高表达精脒合成酶抑制人宫颈癌Caski细胞的生长和迁移-生物化学与分子生物学专业论文.docxVIP

三峡大学硕士学位论文 三 峡 大 学 硕 士 学 位 论 文 Abstract Objective: To study the effect of SPDS overexpression on polyamine content, proliferation and migration of human Caski cervical cancer cells. Methods The SPDS gene was cloned out from the cDNAs derived from the human Caski cervical cacer cell by Nest-PCR and cloned into the eukaryotic expression vector pcDNA3.1(-). Resulted plasmid pcDNA3.1(-)/SPDS and empty plasmid pcDNA3.1(-) were transfected respectively into Caski cells by Lipofectamine-2000 reagent to obtain Caski cell line with SPDS overexpression (Caski/SPDS) and control cell line (Caski/3.1). Western blot and RT-PCR were used to determine the expression of SPDS in the protein and mRNA levels in above cell lines. MTT and FCM were performed to observe the cell proliferation and cell cycle.The polyamine levels were detected by RP-HPLC. The mRNA levels of polyamine biosynthetic enzymes (ODC, OAZI, AMDC, SMS) and catabolic enzymes (SSAT, SMO, APAO) were measured by QT-RT-PCR. Traswell technique was used to assay cell migration of the Caski/3.1 and Caski/SPDS cells. Western blot assay was used to determine Annexin A2 expression in protein level. Results 1) A Caski cell clone with SPDS overexpression (Caski/SPDS) was obtained successfully. The expression of SPDS in mRNA and protein levels was obviously higher in the Caski/SPDS cells than that in the Caski/3.1 cells. 2) The elevated mRNA levels of ODC AMDC SMS SMO SSAT and PAO were observed in the Caski/SPDS cells. 3) The SPDS overexpression could inhibit cell proliferation and effect cell cycle that resulted in an increased cell ratio in G0/G1 phase and decreased cell ratio in S phase. 4) The results of the RP-HPLC shown that, compared with Caski/3.1 cells，putrescine content in the Caski/SPDS cells was dramatically decreased but the contents of spermidine and spermine were both increased in a small range, and the total content of polyamine was reduced. 5) SPDS overexpression dramatically suppressed the Caski/SPDS ce