【作者】 张创业；

【导师】 袁素波； 吴纯启；

【作者基本信息】 中国人民解放军军事医学科学院 ， 卫生毒理学， 2005， 硕士

【摘要】 评价化学物致癌性,主要依靠哺乳动物长期诱癌实验。但其实验周期长,饲养条件要求严格,受试动物的敏感性和给药剂量常影响实验结果。而哺乳动物细胞的体外恶性转化实验既能反映化学物的致癌性,又能弥补动物诱癌实验的不足。因此,作为对长期诱癌实验的补充其结果具有重要参考价值。细胞体外恶性转化实验的起始点是致癌物导致细胞遗传物质改变,终点是细胞成瘤能力的改变,转化过程中则伴随有细胞形态、细胞生长能力、生化表型等的变化。本实验就以致癌物作用人支气管上皮细胞后传代细胞集落的形态学改变为切入点,寻找人源细胞体外转化的早期形态学标志,持续观察由不同形态集落而来的两株细胞随传代其生长特性、细胞构成、细胞遗传物质及细胞成瘤能力的变化,进而探讨肺肿瘤组织发生、人源细胞体外转化的形态学标志和基因组学改变等问题。期望建立一种以人源细胞为实验材料的化学物致癌性评价简化模型。 本研究在我室袁素波、陈光宇、周喆等人先后进行的赛替派(Thiotepa,TEPA)、环磷酰胺(Cyclophosphamide,CP)诱发永生化人支气管上皮细胞株BEAS-2B恶性转化、裸鼠接种成瘤及转化细胞、肿瘤细胞生物学特性和转化机理研究的基础上,分别以遗传毒性致癌物赛替派、丝裂霉素C(Mitomycin C,MMC)、环磷酰胺和非遗传毒性致癌物6-巯基嘌呤(6-mercaptopurine,6-MP)、丁基羟甲苯(butylated hvdroxytoluene,BHT)对BEAS-2B细胞进行染毒,克隆接种。一方面观察比较所产生的集落形态学上的差异,另一方面以电镜、免疫细胞化学染色、核型分析、基因芯片、裸鼠接种成瘤等技术对赛替派染毒BEAS-2B后两种不同形态集落来源的细胞的生长特性、细胞构成、遗传物质改变及其致癌性进行研究。主要实验结果如下: 在遗传毒性致癌物赛替派、环磷酰胺和丝裂霉素C染毒BEAS-2B细胞一定时间后,其传代细胞可形成两种形态差异很大的集落(克隆):一种集落与BEAS-2B细胞集落相似,细胞呈纺锤形、长梭形,胞核卵圆,胞浆少而淡染,细胞排列松散,呈放射状或略呈车轮状,我们称之为松散型集落(S-colony);另一种集落为数比较少,胞体大而圆,胞浆丰富,核嗜碱深染,细胞排列紧密,集落呈圆形边更多还原

【Abstract】 The long-term mammalian induced cancer test is the most classical and reliable method to evaluate the carcinogenicity of chemicals. But it has some disadvantages: the experimental period lasts too long; animal raising condition is strict; the dosage and sensibility of subject animals often affect experimental results. Whereas, the in-vitro mammalian cellular malignant transformation experiment can make up these shortcomings besides evaluating the carcinogenicity of chemicals. So it is an important and valuable complementarity to long-term mammalian induced cancer test. Its initiation point is the change of cellular genetic material induced by carcinogen; its end point is the change of cellular tumorigenicity. The changes of cellular morphology, growth ability and biochemistry phenotype et.al accompany with the transforming process. So, I began my study from the cellular morphologic changes induced by carcinogen, searching for its early-stage morphologic marker.Then,I continuously observe the changes of cellular growth ability,cellulosity,genomics and tumorigenicity of the two cell strains derived from different morphologic colonies.I hope to build a simplified experimental model which use human-originated cells to evaluate chemical’s carcinogenicity.Befor my study, Yuan-subo, Chen-guangyu and Zhou-zhe et.al had made some related researches.They had used thiotepa (TEPA) and cyclophosphamide (CP) to transform the immortalized human bronchial epithelial cell strain BEAS-2B, had inoculated the transformed cells into naked mice which then grew cancer,had researched their cell biology and transformation mechanism.Basing their study,I used the genotoxic carcinogens TEPA, CP,mitomy(?)n C(MMC) and non-genotoxic carcinogens 6-mercaptopurine(6-MP),butylated hydroxytoluene(BHT) to transform the BEAS-2B cells.Then,on the one hand I observed and compared their morphologic differences of transformed colonies;on the other hand I used electron microscope, immunocytochemical stain, karyotype analysis,genechip, inoculation naked mouse et.al techniques to research the two cell strains’ growth properties,cellularity,genomics andcarcinogenicity which derived from different morphologic colonies.The study results as follow:After the genotoxic carcinogens TEPA,CP and MMC induced BEAS-2B cells.its early passage cells can form two different morphologic colonies:One is similar to BEAS-2B’s colony.The cell is fusiform or long-fusiform,nucleus is orbicular-ovate, kytoplasm is poor stain.The colony is loose,scattered and wheel-shaped.So,we named it scattered-type colony(S-colony).The other colony is lesser than S-colony.Its shape is big and round,its kytoplasm is abundant,its nucleus is strong baso-stain.The colony is round and compact.Some cells of this colony grow in ambolayer.So,we named it compact-type colony(C-colony).Whereas,the non-genotoxic carcinogens 6-MP and BHT didn’t induce these changes.The possible reason is that C-colony related to cellular genetic material damage.After the HE stain,we compared the morphologic differences of S-colony cells,C-colony cells and BEAS-2B cells through Mias-300 microimage analytic system.We discovered that the C-colony cells are more larger and more rounder than BEAS-2B cells and S-colony cells.We separated the S-colony and C-colony, subcultured, established two cell strains, and named them BEAS-S and BEAS-C respectively.Their differences exist not only in the cytomorphology but also in the growth properties.The growth rate, cloning efficiency and cellular saturation density of BEAS-2B and BEAS-S cells are bigger than BEAS-C cells’.But only the BEAS-C cells can produce compact-type colonies and transformed focis.Their cell cycle exist differences, too.Coomassie blue stain of the three cell strains showed that the cytoskeleton of BEAS-S and BEAS-C.cells was disordered.lt reflected that thiotepa can change the cellular and colony morphology by impacting their cytoskeleton system.The electron microscope results showed that thiotepa has obvious cytotoxicity.The cellular damage was severe.Edge-collected chromatin and vacuoles existed in nucleus and kytoplasm.The human bronchial epithelial basal cell’s special marker-cytokeratin 340E12 antibody,the goblet cell’s special marker-mucin 5AC(MUC5AC) antibody,the neuroendocrine cell’s special marker— Chromogranin A(ChrA) antibody were used to PowerVision? two step immunocytochemical stain. We discovered that the ChrA didn’t express in the three cell strains.Meanwhile, the MUC5AC was weakly expressed.With the passage,the cytokeratin 34(3E12 expressed increasely in BEAS-C cells as its cellular agglutination and tumorigenicity.But they didn’t change obviously in the BEAS-2B and更多还原