發(fā)布時間：2018-01-24 01:30

  本文關鍵詞： 沙眼衣原體 IL-1α RNA干擾　出處：《中南大學》2012年碩士論文　論文類型：學位論文

【摘要】：沙眼衣原體(Chlamydia trachomatis, Ct)是一種常見的、嚴格細胞內寄生的病原體。Ct侵犯機體可引起泌尿生殖道感染,更為嚴重的是Ct感染與不孕、異位妊娠、宮頸鱗狀上皮細胞癌等疾病發(fā)生相關。Ct可誘導上皮細胞產生IL-1α、IL-6和TNF-a等多種炎癥因子,參與免疫應答,其中IL-1α進一步誘導細胞因子(包括其自身、IL-6和IL-8等),參與Ct感染導致的組織損傷。因此,研究IL-1α在早期炎癥中的作用,為Ct感染的發(fā)病機制、疾病預防和治療提供實驗依據。 目的 1、通過探討IL-1α在Ct感染HeLa229細胞前后,細胞內、外IL-1α的表達情況,明確IL-1α在Ct感染細胞的炎癥早期的表達模式； 2、構建穩(wěn)定表達IL-1αshRNA的細胞系,為后續(xù)研究Ct誘導炎癥機制提供實驗材料。 方法 1、Ct感染Hela229細胞,吉姆薩染色后,光學顯微鏡觀察確定Ct包涵體,以建立Ct感染Hela229細胞的模型； 2、用ELISA檢測IL-1α在Ct感染Hela229細胞內以及培養(yǎng)上清中的表達； 3、利用基因克隆方法構建IL-1α shRNA表達質粒,轉染Hela229細胞,經G418篩選,構建穩(wěn)定表達IL-1α shRNA的細胞系； 4、通過ELISA法檢測各穩(wěn)定轉染重組質粒的細胞IL-1α的表達,篩選出有效抑制內源性IL-1α表達的穩(wěn)定細胞系。 結果 1、光學顯微鏡示細胞內的透明折光體為Ct的包涵體,表明Ct感染Hela229細胞模型已建立； 2、Ct感染Hela229細胞36h,與未感染組相比,胞內IL-1α明顯上升,有顯著差異(P0.05)； 3、成功構建重組質粒pRNAT6.1/Neo-siRNA1、2、3、4(4種IL-1αshRNA質粒),經DNA測序4種重組質粒序列正確； 4、透過熒光顯微鏡可見：90%以上穩(wěn)定轉染細胞有綠色熒光GFP表達； 5、Ct成功感染IL-1αshRNA穩(wěn)定細胞系,鏡下觀察細胞內未著色空洞即Ct包涵體,表明成功建立Ct感染各穩(wěn)定細胞系的模型； 6、各穩(wěn)定轉染的細胞系(Ct感染組和TNF-α刺激組)中,psi4-IL-1α-Hela229細胞(IL-1αshRNA穩(wěn)定細胞系)胞內IL-1α表達量顯著低于對照組psi-Neg-Hela229細胞(P0.05)。 結論 1、IL-1α在Ct感染Hela229細胞早期以細胞內表達為主； 2、成功構建穩(wěn)定表達IL-1αshRNA的細胞系(psi-IL-1α-Hela229細胞),為后續(xù)實驗研究提供了實驗材料。
[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 偽. Many inflammatory factors, such as IL-6 and TNF-a, are involved in the immune response, and IL-1 偽 further induces cytokines (including its own IL-6 and IL-8). Therefore, to study the role of IL-1 偽 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 偽 in HeLa229 cells before and after Ct infection, and to determine the expression of IL-1 偽 in the early stage of inflammation of Ct infected cells. 2. Cell lines stably expressing IL-1 偽 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 偽 in Ct infected Hela229 cells and culture supernatant. 3. IL-1 偽 shRNA expression plasmid was constructed by gene cloning and transfected into Hela229 cells. Cell lines stably expressing IL-1 偽 shRNA were constructed. 4. The expression of IL-1 偽 was detected by ELISA assay, and the stable cell lines which could effectively inhibit the expression of endogenous IL-1 偽 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 偽 increased significantly (P 0.05). 3Recombinant plasmid pRNAT6.1 / Neo-siRNA1 / 2, 2, 3, 4, 4 kinds of IL-1 偽 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 偽 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- 偽. IL-1 偽 shRNA stable cell line of psi4-IL-1 偽 -Hela229 cell line. The expression of IL-1 偽 in the control group was significantly lower than that in the control group (P 0.05). Conclusion 1Interleukin-1 偽 was mainly expressed in Hela229 cells at the early stage of Ct infection. 2. The cell line of psi-IL-1 偽 -Hela229 expressing stably IL-1 偽 shRNA was successfully constructed, which provided experimental materials for further experimental study.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R374

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