kb细胞中spata5l1基因生物学功能的初步分析-preliminary analysis of biological function of spata 5l1 gene in kb cells.docxVIP

PAGE PAGE IV Preliminary Study on Biological Functions of SPATA5L1 Gene in human KB cell Abstract Preliminary Study on Biological Functions of SPATA5L1 Gene in human KB cells Abstract Background and objection: Earlier biochip screening analysis and flowing Western blot assay found that the expression of gene was increased in the KB cell after treated with 2-8 Gy ionizing radiation and/or moderate paclitaxel. The function of this gene in human tumor cell has not been reported yet. The purpose of this project was to realize the function of SPATA5L1 gene by constructing a stable KB cell line with SPATA5L1 over-expression plasmid and applying a variety of experimental methods for detecting the tumor cell biological characteristics. It will be benefit to explore the possible molecular mechanism of the cancer recurrence and metastasis after treatment, find the melecular site for tearget diagnosis and treatment. Method: 1, A recombinant mammalian plasmid pEX-1 carrying a full-length of SPATA5L1 cDNA (pEX-1-SPATA5L1) was transfected into human oral cancer KB cell line with lipofectamine transfection assay. The G418-resistant clones were obtained after incubation in G418-containing medium for three weeks. Alterations of SPATA5L1 mRNA and protein level were monitored by Real-time PCR and Western blot assay. 2, Cell proliferation was assessed by CCK-8 assay. The cell cycle distribution and apoptosis were detected by flow cytometric analysis. Western blot assay was employed to evaluate the alterations of proteins related to cell cycle and apoptosis. 3, Wound healing assay and transwell chamber assay were performed to assess the ability of cell migration. Scanning electron microscope (SEM) as well as optical microscopy was used to observe cell shape changes. Western blot assay was employed to evaluate the alterations of protein related to cell shape changes. Results: 1, The sequence result of pEX-1-SPATA5L1 approved that the gene of SPATA5L1 was inserted into the eu