The Sequence Analysis of RNA Components from Lactobacillus Metabolites and Parts of the Biological Function
|School||Dalian Medical University|
|Keywords||Sequence CPG-ODN Toll Like receptors (TLRs) NF-κB IL-6|
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.