| Background and Objectives:Right now, wound healing is the basic problem we are facing in burns, and how topromote wound repairing after burns is still a major task. Wound healing afterburns is acomplicated progress, it includes varieties of cells, cytokines and the interactions betweencells and cytokines, it also involves cell movement. adhesion. differentiation andproliferation etc. in a word, the two main aspect of these processes are formation ofgranulation tissue and reepithelializing of wound. In the processes ofreepithelializing, thetransfer ability of epidermal cells is considered as importantaddition for wound healing.Current studies suggested that wound will be in a hypoxic state after acute injury. And thishypoxic microenvironment promotes the epidermal cells transferring and woundrepairing.Some experiments in vitro have also indicates that transfer ability of epidermalcells will improve in hypoxic environment. However the mechanism is not clear.Transport process of epidermal cells is a complicated biological behaviour, it involvesthe interaction between epidermal cells and extracellular matrix, it also includescytoskeleton reorganization and cells asymmetric divisions. At every step of cell transport,cytoskeleton especially microtubilin kinetic changes occurs. In addition, it needs a seriousof biochemistry cascade reaction to regulate microtubilin in order to ensure validity andreliability of cell transport.Therefore, we are aimed to simulatee a hypoxic Microenvironmentafter burns to investigate the expression of MAP4(microtubleassociate protein4, MAP4) ofHaCaT cell and its influence for cells transport, as well as its mechanism.On the base of the first stage of research, we develop a anoxia model of HaCaT cell,and observe change of cytoskeletal protein, expression of MAP4and influence for cellstransport of HaCaT cell using cell wound scratch assay, cell migration experiments,immunoprecipitation, immumofluorescence and western blot experiment. Wealsoinvestigate the relationship between MAP4and Tctex-1, as well as their influence for cell transport.Methods: The change of cell migration and microtubilin kinetics of HaCaT cellinhypoxic microenvironment were observed systematicallyPart I SR preparation and C ion injection and observed their microscopic morphology1. The hypoxia model and culture method of HaCaT cell were established: under theway of primed low oxygen (1%O2，94%N2，5%CO2), pour mixed gas into sealed infusionvessel in order establish a hypoxic microenvironment. The method above is feasible andsimple, in addition, cells grows well.2. The time of cell hypoxic treatment were set up:24h, the change of cell migration ofHaCaT cell in hypoxic microenvironment were observed by using two migration models(Wound scratch assay and Tanswell migration experiments). All the data are expressed asmean±std deviation.Statistical analysis was performed using S-PSS version20.0（P=0.05）.3. Three different points of time were set up (0.5h/1h/3h) to observe the change ofskelemin α-tubulin of HaCaT using IF assay, then the dynamic change of microtubilinkinetics were evaluated.Part II The effect of MAP4on microtubilin kinetics and HaCaT cell migrationinhypoxic microenvironment1. Four different point of time were set up:0.5h/1h/3h and24h. The MAP4andphosphorylated MAP4expression in HaCaT cell in hypoxic microenvironment wereinvestigated.2. MAP4high and low expression vector, transmit HaCaT cell were establishedand thestable HaCaT cell models of high and low MAP4expression were built. Then, the changeof cell model migration and microtubilin kinetics were investigatedbyusing wound scratchassay, Tanswell migration experiments and IF assay. Handing time was the same as above,the experiment groups were as follows: control group,MAP4high expression group, lowexpression group and their control group corresponding.Part III The interactions between Tctex-1and MAP4in hypoxic microenvironmentand their impact on microtubilin kinetics regulation and cell migration1. Four different point of time were set up:0.5h/1h/3h and24h. The MAP4, p-MAP4and Tctex-1expression in MAP4high expression, low expression and controlgroups inhypoxic microenvironment were investigated. 2. MAP4high and low expression vector, transmit HaCaT cell were establishedandstable HaCaT cell models of high and low MAP4expression were built. Then, the changesof MAP4, p-MAP4and Tctex-1expression were investigated on Tctex-1high and lowexpression cell using WB experiment. Handing time was the same as above.3. The change of cell migration and microtubilin kinetics on Tctex-1high and lowexpression cell were investigated using wound scratch assay, Tanswell migrationexperimentsand IF assay under the hypoxic microenvironment. Handing time was thesame as above.The experiment groups are as follows: control group, high Tctex-1expression group, lowTctex-1expression group and their control group corresponding.Results:1. The hypoxia model and culture method of HaCaT cell were established successfully.2. The MTT assay and Tanswell migration experiments indicated that the migration ofHaCaT cell was promoted after hypoxia treatment.3. The results of IF experiment showed that the skelemin α-tubulin of HaCaT started todepolymerize and gather around the soma after hypoxia treatment. Meanwhilethecytoskeleton became loose, the cellular morphology began to show pleomorphic, and the cellwas unstable.4. The results of WB experiment indicated that the expressions of MAP4and p-MAP4were increased temporarily after early hypoxia. And then, the expressions of MAP4andp-MAP4were decreased rapidly over time.5. The HaCaT cell models of MAP4high expression and low expression wereestablished successfully. The MTT assay and Tanswell migration experiments indicated thatcomparing to control group, the cellular migration of low expression group was suppressedand had no obviously promotion after hypoxia treatment. However, the cellular migration ofhigh expression group was increased significantly after hypoxia treatment. The results of IFexperiment showed that cell shrinkage appeared in MAP4low expression group afterhypoxia treatment, the depolymerization of skelemin α-tubulin and migration of HaCaTwere not obviously and no soma gathering around was found. The variation trend of skeleminα-tubulin of high expression group was similarto the normal control group in prolongedhypoxia time.6. Comparing to control group, the expressions of MAP4and p-MAP4in high expression group were increased significantly after hypoxia, and both showed the similarchanging trends. Nevertheless, the expressions of MAP4and p-MAP4in low expressiongroup were decreased. And the expression of Tctex-1was decreased temporarily after earlyhypoxia, but it was increased subsequently over time. The expression of Tctex-1wasincreased and decreased in MAP4high expression group and lowexpression grouprespectively.7. Tctex-1high and low expression models of HaCaT cell were established successfully.The migration experiments indicated that comparing to control group, the cellular migrationof Tctex-1low expression group was suppressed.Instead, the Tctex-1low expression groupwas promoted.Similar to the cell of MAP4group, the cellularskelemin α-tubulin of Tctex-1high expression group started to depolymerize and gather around the soma after hypoxiatreatment. Meanwhile the cytoskeleton became loose, the cellular morphology began to showpleomorphic, and the cell was unstable and tended to migrate. The cell shrinkage appeared inTctex-1low expression group, the depolymerization of skelemin α-tubulin was notobviously and no soma gathering around was found, as well as cells were treated by hypoxia.8. The results of WB experiment indicated that the changing trends of MAP4, p-MAP4and Tctex-1in high expression and low expression groups were similar with MAP4highexpression and low expression groups. The expression of MAP4waspromoted in Tctex-1high expression group and decreased in Tctex-1low expression group.Conclusions:The cellular migration can be increased by regulating the expressions of MAP4andTctex-1in the HaCaT cells as well as the cell microtubules dynamics in the hypoxiaenvironment. The possible mechanism is that the expression or activity of MAP4isregulated by hypoxia, and then affecting the expression of Tctex-1in HaCaT cell. Finally,the migration of skin cell is regulated by the factors above. |
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