【作者】 刘春亮；

【导师】 唐立；

【作者基本信息】 大连医科大学 ， 病原生物学， 2011， 硕士

【摘要】 目的:1.分析乳杆菌代谢产物RNA组分的序列,明确该核酸成分的性质2.通过RNA组分在体外对Caco-2细胞TLR-9通路及对细胞增殖影响的研究,探讨其作用机制及对肿瘤细胞的作用3.通过RNA组分对肠道菌群失衡小鼠肠粘膜绒毛形态及派氏结中细胞形态恢复情况的研究,探讨其对肠道机械屏障及免疫细胞的作用方法:1.乳杆菌代谢产物RNA组分序列分析:应用Solexa上机前处理过程对样品RNA进行处理,经过加接头反转录后对连接产物进行PCR扩增,进而对PCR产物做TA克隆测序。(测序委托深圳华大基因科技有限公司进行)2.乳杆菌代谢产物RNA组分对Caco-2细胞TLR9通路及细胞增殖的影响(1)实验分为四组,阴性对照组、阳性对照组、RNA100ug/ml组、RNA200ug/ml组。实验组中加入不同浓度RNA组分,阳性对照组中加入CPG-ODN(2)RT-PCR法检测TLR9、NF-κBp65、IL-6 mRNA水平的变化;免疫组化法检测NF-κBp65蛋白水平的变化;ELISA法检测Caco-2细胞培养液中IL-6的变化(3)MTT法检测RNA组分对Caco-2细胞增殖的影响3.乳杆菌代谢产物RNA组分对肠道菌群失衡小鼠肠粘膜绒毛形态及派氏结中细胞形态的影响(1)SPF级Balb/c小鼠随机分为正常组,重度失衡组,自然恢复组,RNA恢复组。采用抗生素头孢曲松钠灌胃的方式建立小鼠肠道菌群失衡模型后,以RNA组分10mg/kg/天的剂量,连续3天灌胃调节,并从灌服之日起第7天提取标本,检测指标(2)HE染色及透射电镜观察菌群失衡小鼠通过RNA组分恢复后肠粘膜绒毛形态变化(3)透射电镜观察菌群失衡小鼠通过RNA组分恢复后派氏结中细胞形态变化结果:1.乳杆菌代谢产物RNA组分序列分析:所得39条序列,经Blast比对其中包含德氏乳杆菌23S rRNA和鼠李糖乳杆菌tRNA-Leu等2.乳杆菌代谢产物RNA组分对Caco-2细胞TLR9通路及细胞增殖的影响(1)与对照组相比RNA组分组TLR9及NF-κBp65、IL-6在mRNA及蛋白水平的表达量均高于阴性对照组,100ug/mlRNA组表达量低于阳性对照组,200ug/mlRNA组表达量高于阳性对照组(2)RNA组分对Caco-2细胞增殖有抑制作用,显示出明显的时间剂量依赖性3.乳杆菌代谢产物RNA组分对肠道菌群失衡小鼠肠粘膜绒毛形态及派氏结中细胞形态的影响(1)肠粘膜绒毛形态:应用回灌RNA进行恢复后,通过HE染色发现与自然恢复组相比,肠上皮细胞层细胞排列紧密,肠绒毛排列规整,肿胀、断裂消失,绒毛顶端未见坏死脱落的肠上皮细胞,且炎性细胞浸润减少。通过电镜观察发现与自然恢复组相比,微绒毛恢复整齐,细胞间连接更加紧密(2)派氏结中细胞形态:经过回灌RNA进行恢复后,通过电镜观察发现肠壁派氏结重度失衡组与正常组相比较,淋巴细胞之间的间隙明显变大;树突状细胞伪足变短,通过回灌RNA进行恢复后,与自然恢复组相比,淋巴细胞间隙明显缩窄,树突状细胞伪足延伸结论:1.乳杆菌代谢产物RNA组分中包含德氏乳杆菌23S rRNA和鼠李糖乳杆菌tRNA-Leu等2.乳杆菌代谢产物RNA组分对Caco-2细胞TLR通路具有活化作用3.乳杆菌代谢产物RNA组分对人结肠腺癌Caco-2细胞增殖有抑制作用4.乳杆菌代谢产物RNA组分对肠道菌群失衡小鼠肠粘膜绒毛形态及派氏结中细胞形态具有恢复作用更多还原

【Abstract】 Objective:1.The sequence analysis of RNA components from Lactobacillus metabolites .2.The effect of RNA components from Lactobacillus metabolites on Caco-2 cell TLR9 and proliferation influence of Caco - 2 cell .3.The effect of RNA components from Lactobacillus metabolites on intestinal structure of imbalanced flora mice.Methods:1.Sequence analysis: Processing samples RNA applies Solexa pre-treatment process.TA cloning sequencing PCR products after samples RNA reverse transcription add joints connecting. (Sequencing entrust shenzhen huada gene technology Co., LTD)2.The effect of RNA components from Lactobacillus metabolites on Caco-2 cell TLR9 and proliferation.(1)Experiments are divided into four groups and the negative control, positive control, RNA100ug/ml group, RNA200ug/ml group. To join different concentration into RNA Group, join CPG - ODN into positive control.(2)Detecting the change of TLR9, NF-κBp65 ,IL-6 by RT-PCR and Detecting the change of NF-κBp65 protein by Immunohistochemistry, assaying the change of IL-6 in culture of Caco-2 by ELISA.(3)Detecting the effect of RNA components on proliferation of Caco-2 cell line by MTT.3.The effect of RNA components from Lactobacillus metabolites on intestinal structure of imbalanced flora mice.(1)Balb/c mice SPF were randomly divided into four groups, control,severe unbalance group, natural recovery group and RNA recovery group. We established mice model for dysbiosis of Intestinal flora by ceftriaxone, and then detect targets at the seventh day by the secretive RNA of DM9811 10mg/kg / day for 3 days.(2)Observing the bowel structure change of flora imbalances mice after the restoration of RNA components by HE dyed and electron microscopic.(3)Observing the change of Peyer’spatches in flora imbalances mice after the restoration of RNA components by electron microscopic.Results:1.Sequence analysis: Income 39 of the sequence. The samples relevant sequence is Lactobacillus delbrueckii 23SrRNA and Lactobacillus rhamnosus GG tRNA - Leu.2.The effect of RNA components from Lactobacillus metabolites on Caco-2cell line.(1)Compared with control group TLR - 9 NF -κBp65 andIL - 6 in mRNA and protein level is higher than negative control, 100ug/mlRNA group expression is less than positive control group, 200ug/mlRNA group is higher than positive control group.(2)The effect of RNA components on Caco - 2 cell proliferation inhibitory showed time dose dependent obviously.3.The effect of RNA components from Lactobacillus metabolites on intestinal structure of imbalanced flora mice.(1)Intestinal mucosa villi: Through recharge with RNA,and compared with natural recovery, intestinal epithelium cells group cell layers closely-arranged villi with arrangement, neat, swelling, fracture disappear, fluffy top has not seen the necrosis of intestinal epithelium cells off, and inflammatory cells infiltrating decrease. Through recharge with RNA and compared with natural recovery group microvilli restore tidy, cell connections between closer.(2)Peyer’spatches: Compared with severe imbalance flora group and the normal group the lymphocyte clearances between apparent greatens; Dendritic cells pseudopods become shorter, through recharging after recovery, compared with RNA natural recovery group , lymphocytes clearance obviously become narrower, dendritic cells pseudopods outspread.Conclusion:1.Some ingredients of Lactobacillus metabolites RNA component are most similar to Lactobacillus delbrueckii 23SrRNA and Lactobacillus rhamnosus GG tRNA - Leu. 2.The Lactobacillus metabolites RNA components have activatory function on Caco - 2 cells TLR pathways.3.The effect of Lactobacillus metabolites RNA components on human adenocarcinoma Caco - 2 cell line proliferation is inhibition.4.The Lactobacillus metabolites RNA components have recovery function on intestinal flora imbalances mice intestine structural integrity and Peyer’spatches.更多还原