| Objective To observe the effects of recombinant human intestinal trefoil factor(rhITF)on intestinal epithelial cell migration，and explore its possible mechanism.Method1. Experimental medication: using recombinant human intestinal trefoil factor (rhITF) asstimulation drugs, the recombinant protein purity of more than98%, in line with pre-clinical drug research standards.2. Cell model: subcultured human colon cancer cell (Caco-2) for research model, Caco-2cell lines were purchased by the Cell Research Institute of Chinese Academy of Sciences,after correct identification were cultured, and then to the5-10cells were used in thisexperiment.3. Experimental groups: according to the purpose of the experiment, this experimentadopts a plurality of packet mode, the experimental group include:(1) Negative control group and different concentrations of rhITF group;(2) Normalcontrol group and different time points group;(3) Normal control group, rhITF group andrhITF+inhibitor group;(4) Negative control group, rhITF group and rhITF+inhibitorgroup.4. Culture conditions:(1) Negative control group: DMEM medium; rhITF group: given10μg/ml rhITF,25μg/ml rhITF and50μg/ml rhITF after removing the serum;(2)Normal control group: DMEM medium+10%fetal calf serum; different time pointsgroup: the concentration of rhITF was50μg/ml, divided into3h group,6h group,12hgroup and24h group;(3) Normal control group: the same as (2); rhITF group:50μg/mlrhITF; rhITF+inhibitor group:50μg/ml rhITF+50μg/ml genistein;(4) Negative controlgroup: DMEM medium; given50μg/ml rhITF and50μg/ml rhITF+50μg/ml genisteinafter removing the serum.5. Detection indicators:(1) Cell migration: the change of cell migration ability was observed by Transwell;(2) Cell localization and fluorescence expression of adhesion molecule: intestinalepithelial cell localization and fluorescence expression of β-Catenin and E-Cadherin were detected by immunofluorescence;(3) The content of adhesion molecule: the change of β-Catenin and E-Cadherin wereobserved by Western Blot;(4) Detection of adhesion molecule phosphorylation level: the expression ofphosphorylated β-Catenin was detected by Western Blot.Result1. RhITF could promote Caco-2migration, the ability of cell migration along with theconcentration increase. The number of cell in50μg/ml rhITF group was morethan negative control group and10μg/ml rhITF group and25μg/ml rhITF group, thedifference was statistically significant (P<0.01);25μg/ml rhITF group compared with thenegative control group, the difference was statistically significant (P<0.05).10μg/mlrhITF group compared with the negative control group, the difference was notstatistically significant (P>0.05).2. The fluorescence expression and protein expression of β-Catenin and E-Cadherin wereinhibited by rhITF, compared with normal control group,3h group,6h group and24hgroup, the fluorescence expression and protein expression of12h group was the weakest(P<0.05).3. With the stimulation of rhITF, compared with normal control group,3h group,6hgroup and24h group, the protein expression of phosphorylation β-Catenin in12h groupwas highest (P<0.05).4. With the stimulation of rhITF and inhibitor (genistein), the protein expression ofphosphorylation β-Catenin in rhITF group was stronger than normal control group andrhITF+inhibitor group (P<0.05).5. The ability of rhITF promote cell migration was evidently reduced after inhibitingphosphorylation β-Catenin. The number of cell in rhITF group was more than negativecontrol group and rhITF+inhibitor group (P<0.01); rhITF+inhibitor group compared withnegative control group, the difference was not statistically significant (P>0.05).Conclusion1. ITF could promote intestinal epithelial cell migration.2. ITF could reduce cell adhesion and promote cell migration, by inhibiting theexpression of β-Catenin and E-Cadherin.3. The main mechanism of rhITF accelerate cell migration could promote β-Catenin phosphorylation, weaken the crossing-linking of β-Catenin and E-Cadherin, and interferewith cell adhesion. |
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