OH-BBN Induced Balb/c Mice Bladder Cancer And The Initial Establishment Of BBN1617 Cell Lines & Luciferase Labeled Human Bladder Cancer Cell Line EJ-LUC Construction

Objective:At present, transplantable tumor line and tumor strain of mice for efficacy test are limited in our country. In particular, tumor strains of mice with bladder cancer is scarce. The first experiment, through the method of feeding OH-BBN induced Balb/c mice bladder cancer.In order to initially established cell lines BBN1617 of murine bladder cancer, and provide a valuable animal model for basic research in bladder cancer, experimental treatment and anticancer drug screening.The second experiment, by constructing luciferase reporter gene expression plasmid pSin-EF2-luciferase,make the luciferase reporter gene into the EJ cells. After the screening test, to obtain stable expression cell lines, for monitoring orthotopic bladder tumors in mice in vivo imaging.Methods:The first part:OH-BBN induced Balb/c mice bladder cancer and the initial establishment and identification of BBN1617 cell lines1 OH-BBN induced Balb/c mice bladder cancer:6 week old male Balb/c mice, randomly divided into 2 groups, the group induction of bladder cancer as the experimental group (15 mice), given with 0.05% OH-BBN in the drinking water for 24 weeks,replacement of carcinogens in drinking water 2 times a week.Control group (10 mice) given tap water feeding. After the two groups were fed for 24 weeks were fed with tap water.2 The initial establishment and identification of BBN1617 cell lines:After obtained bladder tumor, H&E staining were observed under microscope. By collagenase digestion primary cultured bladder tumor and subculture.3 Preliminary identification of Balb/c mice with bladder cancer cell lines BBN1617:Using cell growth curve and pathological observation method and other methods to determine the cell proliferation and cell cycle.4 By embedding the BBN-induced tumor inoculated in the back subcutaneous of Balb/c mice and subculture. H&E staining observed transplantable tumor.The second part:Luciferase labeled human bladder cancer cell line EJ-LUC1 By PCR method amplified luciferase gene and made it had a specific CGGACTAGT connector,by ligation, transformation, extraction and other methods and cloned into plasmid pSin-EF2 vector for entry clone.2 PCR and sequencing and identify product was correct, then recombinant expression plasmid pSin-EF2-luciferase. Agarose gel electrophoresis and identified and sent to sequence.3 Transfected HEK293T cells and packaged cell then obtained the recombinant lentiviral. After amplified recombinant lentiviral, infected the EJ human bladder cancer cells,screened and test luciferase reporter gene activity. 4 EJ cells in which stable expression of luciferase reporter gene were amplified and cultured in vitro, transplanted into the nude murine bladder cavity via urinary, images collected in vivo imaging system(IVIS).Results:First:OH-BBN induced Balb/c mice bladder cancer and the initial establishment and identification of BBN1617 cell lines1 The rats had been feed to 25 weeks, experimental group tumor formation rate was 66.67%(10/15), the control group tumor formation rate was 0. After the mice were sacrificed and dissected we could observed parts of bladder tumors pale, convex surface, and loss of elasticity, tumor invaded muscle of the bladder could be saw in the stereoscopic microscope were exogenous or invasive growth.2 H&E staining showed tumor cells had 1 or 2 nucleoli, large nucleoli, and had a split-phase, deep cytoplasmic. After bladder tumor cells cultured in vitro and subculture 3 generations, both nucleus and cytoplasm increased(appearance like "oil omelette"). 3 Cell growth curve of rat bladder cancer cells which culture in vitro is shallow "S"-shaped. Cells would enter exponential growth phase in 3 to 5 days.4 Balb/c mice were inoculated subcutaneously tumor block of which OH-BBN-induced bladder cancer could be form tumors about one week. H&E staining and tumor growth curves were cultured in vitro have no significant difference with in primary bladder tumors.Second:Luciferase labeled human bladder cancer cell line EJ-LUC1 PCR amplified product which with specific primers of the luciferase reporter gene, identified by agarose gel electrophoresis, the size of the product is about 1617bp, consistent with the target gene fragment.2 The firefly luciferase was cloned into pSin-EF2 vector and transformed into e.coli competent cells.We selected five ampicillin-resistant bacteria of single colony to shake and culture. After PCR amplified bacterial suspension, identified by agarose gel electrophoresis to obtain 1617bp fragment size. Restructuring plasmid pSin-EF2-luciferase sequencing confirmed that luciferase gene reporter gene sequence is full in line with the result of published Genbank.3 Using promega company U.S. luciferase activity detection kit identified the functional activity of Lentivirus-luci:The result proved that the Lentivirus-luci which constructed in this study could stably express luciferase.4 Intravesical instillation EJ cells with luciferase reporter gene, in vivo imaging systems could clearly see that the bladder tumor cells imaging in nude mice.Conclusion: First:OH-BBN induced Balb/c mice bladder cancer and the initial establishmentand identification of BBN1617 cell lines1 We have successfully obtained Balb/c mice bladder tumors by OH-BBN feeding induced method.2 Bladder tumor were cultured and subculture, and transplanted tumors were passaged. H&E staining confirmed that they were bladder cancer, it is no significant differences between them. 3 Transplanted tumors cells subcultured in vitro and subculture cells of primary bladder cancer has no significantly differents between them. Cell growth curve characteristics were in line with the growth characteristics of malignant cells.Second:Luciferase labeled human bladder cancer cell line EJ-LUC1 Fluorochrome reporter gene plasmid pSin-EF2-luciferase was constructed successfully.2 Agarose gel electrophoresis entry clone products and bacilli PCR products, through identified they were both consistent with the target gene, the size of the products were both the 1617bp.3 The luciferase reporter gene which transfected into the EJ cells can express steadily and can be applied to observe in mice in vivo imaging.4 In vivo imaging systems could clearly see the nude mice bladder cancer EJ-LUC cell imaging.