ABSTRACT
BACKGROUND:Hair folicle stem cels have been confirmed to have stronger proliferative ability than interfolicular epidermal stem cels, which have been an issue of concern in seed cel research. OBJECTIVE:To compare the biological characteristics of rat hair folicle stem cels cultured by tissue explant method and enzymatic digestion method. METHODS: Under stereomicroscope, hair folicles were isolated from the rat whiskers, and then tissue explant method and two-step enzymatic digestion method were employed to culture hair folicle stem cels. Cels were purified using repeated differential adhesion method, and cel growth and morphology were observed periodicaly. Flow cytometry was used to detect the expression of CD34 and β1 integrin in passage 3 hair folicle stem cels. RESULTS AND CONCLUSION:Cels cultured by two-step enzymatic digestion method grew faster with more amount than those cultured by tissue explants method. Flow cytometry showed that the expressions of PE-CD34 and FITC-β1 were (39.52±19.57)% and (93.46±4.73)% for the two-step enzymatic digestion group, and (19.20±11.53)% and (363.57±14.42)% for the tissue explant method, respectively. There was a significant difference between the two methods. In conclusion, these two methods are able to culture high-activity hair folicle stem cels, which can be chosen according to different experimental requirements.
ABSTRACT
BACKGROUND:The proliferation and differentiation of hair-folicle-generating stem cels are influenced by the joint action of their own genes and external signals. Wnt/β-catenin signaling pathway plays an important role in the development of hair folicles, but the detailed mechanisms are not yet clear. OBJECTIVE:To investigate, with interruption of keratinocyte growth factor and lithium chloride, the function and the interrelationship of Wnt/β-catenin signaling pathway with other signal factors when human hair-folicle-generating stem cels differentiate into dermal papila cels or epidermal cels. METHODS: Hair-folicle-generating stem cels were isolated from the bulge and cultivated. Then the growth curve of hair-folicle-generating stem cels was tested and formed in order to observe the cellproliferation ability cultivated at different densities (1×106/L, 1×107/L, 1×108/L, 1×109/L) at each time. Immunoflurorescene staining was performed to identify hair-folicle-generating stem cels and their differentiated cels. Lithium chloride (0, 0.5, 1.5, 10, 25 mmol/L individualy) and keratinocyte growth factor(0, 10, 25, 50, 100 μg/L individualy) were used to induce the differentiation of hair-folicle-generating stem cels. Then, we contrasted and analyzed the proliferation ability in each case, thereby investigating the most appropriate concentration of keratinocyte growth factor and lithium chloride to spur the differentiation of hair-folicle-generating stem cels. At days 3, 5, 7 and 9, we tested and compared the mRNA expressions of β-catenin, APC, GSK-3β, Axin and Lef1 from cels in control group, 10 mmol/L lithium chloride group and 10 μg/L keratinocyte growth factor group. RESULTS AND CONCLUSION:Isolating cultured hair-folicle-generating stem cels stil had a great reproductive activity and multi-lineage potential even after various times subculturein vitro. With higher lithium chloride concentration, the proliferation ability of hair-folicle-generating stem cels declined; while it increased when keratinocyte growth factor concentration increased. In K-SFM medium which contained lithium chloride, the transformation of hair-folicle-generating stem cels was obvious, showing distinct differences among groups. Especialy, the level ofβ-catenin reached the peak when lithium chloride > 10 mmol/L. However, in K-SFM medium which contained keratinocyte growth factor, hair-folicle-generating stem cels differentiated into epidermal cels and the level of β-catenin changed slightly. We found that, while spurring the differentiation of hair-folicle-generating stem cels, lithium chloride could activate Wnt/β-catenin signal pathway and inhibit GSK-3β, a vital component of degradation compound. This facilitated β-catenin expressing in the cytoplasm to translocate into the nucleus. As a result, the transcription of target gene increased. It is the most appropriate concentration to spur hair-folicle-generating stem cels differentiation when lithium chloride level is > 10 mmol/L, but the proliferation ability declines correspondingly. Keratinocyte growth factor, which can facilitate hair-folicle-generating stem cels differentiated into epidermal cels, is a key factor to accelerate proliferation ability and migration of hair-folicle-generating stem cels, re-epithelialization and healing of wound. The mechanisms of hair-folicle-generating stem cels oriented differentiation induced by lithium chloride and keratinocyte growth factor are activating Wnt/β-catenin signal pathway, inducing change of β-catenin expression, and activating the transcription of target gene related to Wnt/β-catenin signaling pathway .
ABSTRACT
BACKGROUND:Results of recent studies demonstrated the modulation of thymosin β4 on hair cycle and regeneration, but the mechanism of action remains unclear. OBJECTIVE:To investigate the mechanism by which thymosinβ4 increases hair regeneration through Wnt signal pathway. METHODS:After the mouse model of depilation was established using rosin/paraffin mixed agents, the experimental animals were randomly assorted to three different groups, including low-dose, high-dose and control groups, and a dose of 0.3μg/50μL, 3μg/50μL thymosinβ4 and PBS was administered on the depilated backs every 12 hours, respectively. Then photography, hematoxylin-eosin staining, immunohistochemistry and in situ hybridization were applied to observe the growth of hair, and the expressions ofβ-catenin and LEF-1 mRNA in different groups at different time were quantitatively evaluated. RESULTS AND CONCLUSION:The hair growth of the low-dose group was faster than that of the other groups. Hematoxylin-eosin staining demonstrated inflammatory cel s infiltration in the dermis after depilation, and the number of hair fol icles that were in the phase of anagen was much more than the other groups as time went by. Immunohistochemistry ofβ-catenin showed the accumulation of intra-cel ularβ-catenin in the low-dose group at the bulge of fol icles assessed by integrated absorbance analysis (P<0.05), so did the in situ hybridization of LEF-1 mRNA. Low-dose thymosinβ4 accelerates hair growth through Wnt signal pathway by elevating the level ofβ-catenin and LEF-1 mRNA.