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CH50, a Recombinant Polypeptiide of Fibronectin, Inhibits the Metastasis of Melanoma B16 Cell in Vivo

Author: ZhuMing
Tutor: FengZuoHua；ZhangGuiMei
School: Huazhong University of Science and Technology
Course: Biochemistry and Molecular Biology
Keywords: Tumor invasion Tumor metastasis Recombinant polypeptide Gene Expression Fibronectin
CLC: Q789
Type: Master's thesis
Year: 2007
Downloads: 51
Quote: 0
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Abstract


Objective To study the polypeptide CH50 mouse melanoma B16 cells in vivo transfer of recombinant fibronectin (FN) , the possible molecular mechanisms to investigate CH50 polypeptide inhibit tumor metastasis . B16 melanoma cells were cultured in vitro , CFSE-labeled with a fluorescent dye , and 24 h after inoculation spleen spleen , liver, lung frozen sections observed tumor cell invasion in the three tissues ; B16 cells inoculated from the spleen , and the establishment of in vivo tumor based on fluid dynamics in vivo gene transfer in animal models ; CH50 polypeptide expression in mice in vivo transfection method for gene therapy ; RT-PCR was used to detect the CH50 mRNA expression in the liver tissue , Western-blot detection CH50 polypeptide expression ; by comparing the original CH50 polypeptide therapeutic effect of differences observed median tumor nodules and metastatic nodules in the number, size , and distribution ; gelatin electrophoresis (zymography) detecting the edge of the tumor tissue of MMP - 2 , MMP- 9 protein expression differences . Results from spleen inoculation labeled with a fluorescent dye CFSE melanoma B16 cells after 24 h , in the spleen to form a fluorescent nodules . 14 days after inoculated from the spleen B16 cells formed in the spleen of the primary tumor , metastatic tumor nodules has formed the surface of the liver to the first 35 days , the animal model can be used for the experimental study of the internal organs (spleen to the liver ) tumor metastasis . 6 h after tail vein injection of pCH510 plasmid in liver tissue detected CH50 mRNA expression was strongest , 24 h , 72 h significantly weakened . Western-blot detected expression of CH50 polypeptide . Naked eye observation the spleen surface of pCH510 group the number of tumor nodules with pcDNA3.1 group , blank control group were not significantly difference , but the the pCH510 group the tumor nodules the size of the pcDNA3.1 group , blank control group significantly reduced compared . No tumor nodules of pCH510 group liver surface , while the surface of the liver of the two control groups were visible metastatic tumor nodules . Three experimental groups of pulmonary surfactant , there was no tumor nodules . Splenic tumor tissue in the two control groups could be detected in the expression of MMP-2 and MMP-9 , while the pCH510 group MMP-2 levels were significantly decreased the expression of MMP -9 was completely inhibited . Conclusion CH50 polypeptide by MMP -2 , MMP - 9 protein expression inhibition to suppress the tumorigenic ability of melanoma B16 cells and in vivo invasion and metastasis .

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