| Object:1. To establish Tca83-GFP-luc monoclonal cell line which stably expressing luciferase by lipofection, and demonstrate that there’s no difference in growth and activity between Tca83and Tca83-GFP-luc, thus create cytology foundation for subsequent bioluminescence imaging in vivo.2. Using Tca83-GFP-luc cell to establish orthotropic and heterotopic models of human tongue squamous cell carcinoma in nude mouse, observing the the biological behavior and condition of lymph node metastasis in vivo and providing a basis for the application of carcinoma models in animals so as to prevent and treat tongue carcinoma.Method:1. Recombinant plasmid containing luciferase gene and GFP gene was transfected into Tca83cells by lipofection, and a monoclonal cell line stably expressing luciferase was screened out by G418. The biological characteristic of Tca83-GFP-luc cells was detected by MTT, flow cytometer and Westernblot. The expressing of luciferase in Tca83-GFP-luc cells was detected by flow cytometer and in vivo imaging system.2. The establishiment of subcutaneous heterotopic models of human tongue squamous cell carcinoma in nude mouse:randomly divided20mouse into two groups, subcutaneously injected cell suspension on hind leg, the experimental group of15mouse with Tca83-GFP-luc cell and the control group of5mouse with Tca83cell to found heterotopic model. Bioluminescence imaging system recorded the growth and metastasis of the tumors in vivo and the luminous intensity was measured. After continuous observation of4weeks, analyzed the relations between tumor size and luminous intensity. Then the nude mice were executed and the tumor tissue sections were observed under fluorescence microscope.3. The establishiment of orthotropic models of human tongue squamous cell carcinoma in nude mouse:randomly divided20mouse into two groups, injected cell suspension on left margin of tongue, the experimental group of15mouse with Tca83-GFP-luc cell and the control group of5mouse with Tca83cell to found orthotropic model. Bioluminescence imaging system recorded the growth and metastasis of the tumors in vivo and the luminous intensity was measured. After continuous observation of4weeks, analyzed the relations between tumor size and luminous intensity.Then the nude mice were executed and the tumor tissue section were observed under fluorescence microscope.Result:1. The luciferase gene can be transfected into Tca83cell by lipofection, and a monoclonal cell line which stably expressing luciferase was acquired after the G418selection. The detection of MTT, flow cytometer and Westerblot demonstrated that Tca83-GFP-luc cell line showed similar biological characteristics to the original Tca83cells. The expressing rate of Tca83-GFP-luc, detected by flow cytometer, reached up to96%. The quantity of cells and luminous intensity reflected positive linear correlation (r2=0.9510, P<0.01)2. Tumor of heterotopic experimental group was formed in6th-11th day after the injection. The rate of tumor formation was73.33%(11/15), While the heterotopic control group formed tumor in7th-9th day with a80%(4/5) formation rate. Tumor of orthotropic experimental group was formed in6th-12th day after the injection. The rate of tumor formation was86.67%(13/15), While the orthotropic control group formed tumor in6th-10th day with a60%(3/5) formation rate.3. In vivo bioluminescence system caught the luminous signals of heterotopic experimental group in the first week. During the observation of four weeks, luminous signals strengthened gradually as the extension of time. The tumor size and luminous intensity reflected positive linear correlation (r2=0.9872,P<0.01).4. In vivo bioluminescence system caught the luminous signals of orthotropic experimental group in the first week. During the observation of four weeks, luminous signals strengthened gradually as the extension of time. The tumor size and luminous intensity reflected positive linear correlation (r2=0.9809,P<0.01) 5. The tumor tissue sections of orthotropic and heterotopic models were under fluorescence microscope. Weak fluorescence was caught in the cytoplasm of Tca83-GFP-luc.Conclusion:1.The luciferase gene can be stably transfected into Tca83cell by lipofection, and a monoclonal cell line which stably expressing luciferase can be acquired after the G418selection and the Tca83-GFP-luc line can be applied to animal bioluminescence imaging in vivo.2. The establishment of a heterotopic mode of human tongue squamous cell carcinoma in nude mouse, and the observation by bioluminescene imaging system in vivo demonstrated that the expressing of luciferase strengthened alone with the growth of tumor size. Mouse tongue carcinoma orthotropic model labeled by luciferase could observe the growth and metastasis of the tumor intuitively, continuously and sensitively. It supplied an important experiment medium in studying development and drug therapy of tongue carcinoma. So it’s a desired animal model for researching the growth and metastasis of human tongue carcinoma in vivo. |
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