| Objective:In our study, human Umbilical Cord-derived Mesenchuymal Stem Ccells (UC-MSCs) were cultured together with the human Sweat Gland Cells (h-SGCs) in the micro-environment that h-SGCs were injured, lead the UC-MSCs differenteiated to h-SGCs. And take advantage of checking antigens expression to discuss the EGF's effect to the diffrentiation of cellular phenotype conversion, and the contribution of extracellulor signal-regulated kinase (ERK) signal pathway in the procession, which might theoretically and practically support the clinical application of human sweat gland tissue restoration.Methods:In our study, the fresh full-term umbilical cord was cutted to segment about 4cm with scissor in asepsis envirment. Washed them cleanlily and eliminated blood vessel in order to avoid the polluting of endodermis cells.Riped the Wharton's Jelly into rops and cuted them to bits about 1mm3. Innoculated these tissue into the plastic culture flask. Indentification of the UC-MSCs via flow cytometry. Cuting the asepsis skin from normal poeple to bits, diped in the culture medium obtaining collagenase II and geted the human sweat gland tissue next day under microscope. Immunohistochemical stain identify the surface antigen expression. The h-SGCs were injured in a hot environment about 47℃for 40 minutes. Then, added medium and the 4rd passage UC-MSCs to 6-well plate well, followed by adding the transwell inserts, and lastly added the medium and cells to the inside compartment, guided the UC-MSCs diffrentiate to h-SGCs. There wree four groups:group 1 is the control:UC-MSCs were culture in the h-SGCs medium, group 2:UC-MSCs were cultured together with injured h-SGCs in the same medium, group 3:UC-MSCs were cultured together with injured h-SGCs in the medium which was added EGF 50mg/ml, group 4:UC-MSCs were cultured together with injured h-SGCs in the medium which was added PD98059 10mg/ml. Indentification the CK7,CK19,CEA of the UC-MSCs via flow cytometry and immunohistochemical staining. Analysing the data with SPSS 16.0.Results:(1) Flow cytometry:①The expression level of CD14,CD19,CD34,CD44,CD45,CD105,CK7,CK19 in UC-MSCs were 0.6%,91.3%,0.6%, 99.1%,0.9%,99.2%,0.8%,0.9%. And the CD29,CD44,CD105 were the symbol antigens of MSCs,the CD14,CD34,CD45 were the symbol antigens of hemopoie stem cell, proved the cells we cultured were UC-MSCs, and the cells didn't express CK7 CK19.②The expression level of CK7 and CK19 between every groups:group1:(2.20±1.51)%, (2.23±0.68)%; group2 (6.37±0.74)%, (5.73±0.32)%; group3:(14.3±0.96)%, (12.57±1.06)%, goup4(14.3±0.96)%, (12.57±1.06)%. Belong to CK7:the goup 1 is similar to group 4 (P=0.999>0.05), group2 compare to group1 (P=0.005＜0.05), group3 compare to grooup 1 (P=0.000＜0.05). Belong to CK19:the goup1 is similar to group 4 (P=0.997＞0.05), group2 compare to group1 (P=0.005＜0.05),group3 compare to group1 (P=0.000＜0.05).(2) Immunohistochemistry staining:The expression of CEA and CK19 of UC-MSCs were in a very low level. As CEA and CK19 were the antigens of h-SGCs, they were in a very high expression level in h-SGCs. Some cells expressed CEA and CK19 in every group, belonged to CEA:group1 (3.33±0.71)%; group2(7.43±1.01)%; group3(17.63±2.31)%; group4 (3.17±0.35)%. The group1 was similar to group4 (P=0.997＞0.05), group2 compared to groupl (P=0.005<0.050), group3 compared to groupl (P=0.001＜0.05)(3) Western-blot:the OD value of every group:group1:(0.64±0.026), group2:(0.79±0.049), group3 (1.20±0.032), group4 (0.62±0.042).The expression of p-ERK in group1 and group4 were similar,but it was obviously in a high level in group3, group2 was also difference to group1.Conclusion:When UC-MSCs were cultured together with injured h-SGCs, the expression of CK7,CK19,CEA in them were increaed, it proved UC-MSCs had the ability to diffrentiate to h-SGCs in the h-SGCs were injured micro-environment. The antigens' expression between every groups and the result of western blot prompted that EGF has positive negative effects on the differentiation, and it has a bearing on ERK signal pathway. |
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