Expression Of Wnt Signals In Rat Tracheal Stem Cell During The Proliferation And Differentiation

IntroductionA stem cell can be defined as protocell which retains a high capacity for self — renewal throughout adult life and usually be considered to have the ability to produce daughters that undergo terminal differentiation. To study the stem cell could explore the puzzle of human development, and could hope to produce the artificial organ as a replacement of the diseased one in the future. Among the a-dult stem cell, hemopoietic stem cell, neural stem cell, epidermal stem cell, heparal stem cell, pancreatic stem cell,prostatic stem cell, stomachal and intestinal stem cell have been demonstrated one after another, but tracheal stem cell is still a concept.Ding and Zhou et al have developed a model of tracheal regeneration induced by fluorouracil ( 5 - FU ) in rats ex vivo, and identified that tracheal stem cells were in the residual cells in G0, which have the capacity to efflux Hoechst 33342 dye, the side population (SP) phenotype can be identified by the expression of Bcrp1/ABCG₂. Various signal pathways are involved in the repair process after injury of the trachea to regulate the migration, proliferation and differentiation of the stem cells. However, there is no report about this at present.The highly conserved Wnt secreted proteins are critical mediators of cell -to - cell signaling during development of animals. Recent research suggested that Wnt signals are involved in the interaction of stem cell and extracellular matrix. Wnt signaling inhibits β - catenin degradation to activate the expression of TCF / LEF transcription factors, hence promote the differentiation of stem cell.We determined the expression of Wnt - 1 ^$ - Catenin^ Tcf -4 Nc - Myc in rat tracheal epithelium during the regeneration after injury by immunohisto-chemistry ^Western - blot and RT - PCR, to explore the mechanisms in the early proliferation and differentiation of the tracheal stem cells.MethodsRat extracorporeal tracheal injury model;Rat tracheal rings were dissociated and cultured on Ham F12 medium. We developed an in vitro injury model of rat tracheal epithelium induced by 5 - FU. The rat tracheal rings were divided into two groups;the treated group were put into Hams F - 12 culture medium with the concentration of 5 — FU 12. 5mg/ml . The culture dishes were incubated at 37 C in a humidified , 5% C02atmosphere. Then exchanged the fresh Hams F -12 culture medium after 12 hours , extracted one ring respectively at 0^6^9A 12^24^48 hours after exchanging medium , fixed for LM. The culture medium was replaced every 24hours.. We apply the same volume of saline taking place of 5 - FU as the controlled group. The other conditions were same as the treated group.2. The wound -repair process were dynamically observed under LM.3. Wnt - 1 expression in tracheal epithelium during the process of regeneration was analyzed by immunohistochemistry and Western blotting.4. (3 - catenin expression levels in normal and injurious groups were analyzed by .immunohistochemistry and Western blotting.5. The expressions of TCF - 4 in normal and injurious groups were detected by Reverse transcriptase poiymerase chain reaction (RT -PCR).6. RT - PCR was applied to detection of c - Myc.Results1. The tracheal epithelium cells were mostly fell off after 12 hours induced by 5 - FU. We observed a few naked nucleus nail - like cells interval distributed on the basement membrane. When removing 5 - FU, the tracheal epitheliumrecovered . The morphology of cells underwent flat after 6 hours , Then we could observe that cuboidal and columnar epithelium appeared, the number of the cells increased, till 48 - 72 hours bronchial epithelium regained pseudostratified mu-cociliary epithelium.2. Immunohistochemistry and Western blotting analysis showed that there were different Wnt - 1 levels at different times after the removal of 5 - FU which in accordance with the change of immunohistochemistry. Wnt - 1 was minimally detected after treatment with 5 - FU for 12 hours, reaching a maximal level at 6 hours after the removal of 5 - FU, and then decreased over time. At 48 hours, very low Wnt — 1 level was detected.3. Immunohistochemistry and Western blotting analysis showed that there were different^ - catenin levels at different times after the removal of 5 - FU which in accordance with the change of immunohistochemistry. 3 -catenin.was minimally detected after treatment with 5 — FU for 12 hours, reaching a maximal level at 12 hours after the removal of 5 - FU, and then decreased over time. At 48 hours, very low ï¿¡ —catenin level was detected.4. The expression of Tcf-4 mRNA levels increased obviously at 12 hours after the removal of 5 - FU, and then decreased gradually with the repair process.5. The expression of c - Myc mRNA levels increased obviously at 6 hours after the removal of 5 - FU, and then decreased gradually with the repair process.Conclusions1. This study applied fluorouracil(5 - FU) as a injury factor,developed an in vitro injury model of rat tracheal epithelium. Just the proliferation and differentiation of tracheal stem cells regenerated tracheal epithelium.2. The expression of Wnt - 1 corresponds with the wound and healing process of tracheal epithelium, suggesting that Wnt -1 may plays the important effect in regulating the tracheal stem cell proliferation and differentiation.3. The increased expression of cytoplasmic (3 - catenin may promote theproliferation of the tracheal stem cell, while inhibite its differentiation .4. The expression of TCF - 4 is time dependence in the tracheal wound healing, suggesting that TCF - 4 is the important positive regulator in the process of the proliferation and differentiation of tracheal stem cells.5. c -myc may participate in the regulation of proliferation N differentiation and repaire of the tracheal epithelium, and its expression could induce the proliferation of the tracheal stem cell, while inhibit its differentiation .