Effect Of Trichostatin A On The Roliferation And Differentiation Of Rat Tracheal Stem Cell

PurposeWe have previously constructed a tracheal injury model induced by 5-fluorouracil (5-Fu) ex vivo to localize the tracheal stem cells and idendified tracheal stem cells exist in the GO cells. To explore the mechanism of involvement of the Wnt/b-catenin pathway during regulated growth of tracheal epithelial stem cells in rats, we used this injury model to observe changes in levels of Wnt1, b-catenin, and cyclinD1 mRNAs, and localization of b-catenin protein during rat tracheal epithelial regeneration. Our findings indicate that the Wnt/b-catenin pathway plays a role in proliferation and differentiation of tracheal epithelial stem cells. We have also observed the dynamic changes of HDACl(histone deacetylase 1) during regulated growth of tracheal stem cells in human tracheal regeneration. This time, we used the most efficacious HDACi TSA(Trichostatin A) to depress the HDAC1. And try to find out the effect of TSA on the proliferation and differentiation of rat tracheal stem cell.Methods1.Preparation of 5-Fu Model and TSA treatmentTwo weeks' Wistar rats provided by the animal center of the China Medical University. The animals were sacrificed by 10% Chloral Hydrate 0.4ml/100g. Then the tracheas were excised aseptically. Then they were douched with PBS and cultured in 1:1 mix of Dulbecco's modified Eagle's medium and Ham's F-12 medium (DMEM/F12) containing 120 mg/ml 5-Fu and 10% fetal bovine serum (FBS) for 12h at 37°C. The tissues were then refreshed by culturing in DMEM/F12 containing 10% FBS and 200nM TSA. Tracheal rings were taken out at 3,6,12,24,48h time points after removing 5-Fu. For western blotting, epithelium of some tracheal rings were stripped under anatomical microscope, snap-frozen and stored at -70°C until use. Some other tracheal rings were fixed in 4.5% formaldehyde, and made into paraffin-embedded tissue sections for hematoxylin-eosin stain and immunohistochemistry observation. Normal tracheal rings were also analyzed as controls.2. Hematoxylin-eosin stain was used to observe the morphological changes during tracheal epithelium regeneration.3. Immunohistochemistry Streptavidin-Peroxidase(SP)immunohistochemical technique was used to detectexpression of HDAC1 in the dynamic process during tracheal epithelium regeneration. PBS replaced the first antibody as negative group.4.Western blot analysis was used to identify changes in levels of HDAC1 protein during tracheal epithelium regeneration.Results1. HE:Without the effect of TSA, We found that the tracheal epithelium desquamated after 5-Fu treatment. The residual were trifle nude-nucleus cells distributed intervally on the basement membrane (GO phase cells) .When removing 5-Fu, the tracheal epithelium began to recover. The tracheal rings were covered with flattened epithelial cells at 3-6h after the removal of 5-Fu.At 12h, most of the epithelial cells were cuboidal cells and merged into pieces,the number of the cells increased.At 24h, pseudostratified mucociliary epithelium appeared in some region of tracheal epithelium,and the ciliated cells can be seen. At 48h after removal of 5-Fu, pseudostratified mucociliary epithelium was restored to its original mode similarly. However, with the effect of TSA, the recovery process was obviously hysteresis. At the 12h point, the epithelium just like the 3h of the epithelium without TSA treatment.2. Expression of HDACl in Tracheal EpitheliumWe found the Expression of HDACl was negative at oh after 5-Fu treatment. only few of the epithelial cells were HDAC1 positive at 3h-6h after the removal of 5-Fu. HDAC1 positive cells increased obviously at 12h. and was in top expression at 24h ,then decreased slightly at 48h.3. Changes in HDAC1 protein Levels during Tracheal Epithelial Regeneration Result of western blotting indicated that there was no detectable level of HDAC1protein in normal tracheal epithelium and in the epithelium at oh after 5-Fu treatment.The level of HDAC1 protein was elevated slightly at 3-6 h after the removal of 5-Fu, reached a top at 24h and then decreased slightly at 48h. TSA obviously inhibit the HDAC1 protein level.Conclusions1. TSA can obviously intercept the injury recovery process of 5-Fu rat tracheal model. The tracheal stem cells were blocked at the proliferation phase.2. Our research aims to provide a new method for the insufficient resource of the stem cells.