本文選題：序列 + CPG-ODN　； 參考：《大連醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的: 1.分析乳桿菌代謝產(chǎn)物RNA組分的序列,明確該核酸成分的性質(zhì) 2.通過RNA組分在體外對Caco-2細(xì)胞TLR-9通路及對細(xì)胞增殖影響的研究,探討其作用機(jī)制及對腫瘤細(xì)胞的作用 3.通過RNA組分對腸道菌群失衡小鼠腸粘膜絨毛形態(tài)及派氏結(jié)中細(xì)胞形態(tài)恢復(fù)情況的研究,探討其對腸道機(jī)械屏障及免疫細(xì)胞的作用 方法: 1.乳桿菌代謝產(chǎn)物RNA組分序列分析:應(yīng)用Solexa上機(jī)前處理過程對樣品RNA進(jìn)行處理,經(jīng)過加接頭反轉(zhuǎn)錄后對連接產(chǎn)物進(jìn)行PCR擴(kuò)增,進(jìn)而對PCR產(chǎn)物做TA克隆測序。(測序委托深圳華大基因科技有限公司進(jìn)行) 2.乳桿菌代謝產(chǎn)物RNA組分對Caco-2細(xì)胞TLR9通路及細(xì)胞增殖的影響 (1)實(shí)驗(yàn)分為四組,陰性對照組、陽性對照組、RNA100ug/ml組、RNA200ug/ml組。實(shí)驗(yàn)組中加入不同濃度RNA組分,陽性對照組中加入CPG-ODN (2)RT-PCR法檢測TLR9、NF-κBp65、IL-6 mRNA水平的變化;免疫組化法檢測NF-κBp65蛋白水平的變化;ELISA法檢測Caco-2細(xì)胞培養(yǎng)液中IL-6的變化 (3)MTT法檢測RNA組分對Caco-2細(xì)胞增殖的影響 3.乳桿菌代謝產(chǎn)物RNA組分對腸道菌群失衡小鼠腸粘膜絨毛形態(tài)及派氏結(jié)中細(xì)胞形態(tài)的影響 (1)SPF級Balb/c小鼠隨機(jī)分為正常組,重度失衡組,自然恢復(fù)組,RNA恢復(fù)組。采用抗生素頭孢曲松鈉灌胃的方式建立小鼠腸道菌群失衡模型后,以RNA組分10mg/kg/天的劑量,連續(xù)3天灌胃調(diào)節(jié),并從灌服之日起第7天提取標(biāo)本,檢測指標(biāo) (2)HE染色及透射電鏡觀察菌群失衡小鼠通過RNA組分恢復(fù)后腸粘膜絨毛形態(tài)變化 (3)透射電鏡觀察菌群失衡小鼠通過RNA組分恢復(fù)后派氏結(jié)中細(xì)胞形態(tài)變化 結(jié)果: 1.乳桿菌代謝產(chǎn)物RNA組分序列分析:所得39條序列,經(jīng)Blast比對其中包含德氏乳桿菌23S rRNA和鼠李糖乳桿菌tRNA-Leu等 2.乳桿菌代謝產(chǎn)物RNA組分對Caco-2細(xì)胞TLR9通路及細(xì)胞增殖的影響 (1)與對照組相比RNA組分組TLR9及NF-κBp65、IL-6在mRNA及蛋白水平的表達(dá)量均高于陰性對照組,100ug/mlRNA組表達(dá)量低于陽性對照組,200ug/mlRNA組表達(dá)量高于陽性對照組 (2)RNA組分對Caco-2細(xì)胞增殖有抑制作用,顯示出明顯的時(shí)間劑量依賴性 3.乳桿菌代謝產(chǎn)物RNA組分對腸道菌群失衡小鼠腸粘膜絨毛形態(tài)及派氏結(jié)中細(xì)胞形態(tài)的影響 (1)腸粘膜絨毛形態(tài):應(yīng)用回灌RNA進(jìn)行恢復(fù)后,通過HE染色發(fā)現(xiàn)與自然恢復(fù)組相比,腸上皮細(xì)胞層細(xì)胞排列緊密,腸絨毛排列規(guī)整,腫脹、斷裂消失,絨毛頂端未見壞死脫落的腸上皮細(xì)胞,且炎性細(xì)胞浸潤減少。通過電鏡觀察發(fā)現(xiàn)與自然恢復(fù)組相比,微絨毛恢復(fù)整齊,細(xì)胞間連接更加緊密 (2)派氏結(jié)中細(xì)胞形態(tài):經(jīng)過回灌RNA進(jìn)行恢復(fù)后,通過電鏡觀察發(fā)現(xiàn)腸壁派氏結(jié)重度失衡組與正常組相比較,淋巴細(xì)胞之間的間隙明顯變大;樹突狀細(xì)胞偽足變短,通過回灌RNA進(jìn)行恢復(fù)后,與自然恢復(fù)組相比,淋巴細(xì)胞間隙明顯縮窄,樹突狀細(xì)胞偽足延伸 結(jié)論: 1.乳桿菌代謝產(chǎn)物RNA組分中包含德氏乳桿菌23S rRNA和鼠李糖乳桿菌tRNA-Leu等 2.乳桿菌代謝產(chǎn)物RNA組分對Caco-2細(xì)胞TLR通路具有活化作用 3.乳桿菌代謝產(chǎn)物RNA組分對人結(jié)腸腺癌Caco-2細(xì)胞增殖有抑制作用 4.乳桿菌代謝產(chǎn)物RNA組分對腸道菌群失衡小鼠腸粘膜絨毛形態(tài)及派氏結(jié)中細(xì)胞形態(tài)具有恢復(fù)作用
[Abstract]:Objective:
1. analyze the sequence of RNA components of Lactobacillus metabolites and identify the properties of the nucleic acid components.
2. through the study of the effects of RNA components on Caco-2 cell TLR-9 pathway and cell proliferation in vitro, the mechanism and the effect on tumor cells were discussed.
3. the effects of the RNA component on intestinal microflora's intestinal mucosal villous morphology and the cell morphology recovery in the Pik junction of the intestinal microflora were discussed, and the effects on the intestinal mechanical barrier and immune cells were discussed.
Method:
Sequence analysis of RNA component of 1. Lactobacillus metabolites: the sample RNA was treated by Solexa preprocessing, PCR amplification was carried out after the joint reverse transcription, and then the TA clone of PCR products was cloned and sequenced. (sequencing of Shenzhen Huada Gene Technology Co., Ltd.)
Effects of 2. RNA components of Lactobacillus metabolites on TLR9 pathway and cell proliferation in Caco-2 cells
(1) the experiment was divided into four groups, the negative control group, the positive control group, the RNA100ug/ml group and the RNA200ug/ml group. The experimental group was added to the different concentration of RNA components, and the positive control group was added to the CPG-ODN group.
(2) the changes of TLR9, NF- kappa Bp65 and IL-6 mRNA were detected by RT-PCR method; the changes of NF- kappa Bp65 protein level were detected by immunohistochemistry; ELISA assay was used to detect IL-6 changes in Caco-2 cell culture solution.
(3) the effect of RNA components on proliferation of Caco-2 cells was detected by MTT.
Effects of Lactobacillus 3. metabolite RNA on intestinal mucosal villi morphology and cell morphology in pare nodes of intestinal flora imbalance
(1) SPF grade Balb/c mice were randomly divided into normal group, severe unbalance group, natural recovery group and RNA recovery group. After using antibiotic ceftriaxone sodium to establish the intestinal microflora imbalance model in mice, the dosage of 10mg/kg/ days in RNA group was used for gastric perfusion, and the samples were extracted from the day of seventh days, and the indexes were detected.
(2) HE staining and transmission electron microscopy were used to observe the morphological changes of intestinal villi after restoration of RNA components in mice with bacterial imbalance.
(3) transmission electron microscopy was used to observe the morphological changes of the cells in the PGE junction after the recovery of RNA components.
Result:
1. sequence analysis of Lactobacillus metabolites RNA: the 39 sequences were compared with Blast, including Lactobacillus Deshi 23S rRNA and Lactobacillus rhamnophilus tRNA-Leu.
Effects of 2. RNA components of Lactobacillus metabolites on TLR9 pathway and cell proliferation in Caco-2 cells
(1) compared with the control group, the expression of TLR9 and NF- kappa Bp65 in the RNA group was higher than that in the negative control group. The expression of 100ug/mlRNA in the 100ug/mlRNA group was lower than that in the positive control group. The expression of 200ug/mlRNA group was higher than that of the positive control group.
(2) the RNA component inhibited the proliferation of Caco-2 cells, showing a significant time dose dependence.
Effects of Lactobacillus 3. metabolite RNA on intestinal mucosal villi morphology and cell morphology in pare nodes of intestinal flora imbalance
(1) the morphology of intestinal villi: after the recovery of RNA, the cell layer cells in the intestinal epithelium were closely arranged, the intestinal villi were arranged regularly, swollen, and broken, and no necrotic intestinal epithelial cells were found at the top of the villi, and the infiltration of inflammatory cells decreased. The microvilli recovered gradually and the intercellular connections were more closely linked.
(2) the cell morphology in piret's knot: after recharge of RNA, it was found through electron microscopy that the severe imbalance between the intestinal wall pies group and the normal group compared with the normal group, the gap between the lymphocytes became significantly larger, and the dendritic cells were short of the pseudo foot. After the recovery of the recharge RNA, the lymphocytic space of the lymphocyte was narrowed and the dendritic shape was obviously narrowed. Cell pseudo foot extension
Conclusion:
1. Lactobacillus metabolite RNA contains Deshi Lactobacillus 23S rRNA and Lactobacillus rhamnophilus tRNA-Leu.
2. RNA component of Lactobacillus metabolite has activation effect on TLR pathway of Caco-2 cells.
3. Lactobacillus metabolite RNA has inhibitory effect on the proliferation of human colon adenocarcinoma Caco-2 cells.
4. the RNA components of Lactobacillus metabolites restored the morphology of intestinal mucosal villi and the morphology of cells in PASH nodes in mice with intestinal flora imbalance.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R378

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