The Research On The Transplantationtreatment Of Non-cultured Autologous Epidermal Cell For Burns

objective:1.In order to find the optimal isolation condition,we take different concentration and different time to isolate epidermal cell from rat skin.2.After obtaining the optimal concentration of digestive enzyme and digestion time from thesis one,we try to make aware correlation between skin area and the number of epidermal cells and get the area of epidermal cell 3.We make the rat burn model basing the former work.We observe the effect of treatment of burns by transplanting epidermal cell with different density in order to investigate the possibility and optimum transplantation density.Methods:1.Three different concentration enzyme and three different digestion time,9 groups,to detect the optimal isolation condition. The epidermal cells are identified with immunohistochemistry,colored by 0.4% trypan-blue exclusion and then count the cell number.MTT method investigates the number of functional cells indirectly.The optimum digestive ferment concentration and digestion time are determined by crosscut analysis.2.Digesting different area of rat skin under the optimal isolation condition,5 groups.The correlation between skin area and the number of epidermal cells is analyzed by statistical analysis.We get the area of epidermal cell through Micro-measure device,observing the epidermal cells cultured in 24-Well plates.3.We make the rat burn model,dividing into treatment groups(A,B,C),control group(D).The treatment groups takes different cell transplantation density.GroupA:6.0×10⁴/cm²;groupB:1.2×10⁵/cm²;groupC:2.4×10⁵ c/m².The healing status,concentration proportion and the number of cicatricle healing in each group are observed and recorded.Results:1.The cell isolated is proved by immunohistochemistry.The epidermal cells show elliptic before adhering,irregular shapes after adhering.The cultured one looks like pave-stone.It's the typical characteristics of the epidermal cell.The epidermal cell is colored by 0.4%trypan-blue exclusion and then count the cell number;the number of functional cells is detected by MTT.These two results are both analyzed by crosscut analysis.Both of them indicate that the best isolation condition is 0.25%Trypsin-0.04%EDTA-Na₂,16h.2. The correlation between skin area and epidermal cells is analyzed by statistical method:linear correlation,linearity equations exists.The epidermal cells are cultured in 24-Well plates.We get the cell parameters through Micro-measure device,and cell area after calculating:6540±450um².3.One of group A is dead after transplantation; One of group B and C is unsuccessful because of infection.The number of cicatricle healing:group A:9;B:1;C:0;D:15.The concentration proportion:group A:34.3%; B:20.1%;C:18.9%;D:84.5%.Statistical Analysis:Cicatricle healing:the difference between group B and C is not statistically significant,while the else differences are statistical significant;In the construction of wounds,the difference between group B and C is not statistically significant,while the else differences are statistical significant.Conclusions:1.The digestion method of Trypsin-EDTA is successful in isolating epidermal cell.The cell is identified.Both appearance and function of cell are normal through culture.The result of crosscut analysis indicates:The digestion method of 0.25%Trypsin-0.04%EDTA-Na₂,16h cold digestion and corium curettement is a kind of practical optimum method to isolate epidermal cell.2.The skin area is linear correlated with the number of epidermal cells,and we get the linear equation:y(number of cell×10⁵)=6.445×x+0.17.3.The making of rat burn model is successful,it's feasible that the transplantation treatment of non-cultured autologous epidermal cells for burns.The suitable transplantation density is 1.2×10⁵/cm².The corresponding between skin area and treatment area:1cm² can heal 5.5cm² excision of eschar area.