Effect Of Glycation Of Collagen Ⅳ On Biological Character Of Epidermal Stem Cells

[Objectives]TO investigate the effect of glycation of collagenⅣon epidermal stem cells,such as adhesion,proliferation,apopt-osis.[Methods]Epidermal stem cells were cultured by fast adhesion with collagenⅣ.After epidermal stem cells were cultured,keratinocyte 19 and integrinβ1 were identified by immunocytochemistry and immunofluorescence which are markers of epidermal stem cells.Then they were seeded on culture plates which were coated with normal collagenⅣand glycated collagenⅣ(7 days,14 days, 21 days).After fifteen minutes,three ports of cells of every groups were taken out,then the cells that didn't adhere to collagenⅣwere discarded and compared their adhesion rate.The second aspect,the epidermal stem cells of second generation were seeded at a density of 5×10~3 cells on culture plates which were coated with normal collagenⅣand glycated collagenⅣ.After 3hours,2days,4days,6days,8days,10days,12days and 14 days,5 ports of cells were taken out to measure OD by MTT to draw pictures of growth curve.ON the third way,the epidermal stem cells of the second generation were seeded at a density of 200 cells on culture plates Of 6 wells,after 6 days and 10 days,the AnnexinⅤof two groups were determind by flow cytometry[results]The expression of integrinβ1 were detected with immunofluorescence. K19 serving as first antibody,SP method of immunocytochemistry showed that the isolated cells positively stained with grown-yellow were epidermal stem cells.After 7 days,the fluorescence value of collagenⅣwith thick glucose is 1.864±0.126,while the normal collagenⅣis 0.699±0.0831.After 14 days,the glycated group is 3.051±0.112,while the normal group is 0.765±0.0927.After 21 days,the glycated group is 4.416±0.1378,while the normal group is 0.8254±0.058.The second index is adhesion rate.After 7 days's effect of high glucose on collagenⅣ,there is no statistical difference between the glycated group and the normal group.P＞0.05.After 14 days,the adhesion rate of glycated group is lower than that of normal group.P＜0.05.After 21 days,the adhesion rate of the former is much lower than the latter.P＜0.05.there is obvious difference between the glycated group and the normal group.So we can see that the adhesion rate is lower and lower with the deep of the glycation.The third index is the clone production rate.The clone production of the glycated group is(38.5±1.9)%,while the normal group is(32.7±1.5)%.P＜0.05.there is statistical difference between the glycated group and the normal group.The last index is the apoptosis.On the sixth day,the positive rate of the glycated group is(16.22±0.7912)%,while the normal group is(10.7±0.9438)%.On the 10th day,the positive rate of the former is(8.266±0.8177)%,while the latter is (4.66±0.7368)%.P＜0.05[conclusion]With the deep of the glycation of collagenⅣ,the adhesion and proliferation rate of epidermal stem cells are lower and lower,while the apoptosis rate is higher and higher.Glycation of collagenⅣinhibited the adhesion and proliferation of epidermal stem cells and accelerate the apoptosis, which may be one of the reasons why the diabetic wound is difficult to heal.