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

【摘要】：沙眼衣原體(Chlamydia trachomatis, Ct)是一種常見(jiàn)的、嚴(yán)格細(xì)胞內(nèi)寄生的病原體。Ct侵犯機(jī)體可引起泌尿生殖道感染,更為嚴(yán)重的是Ct感染與不孕、異位妊娠、宮頸鱗狀上皮細(xì)胞癌等疾病發(fā)生相關(guān)。Ct可誘導(dǎo)上皮細(xì)胞產(chǎn)生IL-1α、IL-6和TNF-a等多種炎癥因子,參與免疫應(yīng)答,其中IL-1α進(jìn)一步誘導(dǎo)細(xì)胞因子(包括其自身、IL-6和IL-8等),參與Ct感染導(dǎo)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(jī)制、疾病預(yù)防和治療提供實(shí)驗(yàn)依據(jù)。 目的 1、通過(guò)探討IL-1α在Ct感染HeLa229細(xì)胞前后,細(xì)胞內(nèi)、外IL-1α的表達(dá)情況,明確IL-1α在Ct感染細(xì)胞的炎癥早期的表達(dá)模式； 2、構(gòu)建穩(wěn)定表達(dá)IL-1αshRNA的細(xì)胞系,為后續(xù)研究Ct誘導(dǎo)炎癥機(jī)制提供實(shí)驗(yàn)材料。 方法 1、Ct感染Hela229細(xì)胞,吉姆薩染色后,光學(xué)顯微鏡觀察確定Ct包涵體,以建立Ct感染Hela229細(xì)胞的模型； 2、用ELISA檢測(cè)IL-1α在Ct感染Hela229細(xì)胞內(nèi)以及培養(yǎng)上清中的表達(dá)； 3、利用基因克隆方法構(gòu)建IL-1α shRNA表達(dá)質(zhì)粒,轉(zhuǎn)染Hela229細(xì)胞,經(jīng)G418篩選,構(gòu)建穩(wěn)定表達(dá)IL-1α shRNA的細(xì)胞系； 4、通過(guò)ELISA法檢測(cè)各穩(wěn)定轉(zhuǎn)染重組質(zhì)粒的細(xì)胞IL-1α的表達(dá),篩選出有效抑制內(nèi)源性IL-1α表達(dá)的穩(wěn)定細(xì)胞系。 結(jié)果 1、光學(xué)顯微鏡示細(xì)胞內(nèi)的透明折光體為Ct的包涵體,表明Ct感染Hela229細(xì)胞模型已建立； 2、Ct感染Hela229細(xì)胞36h,與未感染組相比,胞內(nèi)IL-1α明顯上升,有顯著差異(P0.05)； 3、成功構(gòu)建重組質(zhì)粒pRNAT6.1/Neo-siRNA1、2、3、4(4種IL-1αshRNA質(zhì)粒),經(jīng)DNA測(cè)序4種重組質(zhì)粒序列正確； 4、透過(guò)熒光顯微鏡可見(jiàn)：90%以上穩(wěn)定轉(zhuǎn)染細(xì)胞有綠色熒光GFP表達(dá)； 5、Ct成功感染IL-1αshRNA穩(wěn)定細(xì)胞系,鏡下觀察細(xì)胞內(nèi)未著色空洞即Ct包涵體,表明成功建立Ct感染各穩(wěn)定細(xì)胞系的模型； 6、各穩(wěn)定轉(zhuǎn)染的細(xì)胞系(Ct感染組和TNF-α刺激組)中,psi4-IL-1α-Hela229細(xì)胞(IL-1αshRNA穩(wěn)定細(xì)胞系)胞內(nèi)IL-1α表達(dá)量顯著低于對(duì)照組psi-Neg-Hela229細(xì)胞(P0.05)。 結(jié)論 1、IL-1α在Ct感染Hela229細(xì)胞早期以細(xì)胞內(nèi)表達(dá)為主； 2、成功構(gòu)建穩(wěn)定表達(dá)IL-1αshRNA的細(xì)胞系(psi-IL-1α-Hela229細(xì)胞),為后續(xù)實(shí)驗(yàn)研究提供了實(shí)驗(yàn)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(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R374

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