Study Of Dermal Papilla Cells And Some Of Their Growth Factors

Objective: Hair dermal papilla cells (DPCs) are specialized mesenchymal cells that exist in the dermal papilla with an essential role in hair formation, growth, and cycling. The structural or functional abnormality of DP could lead to the occurrences of various hair diseases. As DPCs are concealed in the dermal papilla, their isolation and culture therefore become difficult. However, because of the important physiological role in hair growth, the research of DPCs has been continually popular and even becomes the new focus in the field of hair formation, growth, and cycling. Cultured DPCs resemble fibroblast in appearance, but their essence is different. On growth pattern, the cultured DPCs exhibit a polygonal cellular morphology and a tendency to form multi-layered cluster, i.e. aggregative growth pattern. DPCs tend to gradually lose their aggregative capacity after being passaged, and after the seventh passage, the subcultures tend to fail to coagulate. The aggregative behavior is one of the significant characteristics of DPCs, which is associated with their biological functions. Scientific research showed that the subsequent passages of human DPCs, which were cultured with conditioned medium from their early passages, exhibit the aggregative growth pattern. And growth factors in DPCs were stronger expressions in early passages compared with them in late passages. These results indicate that the early passages human DPCs cultured in vitro might secrete growth factors which have some biological activities. The previous research show, DPCs produce many growth factors, including vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), transforming growth factor-β(TGF-β), insulin-like growth factors-Ⅰ(IGF-Ⅰ) etc. However, which kind(s) of the growth factors play what kind(s) of specific role in the hair follicle biology are worth study. Studies on the growth factors of DPCs will contribute to understanding the roles they play in the growth process of hair follicle, which will gear up for the treatment of hair follicles diseases and the progress of skin tissue engineering. The objective of our experiment is to get enough DPCs by improving the traditional isolation methods; investigate the expressions of stem cell factor (SCF) and vascular endothelial growth factor (VEGF) in different passages of cultured DPCs and observe the growth curves of DPCs in vitro culture with the effect of SCF or VEGF.Methods:1 Culture and Observe DPCs. The method for isolation of DPCs was improved from two steps enzyme method. Enough DPCs were gained by this method. DPCs were cultured in DMEM medium supplemented with 10% heat inactivated fetal bovine serum and in a humidified incubator at 37℃, 95% air, 5% CO2. The morphological changes of cells were detected by the reverse microscopy.2 DPCs were identified by immunochemistry withα-SMA antibody, blue special and PAS stain.3 The expressions of VEGF and SCF in different passages of cultured DPCs were investigated by immunochemistry staining and FCM.4 The growth curve of DPCs in vitro with the effect of SCF or VEGF was assessed by MTT.5 Statistic analyses: Data was analyzed by spss13.0 for windows.Results:1 The method for isolation of DPCs was improved from two steps enzyme method, which not only saved specimens but also reduced the work load significantly. Successful separation by dispase and collagenase brought about the DPCs of high purity, rapid adherence. The cells exhibit the characteristic of aggregative behavior, which could be phased out along with their cultivation. And after the seventh passage, the subcultures tend to fail to coagulate.2 The cells were positive to smooth muscle actin alpha, presented metachromatism by blue special staining and were positively stained with PAS stain.3 The expressions of SCF and VEGF in different passages of cultured DPCs were investigated by immunochemistry staining. SCF and VEGF proteins were expressed in the intracytoplasm of cultured DPCs, with their stronger expressions in early passages compared with them in late passages.4 The expressions of SCF and VEGF in different passages of cultured DPCs were investigated by FCM. The average channel values of SCF and VEGF were significant differences among 3 passages, 6 passages and 9 passages ( p<0.05 ). With an increase in the number of cell passages the growth factors decreasing.5 Compare the control group with the groups SCF and VEGF, it showed that the lag phase was decreased with the entry into the logarithmic growth phase early and the OD value was increased ( p<0.05 ). The groups SCF and VEGF promoted the growth of DPCs.Conclusion:1 The method for isolation of DPCs was improved from two steps enzyme method, which provided us with enough DPCs needed. Successfully separated and cultured from a small number of samples, the high purity DPCs showed the typical aggregative growth pattern.α-smooth muscle actin expression was positive, DPCS grew well in DMEM medium. Improved two steps enzyme method is a simple, effective and economical isolation and culture method for DPCs.2 The expressions of SCF and VEGF in early passages of cultured DPCs were stronger than in late passages. With the increases of DPCs passages SCF and VEGF expressions gradually weakened.3 The growth factors including SCF, VEGF were proved to stimulate the proliferation of DPCs.