Study On The Cell Strains Derived From Different Morphologic Colonies In The Process Of Malignant Transformation Of Immortalized Human Bronchial Epithelial Cells

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...