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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

Author: ZhaoXian
Tutor: WangJianSong
School: Kunming Medical College
Course: Surgery
Keywords: Balb / c mice Bladder cancer Cell lines Luciferase Lentiviral Plasmid Vivo imaging technology
CLC: R737.14
Type: Master's thesis
Year: 2011
Downloads: 73
Quote: 0
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Abstract


OBJECTIVE: The existing portable tumor strain and for efficacy trials of mouse tumor lines, especially the scarcity of murine bladder tumor cell strains. The Experiment 1 Balb / c mice bladder cancer induced by feeding the OH-BBN method, the initial establishment of Balb / c mice bladder cancer BBN1617 of for bladder tumors basic research, experimental therapeutics and new cancer drug screening provides a valuable in animal models. Experiment 2 Construction of luciferase reporter gene recombinant expression plasmid pSin-EF2-luciferase, the luciferase reporter gene is transferred into the EJ cells detected after screening to obtain a stable expression cell strain for mouse bladder in situ transplanted tumors in vivo imaging monitoring. Methods: OH-BBN-induced OH-BBN induction of Balb / c mice with bladder cancer initially established and BBN1617 cell lines 1.Balb / c mouse bladder tumor: 6-week-old male Balb / c mice were randomly divided into 2, bladder cancer induced experimental group (15), 24 weeks given drinking water containing 0.05% OH-BBN, carcinogens in drinking water is changed 2 times a week. The control group (10), to give the water feeding. Two groups after 24 weeks of feeding water feeding. 2.Balb / c bladder cancer BBN1617 in mice cell lines initially established: for bladder tumor tissue, HE staining were observed, collagenase digestion and bladder tumors in primary cultured and subcultured. Preliminary identification of the 3.Balb / c bladder cancer BBN1617 in mice cell lines: cell growth curve and pathological observation method to determine cell proliferation and growth cycle. Embedding OH-BBN-induced tumor inoculation in Balb / c mice subcutaneously subculture, observation of xenografts HE staining. , Luciferase-labeled human bladder cancer cell lines EJ-LUC construct using the method of PCR amplification of the luciferase gene so that it has specific CGGACTAGT connector, to connect to, transformation, plasmid was extracted and cloned into pSin-EF2 carrier to obtain entry clone. Product after PCR and sequencing identified to construct the recombinant expression plasmid pSin-EF2-luciferase agarose gel electrophoresis and sequenced. 3 HEK293T packaging cells transfected with the recombinant lentivirus. Recombinant lentivirus infection of human bladder cancer EJ cells after amplification, screening and detection of luciferase reporter gene protein activity. Will stabilize EJ cells in vitro expression of the luciferase reporter gene were cultured, transurethral perfusion image acquisition to the bladder cavity of nude mice in vivo imaging system. : OH-BBN-induced Balb / c mice bladder, and BBN1617 of cell lines established feeding to 25 weeks into the tumor rate was 66.67% (10/15), control tumor formation rate. The mice were killed and dissected visible bladder tumor foci parts pale, convex surface, and loss of elasticity. Stereological microscope, tumor-infiltrating bladder muscle, were exogenous or infiltrative growth. HE staining cell tumors 1-2 nucleoli, nucleoli, split cytoplasm deeply stained. Primary cultured bladder tumor cells subcultured three generations after the nucleus and cytoplasm were increased (\3 in vitro murine bladder cancer cell growth curve pale \4.Balb / c mice were inoculated subcutaneously with OH-BBN-induced bladder cancer tumor mass about 1 week to tumor xenografts HE staining and in vitro growth curve is no significant difference in the primary bladder tumor. Second, luciferase-labeled human bladder cancer cell lines the EJ-LUC build 1.PCR amplified with specific primers luciferase reporter gene, agarose gel electrophoresis product about the size of 1617bp, with the target gene fragment the same size. The beetle luciferase was cloned into pSin-EF2 vector, and transformed into E. coli competent cells. The selection of five ampicillin-resistant single colony shake culturing 1617bp size fragments were PCR agarose gel electrophoresis. Recombinant plasmid pS in the the-EF2--luci fErase sequencing of the results confirmed that the target gene luciferase reporter gene sequence with GenBank published results fully in line. 3 using the Promega luciferase activity assay kit for the identification of the the Lentivirus-luci function activity: Lentivirus-luci constructed in this study results demonstrate stable expression of the luciferase enzyme. EJ cells, in vivo imaging system. Intravesical containing the luciferase reporter gene can clearly see the imaging of tumor cells in nude mice bladder. Conclusion: OH-BBN-induced Balb / c mice bladder, and BBN1617 of cell lines to the initial establishment of OH-BBN feeding method can be successfully induced Balb / c mice bladder tumor. 2-induced bladder tumor in vitro culture, subculture and subcutaneous tumor, HE staining showed that the induced tumors and subcutaneous tumors are bladder cancer. 3 subcutaneously transplanted tumor passaged culture passaging of the cells with primary bladder cancer cells without significantly different culture, cell growth curve are found. Malignant cell growth characteristics. , Luciferase-labeled human bladder cancer cell line EJ-LUC build a successful build luciferin pigment enzyme Report gene recombinant plasmid pSin-EF2-luciferase. 2 the entry clone products and broth PCR products of agarose gel electrophoresis, identified with the target the same gene, are 1617bp size of the product. Dye enzyme Report. Luciferin constructed recombinant plasmid pSin-EF2-luciferase luciferase reporter gene transfected into a the EJ cell and stable expression can be used for mice in vivo imaging observation. 4 EJ-LUC cells in nude mice intravesical perfusion in vivo imaging.

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CLC: > Medicine, health > Oncology > Genitourinary tumors > Urinary tumors > Bladder tumor
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