Igfbp5 In Osteosarcoma Growth And Metastasis Mechanism Of Study

PART ONE LUCIFERASE TAGGED MG63 CELL LINE AND ESTABLISHMENT OF SUBTYPE OF THE CELL LINEObjective: using the luciferase to tag the MG63 cell line by retro-virus, culture the cells which got from the lung metastasis sites, then got the cell lines of MG63.1 and MG63.2.Methods: culture the MG63 cell lines in vivo, using the cells which was tagged by luciferase, the cells were injected in the intra-tibia, about 5weeks, when we found the signal from the lungs if we made the image by Xengon machine, the lung tumors which were metastasized from the legs were taken and cultured in the incubator, this is the cell line which we definite as MG63.1, repeated the procedure and then we got the cell line MG63.2.Results: (1)Using the classifying method by antibiotic BSD, we successfully got the cell line MG63-luc. (2)Successfully established the cell lines MG63.1 and MG63.2 by injecting the cell line MG63-luc to the intra-tibia of the 4 weeks old nude mice.Conclusion: Successfully established the cell line MG63-luc which tagged luciferase and the subtype cell lines MG63.1 and MG63.2, and this made us easier further research on the metastasis of the osteosarcoma. PART TWO THE IN VITRO STUDY OF THE SUBTYPE CELL LINE OF MG63Objective: through series methods of studying the metastasis ability, compare the metastasis ability between MG63 and MG63.2 and prove the MG63.2 has a stronger ability to metastasis than MG63.Methods: Draw the proliferation curve of MG63 and MG63.2, wound healing test, cell adhesion test, matrigel invasive test, cell migration test, and the seven genes MMP2,MMP9,TIMP1,TIMP2 and Ezrin which had relationship with metastasis run the RT-PCR and western-blotting to test the expression of RNA and protein.Results: (1) Comparing with the MG63, MG63.2 grew slower and the cell cycle was a little longer. (2)Cell adhesion test showed MG63.2 had a stronger ability than MG63. (3)Matrigel migration test showed MG63.2 had a stronger ability than MG63. (4) Both the RT-PCR and western-blotting test showed that the expression of MMP2 and MMP9 decreased while the TIMP2 and ezrin increased in these two cell lines.Conclusions: Through the test of cell proliferation curve, wound healing test, cell adhesion test, matrigel invasive test and RT-PCR and western-blotting of 5 genes MMP2, MMP9, TIMP1, TIMP2, ezrin showed that MG63.2 definitely had the ability to metastasis than MG63 in vitro. PART THREE THE IN VIVO STUDY ON THE SUBTYPE CELL LINE OF MG63Objective: To study the tumor growth and the ability to metastasis by the animal model which were established by injecting the cells to the intratibia between the MG63 and MG63.2.Methods:Inject the suspended cells MG63 and MG63.2 diluted by PBS to the intra-tibia of the nude mice. Observe the tumor grow and lung metastasis. HE staining proved that the MG63.2 cans metastasis to the lungs.Results:(1) The model which can be formed by injecting the cells to the intra-tibia was stable and reliable. (2) The tumor growth in the primary site showed the MG63.2 grew a little faster than MG63. (3)Xengon image showed MG63.2 had the stronger ability to form the tumor metastasis. And there was significant difference between these two groups. (4)HE staining of the histology showed the quantity of the tumor metastasis was more in the MG63.2.Conclusions: MG63.2 had the stronger ability of tumor forming and metastasis than MG63. PART FOUR THE RESEARCH ON THE EFFECT OF TUMOR GROWTH AND METASTASIS IN VITRO AND IN VIVO STUDY FOR THE GENE IGFBP5Objective: To construct the ad-easy virus of IGFBP5 and RNA interference virus, do the research on effect of the tumor growth and metastasis for the gene IGFBP5.Methods:Construct the ad-virus of IGFBP5 and its RNA interference virus, infected the mefs,C3H10 and C2C12 three cell lines, read the ALP reading, analysis the effect on the differentiation of IGFBP5. Draw the proliferation curve for the 143B,MG63-P,MG63.1 and MG63.2 after the IGFBP5 virus infecting. Study the matrigel migration ability for the IGFBP5 virus infected cell lines. Measured the tumour growth and Xengon image analysis the signal express in vivo. Counted the quantity of the metastasis in the lungs and study the effect of IGFBP5 in the metastasis.Results:(1)ALP reading proved that IGFBP5 can promote C3H10, mefs and C2C12 differentiation and inhibit the proliferation. For the SiIGFBP5, it is opposite. (2) IGFBP5 can inhibit the osteosarcoma cell lines 143B-luc, MG63, MG63.1and MG63.2 growth while the SiIGFBP5 was the opposite. (3)Wound healing test, matrigel invasive test proved that IGFBP5 can inhibit the tumour invasive while the SiIGFBP5 was the opposite. (4)Tumour measurement and Xengon image showed that IGFBP5 can inhibit the tumour growth, while the SiIGFBP5 is the opposite. Conclusions: IGFBP5 can inhibit the tumour growth, promote the tumour differentiation. It played an important role in the tumour growth and metastasis.