Study On The Expression Of IL-1α In The Chlamydia Trachomatis Infected Hela229Cells And Establish The Stable Cell Lines Of IL-1α ShRNA

Chlamydia trachomatis (Ct) is a common pathogen which is endotrophic obligatory parasitism. Human infected of Ct can lead to genitourinary infections, and subsequent complications such as infertility, ectopic pregnancy and cervical squamous carcinoma. Ct induce epithelial cells to produce many proinflammatory cytokines such as IL-1、IL-6and TNF-α et al. IL-1α is one of the pyrogen in inflammation and immunal regulation. The study of IL-1α on primary inflammation can establish the experimental foundation for mechanisms, therapy and prevention of Chlamydia.Objective1. To investigate the expression level of IL-la in Ct infected HeLa229cells and identify the mode of IL-la expression early in Ct infected period;2. To set up a stage for studying the mechanism of Ct induced inflammation through silence of IL-1α in HeLa229cells.Methods1. Treat HeLa229cells with Ct, then Giemsa’s staining and observed by light microscopy;2. Through ELISA Compare the intracellular and extracellular production of IL-la production after Ct infected HeLa229cells;3. Establish pRNAT-shRNA IL-1α plasmids (IL-la shRNA plasmids), Silence the endogenous expression of IL-la, to establish stable cell lines of Hela229, through gene cloning technology;4. Through ELISA Compare each stable cell line’s production of IL-la, and chose the optimal cells of which IL-1α is inhibited.Results1. Inclusion bodies are found by the optical microscope in the model of Chlamydia trachomatis infected cells;2. The intracellular production of IL-la is higher than the extracellular group induced by Ct (P<0.05);3. Through the fluorescence microscope:Above90%cells are transfected successfully and exhibit green fluorescence, And the black holes in Ct infected cells is the inclusion bodies;4. The DNA sequence of four plasmid is correct via DNA verification;5. ELISA results show:The IL-la production of psi4-IL-1α-Hela229cell is lower than other four groups cells after antigen (Ct and TNF-a two groups) stimulation (P<0.05).Conclusions1. Intracellular IL-la of Hela229cells is major in the early infective stage by Ct;2. Establish the model of Ct infected the psi-IL-1α-Hela229cell. Silencing IL-la stable cell line may be developed into a potential tool for the next studies.