| Objective:As a subsidiary organ of skin, hair has an important function of physiology and sociology. Missing or abnormal distribution of hair brings great psychological burden to patients. The current research of hair growth mechanism has focused on hair follicles. Dermal papilla cells (DPCs) are a group of dermis-derived cells located at the bottom of follicle, which are specialized fibroblasts. The salient biological characteristics of DPCs are manifested aggregation of growth pattern and the ability to induce hair follicle formation in vivo and in vitro. DPCs occur at the central link in the morphology of the hair follicle and its cyclical growth regulation. This feature will disappear after the cells were passed on several times. A number of studies have shown that the biological functions of DPCs have a lot to the aggregation property. The biological functions are completed through a series of signaling molecules pathway and a variety of cell growth factors. Insulin-like growth factor-1(IGF-1) is similar with insulin that can stimulate the melanocytes, epithelial cells and hair follicle growth. Fibroblast growth factor 7 (FGF7) also known as keratinocyte growth factor, can stimulate keratinocytes and other epithelial cell proliferation.DPCs are not only essential for the induction and maintenance of the cyclical nature of hair follicle cycle, but also for the size and anagen length of hair follicles. Licorice is frequently used in traditional Chinese medicine to promote hair growth. It is demonstrated that licorice can promote hair follicles growth in vitro. Stroner Neo-MinophagenC (SNMC), the essential component of which is glycyrrhizin compounded with glycine and cysteine, is widely used in the treatment of skin diseases and liver disease. Clinical trials found that SNMC is effective for alopecia areata. The mechanism is not clear, its effect on cultured human DPCs has not been reported yet. Glucocorticoid applied for treatment of alopecia areata have been more than 60 years so far, which is one of the most commonly used, reliable and effective methods. Foreign doctors often take the corticosteroid injection as the first treatment for adults with limitated alopecia areata. The mechanism may be related with anti-inflammatory and immune suppression, but the role to human DPCs has not been reported.To observe the biological characteristics of DPCs in vitro as well as its correlation with IGF-1 and FGF7, and the effects of SNMC and dexameth on the activity of DPCs, we have managed to separate and culture dermal papilla, and detect two factors in the aggregative, non-aggregative growth of DPCs, and evaluate the proliferative effect of IGF-1, SNMC and dexameth on DPCs.Methods:1 Isolation and culture of dermal papilla cellsDermal papillas were isolated from human scalp hair follicles by digestion with dispase and collagenase D and cultured. The morphology and growth of DPCs were observed in vitro.α-smooth muscle actin (α-SMA) was detected by immunohistochemical staining to identify DPCs.2 The expression of IGF-1,FGF7 protein in different passages of cultured DPCs were detected by immunohistochemical method.3 The expression level changes of IGF-1, FGF7 in different passages of cultured DPCs were detected by FCM.4 The FCM was also used to detect the changes of the apoptosis rate and cell cycle in different passages.5 Effect of different concentration of IGF-1(2.5～100ng/ml) on the cell proliferation of DPCs were measured by MTT.6 Effect of different concentration of SNMC and dexameth (0.5～ 100ng/ml) on the cell proliferation of DPCs were measured by MTT.7 Statistic analysis: data were analyzed by statistical software Spss13.0 for windows, using t-test, one-way ANOVA analysis, with significance of difference standardα=0.05.Results:1 Isolation, cultivation and identification of human DPCs.1.1 Isolation and cultivation of human DPCsThe dermal papillas obtained by "two-step enzymatic digestion" were round or oval in the inverted microscope observation. They were adherent next day, and then DPCs could be observed move out to the surrounding in the radiative form. The majority cells were confluent after 2～3 weeks. Observed under low power lens, the cells were swirling growth, with aggregative growth tendency. The cells elongated spindle. Cells were adherent the next day after digestion, aggregative growth patten was formed again with the increase of cells number. Cell proliferation also accelerated after passage, especially in 3rd to 5th generations, and aggregative growth also could be observed. Continue passed on, the cells become multi-level, spindle or elongated fibroblast-like, the aggregative growth gradually disappeared. The cells growed more slowly, passaged longer.1.2 Identification of human dermal papilla cellsα-SMA was expressed in DPCs by immunohistochemistry method.2 The expression of IGF-1, FGF7 protein in 3rd and 9th generations of cultured DPCs by immunohistochemical methodIGF-1 and FGF7 were expressed in the cytoplasm, coloring ranging from yellow to brown. The expression of IGF-1 in 3rd, 9th generation DPCs after immunohistochemical staining were +～++, +; FGF7 were +～++,±～+。3 The expression level changes of IGF-1, FGF7 in different passages of cultured DPCs by FCMThe results showed that fluorescence intensity of IGF-1 in 3rd and 9th generations were 270.98±26.84, 232.14±14.80 respectively, the former exceeded the latter, but the statistical analysis showed that there was no significant differences (P>0.05). The fluorescence intensity of FGF7 were 373.51±24.97, 270.91±32.83 respectively, the former exceeded the latter, statistical analysis showed a significant difference between them (P <0.05).4 Apoptosis and cell cycle distribution of human DPCs were detected by FCMThe G1-phase ratio of 3rd and 9th generation cells were 55.53±3.34%, 57.53±6.84% respectively; the S-phase ratio of cells were 34.87±3.18%, 34.10±6.36%; the G2-phase ratio were 7.82±3.37%, 8.40±3.19%. The apoptosis of the two generations were 1.36±0.24, 1.28±0.36 respectively. There were no significant differences between the two passages in each phase (P> 0.05).5 Effect of different concentration of IGF-1(2.5～100ng/ml) on the cell proliferation of DPCsThe DPCs of logarithmic 4th passage which cultured with different concentrations of recombinant human IGF-1 after 72h were observed under light microscope, no obvious morphological difference among them. The results of MTT showed that, compared with the control group, each experimental group could significantly promote the proliferation of DPCs (P <0.05). 2.5ng/ml was the most significant and there were no significant difference among the rest four groups (P> 0.05).6 Effect of different concentration of SNMC (0.5～100μg/ml) on the cell proliferation of DPCs.The DPCs of logarithmic 4th passage which cultured with different concentrations of recombinant human IGF-1 after 72h were observed under light microscope, no obvious morphological difference among them. The results of MTT showed that, the effect of the proliferation was dose-dependent with concentration of 0.5～20μg/ml. 20μg/ml could significantly promote the proliferation of DPCs (P <0.05).The effect was weakened when the concentration further increased, and there were no significant difference among the rest groups (P> 0.05).7 Effect of different concentration of dexameth (0.5～100μg/ml) on the cell proliferation of DPCsThe DPCs of logarithmic 4th passage which cultured with different concentrations of dexameth after 72h were observed under light microscope, no obvious morphological difference among them. The results of MTT showed that, the effect of the proliferation was dose-dependent with concentration of 0.5～2.5μg/ml, of which 2.5μg/ml could significantly promote the proliferation of DPCs (P<0.05). The effect was weakened when the concentration further increased, and there were no significant difference among the rest groups (P> 0.05).Conclusions:1"Two-step digestive treatment with dispase and collagenase D"was an efficient and rapid method to isolate and culture human hair dermal papillas.2 Aggregative growth property was visible in cultured DPCs before 6th generation, this feature gradually disappeared with DPCs were passed on.3 The cell cycle and apoptosis of DPCs with aggregative and non-aggregative growth were no significant differences, indicating that cultured DPCs maintained at a stable proliferation of the state.4 FGF7 in DPCs with aggregative growth expressed significantly higher than cells with non-aggregative growth, suggesting that DPCs promote hair matrix cell proliferation and cornification may be related to FGF7.5 IGF-1 can significantly stimulate the proliferation of DPCs, but also regulated by IGF bingding protein. IGF-1 in the DPCs with aggregation growth expressed higher than that of non-aggregation growth, but no statistically significant difference. It remains to be further explored that the mechanism of IGF-1 promoting the growth of hair follicle.6 The mechanism of SNMC and dexameth may also induce hair growth by regulating the activity of DPCs in addition to anti-inflammatory, immune regulation and suppression.
|
PDF Full Text Request