发人支气管上皮恶性转化成瘤细胞的基因突变

目的　旨在了解转化细胞在成瘤过程中的p15 ,p16 ,p5 3和K ras基因编码序列突变情况。方法　运用逆转录聚合酶链反应 (RT PCR)技术和DNA测序技术。结果　转化各阶段细胞p15和K ras基因编码序列为野生型。在转化进程中 ,各转化细胞均携带与细胞永生化有关的p5 3第 4 7密码子CCG→TCG(脯氨酸→丝氨酸 )转换 ,在此基础上无新的错义突变发生。p16基因在BEAS 2B细胞和BEAS STE细胞中均为野生型 ,在BEAS TTb细胞中第 4密码子发生TCG→TAG(丝氨酸→终止密码子 )碱基转换 ,结果为无义突变 ,第 19,5 2密码子分别发生GAG→AAG (谷氨酸→赖氨酸 )转换 ,GCG→ACG(丙氨酸→苏氨酸 )转换的错义突变。BEAS TTc细胞中第 19密码子发生GAG→AAG(谷氨酸→赖氨酸 )转换的错义突变 ,而没有可检测的BEAS TTa细胞的mRNA存在。结论　p16基因的突变或表达缺失与恶性转化细胞的成瘤过程相关。