| ObjectiveIn recent years, the incidence of lung cancer rises year by year. Non-small cell lung cancer (NSCLC) accounts more than 80% of all cases of lung cancer. And because of the distant metastasis, 5 years survival rate of lung cancer patients is only about 15%. Although majority of the tumor come from monoclone, as a result of the tumor genetics instability and the target organ selective action, the performance of tumors variate unceasingly and some subclone obtains the metastatic phenotype. During the process, many changes of gene expression are very important for the metastatic phenotype. Finding these genes, detecting the mechanism, designing the block spot and inhibiting tumor metastasis are much meaningful for theory and clinical application.We established a mouse model of lung cancer with metastasis by using 95 D cell line which expressing GFP(Green fluorescent protein) stably, and purified the tumor cells in the subcutaneous primary lesion and the metastatic lung lesion with FACS (fluorescence-activated cell sorting). We studied and analyzed the gene expression profiles of the two lesions by using the technology called SAGE (serial analysis of gene expression) for screening the differently expressing genes for further studying the metastatic mechanism of NSCLC.Method1. 95D cell line was infected by the retrovirus which mediating expression of an enhanced green fluorescent protein gene and selected by G418 (neomycin). The expression of GFP of the infected cells was observed by fluorescence microscopy and detected by flow cytometry. Multiple biological behaviors, such as the growth curve and cell adherent rate were compared between the infected and uninfected cells.2. Nude mice were subcutaneously inoculated with 95D/GFP-1.And the mice were visualized with fluorescent stereo microscope and anatomized after 2 months to find the metestatic lesion. Then purified the tumor cell in the lesion with FACS.3. Establish two expression sequence tags libraries of the primary lesion and lung metastatic lesion with SAGE method. Use the SAGEMAP database of NCBI(National Center of Biotechnology Information) and compare the two libraries to screening the differential expression genes.Result1. 95D cell line can be infected efficiently by retrovirus. The selected cellls can express the GFP stably, efficiently and persistently. There was no significant difference between infected cells and uninfected cells in their biological behaviors.2. Nude mice were subcutaneously inoculated with 95D/GFP-1 cells .The lymphonode, lung and liver metastatic lesions can be finded.The GFP can de detected in primary lesion, lung metastatic lesion and metastatic lymphonode.3. The tumor cells in primary and metastatic lung lesion were purified by FACS.Two SAGE tags libraries of the two lesions were established, including 5420 and 5016 long sage tags. We compared the two libraries and got 20 down-regulated genes such as CD9,COMMD6,CD82. And 33 up-regulated genes such as TMSB4,IRAK1, ICMT of the lung metastatic lesion.Conclusion1. The establishment of 95D/GFP-1 can offer a useful cell line to investigate the mechanisms of tumor's infiltration and metastasis.2. Subcutaneous inoculation of 95D/GFP-1 cell line in nude mice can form metastatic lesions in liver, lung, and lymphonode. We successfully established an animal model for lung cancer cell metastasis.3. Two LongSAGE libraries of subcutaneous primary lesion and metastatic lung lesion were constructed. We got 53 differentially expression genes through comparing the two libraries. The change of these genes'expression may relate with the metastatic potential of lung cancer. We will further study the mechanism of these genes with NSCLC metastasis.
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