IL-1α在沙眼衣原体感染Hela229细胞中的表达情况及稳定表达IL-1α shRNA细胞系的构建

发布时间：2018-01-24 01:30

本文关键词： 沙眼衣原体 IL-1α RNA干扰　出处：《中南大学》2012年硕士论文　论文类型：学位论文

【摘要】：沙眼衣原体(Chlamydia trachomatis, Ct)是一种常见的、严格细胞内寄生的病原体。Ct侵犯机体可引起泌尿生殖道感染,更为严重的是Ct感染与不孕、异位妊娠、宫颈鳞状上皮细胞癌等疾病发生相关。Ct可诱导上皮细胞产生IL-1α、IL-6和TNF-a等多种炎症因子,参与免疫应答,其中IL-1α进一步诱导细胞因子(包括其自身、IL-6和IL-8等),参与Ct感染导致的组织损伤。因此,研究IL-1α在早期炎症中的作用,为Ct感染的发病机制、疾病预防和治疗提供实验依据。目的 1、通过探讨IL-1α在Ct感染HeLa229细胞前后,细胞内、外IL-1α的表达情况,明确IL-1α在Ct感染细胞的炎症早期的表达模式； 2、构建稳定表达IL-1αshRNA的细胞系,为后续研究Ct诱导炎症机制提供实验材料。方法 1、Ct感染Hela229细胞,吉姆萨染色后,光学显微镜观察确定Ct包涵体,以建立Ct感染Hela229细胞的模型； 2、用ELISA检测IL-1α在Ct感染Hela229细胞内以及培养上清中的表达； 3、利用基因克隆方法构建IL-1α shRNA表达质粒,转染Hela229细胞,经G418筛选,构建稳定表达IL-1α shRNA的细胞系； 4、通过ELISA法检测各稳定转染重组质粒的细胞IL-1α的表达,筛选出有效抑制内源性IL-1α表达的稳定细胞系。结果 1、光学显微镜示细胞内的透明折光体为Ct的包涵体,表明Ct感染Hela229细胞模型已建立； 2、Ct感染Hela229细胞36h,与未感染组相比,胞内IL-1α明显上升,有显著差异(P0.05)； 3、成功构建重组质粒pRNAT6.1/Neo-siRNA1、2、3、4(4种IL-1αshRNA质粒),经DNA测序4种重组质粒序列正确； 4、透过荧光显微镜可见：90%以上稳定转染细胞有绿色荧光GFP表达； 5、Ct成功感染IL-1αshRNA稳定细胞系,镜下观察细胞内未着色空洞即Ct包涵体,表明成功建立Ct感染各稳定细胞系的模型； 6、各稳定转染的细胞系(Ct感染组和TNF-α刺激组)中,psi4-IL-1α-Hela229细胞(IL-1αshRNA稳定细胞系)胞内IL-1α表达量显著低于对照组psi-Neg-Hela229细胞(P0.05)。结论 1、IL-1α在Ct感染Hela229细胞早期以细胞内表达为主； 2、成功构建稳定表达IL-1αshRNA的细胞系(psi-IL-1α-Hela229细胞),为后续实验研究提供了实验材料。
[Abstract]:Chlamydia trachomatis( CTS) is one of the most common pathogens in chlamydia trachomatis.Chlamydia trachomatis.Ct, a strict intracellular pathogen, can cause genitourinary infection. What is more serious is that Ct infection is associated with infertility, ectopic pregnancy, cervical squamous cell carcinoma and other diseases. Ct can induce epithelial cells to produce IL-1 伪. Many inflammatory factors, such as IL-6 and TNF-a, are involved in the immune response, and IL-1 伪 further induces cytokines (including its own IL-6 and IL-8). Therefore, to study the role of IL-1 伪 in early inflammation, to provide experimental basis for pathogenesis, disease prevention and treatment of Ct infection. Purpose 1. To investigate the expression of IL-1 伪 in HeLa229 cells before and after Ct infection, and to determine the expression of IL-1 伪 in the early stage of inflammation of Ct infected cells. 2. Cell lines stably expressing IL-1 伪 shRNA were constructed to provide experimental materials for further study on the mechanism of Ct induced inflammation. Method (1) Ct was infected with Hela229 cells, and the inclusion bodies of Ct were determined by optical microscope after Gimsa staining to establish the model of Ct infection with Hela229 cells. 2. ELISA was used to detect the expression of IL-1 伪 in Ct infected Hela229 cells and culture supernatant. 3. IL-1 伪 shRNA expression plasmid was constructed by gene cloning and transfected into Hela229 cells. Cell lines stably expressing IL-1 伪 shRNA were constructed. 4. The expression of IL-1 伪 was detected by ELISA assay, and the stable cell lines which could effectively inhibit the expression of endogenous IL-1 伪 were selected. Results 1. The transparent refraction in the cells was shown to be the inclusion body of Ct by optical microscope, which indicated that the model of Ct infection with Hela229 cells had been established. (2) Hela229 cells were infected with Ct for 36 h. Compared with the control group, the intracellular IL-1 伪 increased significantly (P 0.05). 3Recombinant plasmid pRNAT6.1 / Neo-siRNA1 / 2, 2, 3, 4, 4 kinds of IL-1 伪 shRNA plasmids were successfully constructed, and four recombinant plasmids were sequenced by DNA. 4The green fluorescent GFP expression was observed in over 90% of the transfected cells by fluorescence microscope. IL-1 伪 shRNA stable cell lines were successfully infected with 5 Ct. The unstained cavities in the cells, Ct inclusion bodies, were observed under microscope, which indicated that the model of Ct infection in various stable cell lines was successfully established. 6, the stable transfected cell lines were infected with Ct and TNF- 伪. IL-1 伪 shRNA stable cell line of psi4-IL-1 伪 -Hela229 cell line. The expression of IL-1 伪 in the control group was significantly lower than that in the control group (P 0.05). Conclusion 1Interleukin-1 伪 was mainly expressed in Hela229 cells at the early stage of Ct infection. 2. The cell line of psi-IL-1 伪 -Hela229 expressing stably IL-1 伪 shRNA was successfully constructed, which provided experimental materials for further experimental study.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R374

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