| Objective: 1. OLC1 related information were obtained by bioinformatics method. 2. H1299-OLC1 stable cell line using limiting dilution screening methods was formed. OLC1 as candidate proto-oncogene and some functional research have been performed. The series studying inducing the cells on H1299, H1299-OLC1, H1299-pcDB examined with MTS assay and colony forming assay, cell cycle on different time interval were test by flow cytometry etc. 3. H1299-OLC1 as model cell line, OLC1 as drug target, 1180 small molecular compounds as candidate drug for screening were also studied and drug small molecular compounds'screening experimental platform was constructed. 4. Effect on proliferation of lung cancer stable expression cell line of four kinds of anticancer drugs and Xinjiang Pleurotus Ferulae's extracts were detected.Methods: 1. Prediction and analysis of OLC1's chromosome location, amino acid sequences, protein domain, homologous sequences information by bioinformatics was practiced; 2. Research of OLC1 stable expressed cell lines on H1299 cells by limiting dilution method; western Blotting Detection OLC1 protein's over expression were identified and functional identification on OLC1 as a candidate pro-oncogene was characterized; MTS proliferation assay were used to record H1299, H1299-OLC1, H1299-pcDB cells growth condition and the growth of those cells was also examined with colony forming assay; the H1299-OLC1 and H1299-pcDB cell cycle in 24hr, 48hr and 72hr interval was demonstrated with flow cytometry. 3. 1180 candidate drug's screening with fluorescence probe method, Calcium AM as fluorescence probe, colorimetric method was performed by GENios Pro reader, the green fluorescence intensity were tested at 535nm, excited wavelength at 484nm. Cell morphology was observed by fluorescence convert microscope. 4. OLC1 as drug target experiment: MTS cell proliferation assay applied on Xinjiang Pleurotus ferulae's extracts and four kinds of anticancer drugs were carried out. Results: 1. OLC1 located on 16q22.3 region, coded 364 amino acids, no transmembrane domain and singal peptide. OLC1's subcellular location: nuclear 56.5%, cytoplasmic 30.4%, mitochondrial 13.0%. And it has two conserved domains, DUF292 domain (33-139) and praline-rich domain (220-278) was observed respectively. It also widely distributed in many kinds of tumor cells and tissues was found. 2. H1299-OLC1 cells show transformed cells'characteristics, such as proliferation speed up, Compared to H1299-pcDB, the numbers of colonies significantly increased than those of H1299-pcDB(P<0.05), G1 phase cells rate increased significantly than that of H1299-pcDB(P<0.05) after 24hr, 48hr, 72hr different interval ; 3. Calcium AM assay drug screening platform were constructed, and 6 kind of small molecular compounds at 1000nm concentration could inhibit H1299-OLC1 cell line's proliferation significantly and were selected as candidate drug by primary screening; 4. H1299-OLC1 is the best drug resistance among three kind of cell lines; 5. Xinjiang Pleurotus ferulae's extracts significantly inhibited H1299-OLC1 cell lines proliferation;Conclusions: 1. OLC1 was widely distributed in many kinds of tumor cells and tissues. It also has revealed special conserve domains and functional sites. The bioinformatics analysis results suggest that OLC1 may be a very important functional gene. 2. Limiting dilution method was applied to screen monoclonal stable cell lines and have got four kind of H1299 cell model; 3. OLC1 showed some pro-oncogene characteristics in vitro experiment; 4. Calcium AM assay drug screening platform were constructed; 5. 6 kind of small molecular compounds were selected as candidate drug by primary screening; 6. Xinjiang Pleurotus ferulae's extracts significantly inhibited H1299-OLC1 cell lines proliferation at 2.5% concentration was observed. |
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