关键词: 宫颈癌, TPX2-shRNA, 慢病毒

Abstract: Objective To construct the Lentiviral RNA interference vector targeting TPX2 and obtain the human cervical cancer hela cell line stably infected by TPX2-shRNA ,which laid research foundation for studying the relationship between human cervical carcinoma and TPX2 gene. Methods By targeting TPX2 gene, four double-stranded DNA hairpin structures corresponding to shRNA were designed, synthesized and connected with Pglv2-U6-Puro to construct the recombinant plasmids.Then these recombinant plasmids were transformed into DH5α competent cells. The positive clone was extracted and transfected into 293T cells for virus packages after sequenced correctly.Human cervical carcinoma HeLa cell line infected by these recombinant Lentiviral was screened by Puromycin, then stable cell lines was obtained.The silencing effect of TPX2 in HeLa cell was detected by Real-time fluorescent quantitative PCR and Western blot. Cell cycle and cell apoptosis wer detected by Flow cytometry.Results Sequencing results confirmed that 5 Lentiviral is packaged successfully. The steady cell lines transfered TPX2-shRNA was screened with 0.4ug/ml puromycin. HeLa cells infected by recombinant lentivirus all play the gene silencing effect especially in the group of TPX2-shRNA-1.In the group of TPX2-shRNA-1,TPX2mRNA (0.21 ± 0.07) and protein(0.19 ± 0.28) relative expression levels are lower than those in the control group (1.08 ± 0.07) (p<0.01)and(0.64 ± 0.03)(p<0.01)respectively;G2 and S-phase cells are higher than those in the control group (p<0.05)and the apoptosis rate was significantly more than those in the control group (p<0.05).Conclusions.The effective TPX2 genetic interference sequence was Obtained, Lentiviral vectors carrying TPX2shRNA was successfully constructed,and the HeLa cell line with TPX2 Silenced were successfully screened,which lay the research foundation for the role of TPX2 in cervical cancer.

Key words: cervical cancer, TPX2-shRNA, lentivirus