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Effects and Mechanisim of TNF-α on Intestinal Epithelial Cellular Permeability

Author: WangGuoLi
Tutor: YinFei
School: Central South University
Course: Pediatrics
Keywords: Tumor necrosis factor -α Caco-2 cells Actin NF-κB Paracellular permeability
CLC: R363
Type: Master's thesis
Year: 2010
Downloads: 74
Quote: 0
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Research background and purpose of intestinal mucosal barrier is one of the body's most important immune defense barrier, it body and exogenous substances in the intestinal isolate, to avoid pathogenic microorganisms invasion and antigen molecules injury. Intestinal mucosal barrier of the intestinal epithelial barrier, immune barrier and microbial barrier, intestinal epithelial cell barrier is the most important barrier is the basis of selective permeability of intestinal mucosal barrier. Study found increased intestinal epithelial cell barrier permeability to participate in a variety of diseases (such as inflammatory bowel disease, sepsis, burns, etc.), and in these cases, the serum levels of tumor necrosis factor-α (TNF-α) was significantly higher The extent of the damage, and intestinal epithelial barrier was positively correlated with anti-TNF-α antibody can restore the damaged intestinal epithelial barrier. TNF-α is therefore considered important factors to increase the intestinal epithelial cell barrier permeability. Although there has been formed consensus TNF-α can increase the permeability of the intestinal epithelial barrier, but its specific mechanism of action and the way is still controversial. Early that is closely related to apoptosis induced by TNF-α and TNF-α cause intestinal epithelial barrier injury in recent years tended to apoptosis-independent. TNF-α caused specific control aspects of the intestinal epithelial cell barrier permeability increase is not very clear, it may be associated with the protein kinase C (PKC), nuclear factor κB (NF-κB). Myosin light chain kinase (MLCK ), mitogen-activated protein kinase (MAPK) signaling pathway. Therefore, this study establish the Caco-2 cell lines in vitro intestinal epithelial barrier model, the impact of its permeability observed TNF-α, and explore TNF-α lead to possible mechanisms of intestinal epithelial cell barrier permeability increase. Research methods in vitro intestinal epithelial barrier model application of Caco-2 cell lines in vitro intestinal epithelial cell barrier model, application transepithelial resistance (transepithelial electrical resistance, TEER) indicators detect intestinal epithelial barrier formation. 2 plasmid transfection and packet be Caco-2 cells to close monolayer, serum-free medium 24h, according to the presence or absence of NF-K B inhibition transfection plasmid (Mu-IκB) will be divided into the Mu-IκB plasmid transfection group (M -TNF-α group) and Mu-IκB plasmid transfection group (TNF-α group). Further, according to the TNF-α group pretreatment time, were divided into M-of TNF-α0 3,6,12,24 h; TNF-α0, killed at the 3rd, 6th, 12th, 24th h 5 subgroups, each subgroup in its the corresponding point of time is added to the base side in Transwell 100μg / L TNF-α were incubated for 0,3,6,12,24 h, wherein Oh subgroup not TNF-α stimulation. 3 TNF-α on intestinal epithelial cell barrier permeability join 100μg / L TNF-α for different time (0,3,6,12,24 h), observation groups Caco-2 cells barrier TEER changes. 4 TNF-α on NF-κB activation in intestinal epithelial cells joined 100μg / L TNF-α role (0,3,6,12,24 h), luciferase reporter gene assay Caco-2 cells NF-κB activity. 5 TNF-α on intestinal epithelial cells of myosin light chain (myosin light chain, MLC) phosphorylation Join 100μg / L TNF-α for different time (0,3,6,12,24 h) using Western blotting. 6 TNF-α (western blot) detection MLC phosphorylation protein expression levels of intestinal epithelial cells F-actin added 100μg / L TNF-α for different time (0,3,6,12,24 h), rhodamine - direct phalloidin staining immunofluorescence change of F-actin in Caco-2 cells under the microscope. Results 1 TNF-α induced caused by the permeability changes of TNF-α in intestinal epithelial cells Caco-2 cells TEER reduce and having a time-dependent manner. TNF-α after 3h, TEER values ??decreased with the TNF-α0h group difference was significantly decreased (P lt; 0.05), TNF-α role 24h minimize the TEER is of TNF-α0h group 68.7%. Mu-IκB (transfected with plasmid) the inhibition of NF-κB activity, TNF-α caused by the Caco-2 cells TEER reduced compared to the respective sub-group, each sub-group of the M-TNF-α and TNF-α, the TEER decline significantly reduced (P lt; 0.05). 2 TNF-α induced intestinal epithelial cells of NF-kappa B's activity changes of TNF-α3h 6h, 12h group NF-κB activity increased, and of TNF-α0h group comparison there are significant differences (all P lt; 0.05), which of TNF-α12h The highest activity of NF-κB subgroup, with TNF-α0h, 3h, 6h, 24h subgroups more significant difference (P lt; 0.05). Transfection Mu-IκB plasmid of TNF-α role at 3h NF-κB activity start higher, with the M-of TNF-α0h group comparison there is a significant difference (P lt; 0.05), with the role of time extension, of NF-κB activity further increased , 12h the highest, followed by the decline in the activity of NF-κB. The various subgroups corresponding subgroup of M-TNF-α and TNF-α compared with the NF-κB activity increased amplitude were significantly lower (P lt; 0.05). 3 TNF-α induced intestinal epithelial cell MLC phosphorylation levels of the role of TNF-α, 6h, MLC phosphorylation levels increase with the TNF-αOh group transferred Mu = IκB plasmid, the role of TNF-α 6h MLC phosphorylation levels no significant change in the role 12h before MLC phosphorylation. Reduce the activity of NF-κB, TNF-α after MLC phosphorylation caused delay. 4 TNF-α induced F-actin in intestinal epithelial cells normal CaCo-2 cells, closely connected to the structural integrity of the banded structure was dense. Add 100μg / L role of TNF-α 12h extracellular week dense band edges become rough and irregular the weakened cell staining, 24h, cells peripheral outline blurred, the gradual emergence of a sawtooth-like fracture tends fine disintegration dissipated. The Mu-IκB plasmid transfection, M-TNF-α3, 6,12 h group F-actin in Caco-2 cells, no significant change M-TNF-α24h in group cells peripheral dense band edges start to become rough and irregular cell fluorescence staining decreased. Research conclusions intestinal epithelial cell MLC phosphorylation and F-actin reorganization is one of the mechanisms of TNF-α caused the increased permeability, this process may be regulated by NF-κB.

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