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Effect of Human Papillomavirus Type 16 E5 Gene Transfected to Human Oral Epithelial Cell on the Bilogicol Behaviors of Human Oral Epithelial Cell

Author: YangWenYu
Tutor: ChenChuanJun；JiangTong；JiangXinQuan
School: Anhui Medical University,
Course: Clinical Stomatology
Keywords: Human papilloma virus HPV16 E5 gene Transfection Human oral epithelial cells Cell adhesion Cell spreading Cells
CLC: R780.2
Type: Master's thesis
Year: 2010
Downloads: 46
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Abstract

Purpose of human papilloma virus (human papilloma virus, HPV) is strictly addicted epithelial viruses. People are its only host. Cervical HPV and women not only benign and malignant tumors are directly linked, but also often leads to the oral mucosa, esophagus, pharynx, esophagus and conjunctiva and other parts of the lesion. High-risk subtypes of the virus, and also tend to malignant squamous epithelial cells closely related. E5 is typically found in high-risk HPV, causes benign tumors in the low-risk HPV types are often the default, this feature may be high-risk HPV potentially carcinogenic effects of contact. Most of the current research focus on HPV HPV oncogenes E6, E7 commenced and rarely reported for the E5 gene. The experiment was constructed E5 eukaryotic expression vector was transfected into human oral epithelial cells, and epithelial cell biological characteristics related to a preliminary analysis, the results will be in-depth study of gene function E5 foundation. Methods ① E5 gene PCR amplification and cloning: By comparing high-risk HPV sequences of the E5 and downstream conserved designed a pair of primers (each containing BamHI, Hind Ⅲ Fu storm compulsory restriction sites), PCR reaction, recovered and purified product obtained with restriction sites E5 gene, sequence analysis; connected to the HPV16 E5 plasmid pGEM-T, plasmid pGEM-T-E5, double digestion and sequencing. ② eukaryotic expression vector: Design with a pair of restriction sites (Hind Ⅲ, BamH Ⅰ) of HPV E5 primers were recovered and purified digested under the HPV16 E5 DNA and eukaryotic expression vector pLEGFP-N1l ligated recycling product construct eukaryotic expression vector pLEGFP-E5, the use of PCR amplification, restriction enzyme digestion and sequencing methods were identified. ③ the use of PA317 packaging cell system packaged into retroviral and infect NIH3T3 cells to determine the virus titer. ④ human oral epithelial cells in primary culture cells seeded and using immunohistochemical methods were identified to determine their epithelial origin, the use of retroviral will HPV16 E5 gene transfected into human oral epithelial cells, and the use of PCR, RT -PCR detection method other confirm expression ⑤ using the MTT assay, cell attachment and spreading test, study HPV16 E5 gene on human oral epithelial cell biology traits. Results ① construct containing HPV16 E5 gene eukaryotic expression vector pLEGFP-E5. Restriction enzyme digestion and DNA sequencing revealed, E5 is properly inserted into the multiple cloning site of the plasmid vector within. ② the pLEGFP-E5 transfected PA317 packaging cells obtained retrovirus eukaryotic cells can be transfected. ③ The HPV16-E5 gene was transfected into human oral epithelial cells by PCR and RT-PCR validation E5 gene can be expressed in transfected cells biological behavior tests showed: HPV16 E5 gene can promote human oral epithelial cell adhesion and proliferation (P lt; 0.05), while no significant effect on cell spreading (P gt; 0.05) Conclusion HPV16 E5 gene can increase the activity of human oral epithelial cells and promote cell adhesion and proliferation, extend its life cycle.

Effect of Human Papillomavirus Type 16 E5 Gene Transfected to Human Oral Epithelial Cell on the Bilogicol Behaviors of Human Oral Epithelial Cell

Abstract

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