Effect And Regulation Of Saccharomyces Cerevisiae Cell Wall On The Expression Of SBD-1 In Ruminal Epithelial Cells Of Sheep

There are abundant species and numerous quantity of microbe in ruminant rumen,which intimately contacted with the muscosal epithelium of rumen.The imbalance in flora of rumen easily caused disease.Antibiotics are still the first choice to kill the microbe for now.However,with the abuse of antibiotics,the negative effects became increasingly acute.Defensins can not only kill bacteria,fungus,and virus directly but also show other various biological functions including regulating inflammatory response,accelerating chemotaxis of immune cell,and promoting cell differentiation.Defensins generally participated in anti-inflammation mechanism against pathogenic microorganism.The early researches had demonstrated that Saccharomyces cerevisiae(S.cerevisiae)could facilitate the expression of defensin in sheep rumen epithelia cells(SRECs).To confirm the effective constituent of S.cerevisiae inducing defensin expression,the cell wall of S.cerevisiae was used to explore the effect of defensin expression in ovine rumen epithelial cells in this research and the signal pathway inducing the expression of defensin by S.cerevisiae was researched,which could provide the theoretical foundation for exploiting biological products such as probiotics for prevention and treatment of ovine digestive tract diseases.The main thesis of this research includes the following aspects:1.The culture system in vitro of SRECs was establishd and identified by immunocytochemistry(ICC).The positive results of ICC showed that the purity of epithelium cells met the requirements of present research.The result of CCK-8 indicated that growth characteristics of SRECS accorded with cell rhythm.The results of real-time label-free cell growth assay showed that the third-passage SRECs met the requirements with a density of 1.63×10~5 cells/mL for 96 h.2.The 26S rDNA sequence of the cultured yeast was amplified by PCR and its sequence showed 100%homology with the sequence of standard S.cerevisiae strain.By plotting the growth curve of S.cerevisiae,it was determined that the S.cerevisiae culture time entered the plateau at 12 h.The disruption for cell wall was proceeded when they just entered the stationary.The shape of the S.cerevisiae before and after breaking wall were observed by the scanning electron microscopy.It confirmed that the cell wall was successfully obtained;3.The extracted cell wall was fully washed and its component was identified through Kjeldahl nitrogen,acid hydrolysis,and HPLC.The result confirmed the components of extracted cell wall agree with constituents of S.cerevisiae,which could replace whole cell wall to proceed following experiment.4.Induction experiments showed that the expression level of sheep beta defensin-1(SBD-1)in SRECs was dependent on the concentration and time of cell wall.Adding different concentrations of S.cerevisiae cell wall(0,25,50,100,200,400?g/mL)to culture SRECs with different times(0,2,4,8,12 and 24 h)could promote the mRNA expression of SBD-1 in different degrees.The 200?g/mL cell wall with cluture for 12 h induced a extremely significant higher mRNA expression level of SBD-1 compared with the blank control(P<0.001).5.Treatment with TLR2 inhibitors followed by induction of cell wall of S.cerevisiae on SRECs demonstrated that the expression of SBD-1 in SRECs was related to membrane receptor TLR2.Treatment with MyD88 inhibitors followed by induction of cell wall of S.cerevisiae on SRECs demonstrated that the intracellular adaptor protein MyD88 was involved in SBD-1 expression.Treatment with NF-?B inhibitors followed by induction of cell wall of S.cerevisiae on SRECs demonstrated that SBD-1 expression in SRECs was induced via NF-?B signaling pathway by S.cerevisiae cell wall.The results indicated that S.cerevisiae cell wall could induce the differential expression of SBD-1,which could be activated through TLR2-MyD88-NF-?B pathway.