A Primary Study On The Characterization Of Bulge-originated Cells From Hair Follicles And The Role Of β-Catenin In Hair Follicle Growth And Differentiation

Hair follicle is an important and complex epidermal appendages of skin, composed of hair shaft, inner root sheath and outer root sheath. The bulge is a part of the outer root sheath, which was contiguous with the epidermis. It is located below the opening of the sebaceous duct and serves as the attachment site of arrector pili muscle. Previous investigators using [~3H] thymidine to evaluate label retention in murine haired epidermis discovered that the majority of label-retaining cells (LRCs) in the skin were restricted to the bulge region of the hair follicles. As the bulge represent a slow-cycling population and may regenerated complete hair follicle after X-irradiation or after removal of the lower parts of the hair follicles, it has been proposed that these cells represent multipotent stem cells in the skin, termed as hair follicle stem cell (HFSC ) . A great deal of investigation revealed that the stem cells resided in the upper follicle of the bulge area had a proven role in hair follicle cycling and organ formation. Moreover, the bulge, with pluripotent differentiation potential, is able to not only migrate downwards to become the matrix keratinocytes of bulb, but also migrate upwards to maintain the epidermis and involve in the formation of hair follicles and sebaceous gland. At present, people universally accepted the location of HFSC, and believed that the HFSC population with greatest proliferative potential occur in the bulge region. But up to now, the exact characteristic of cells from bulge region has not been examined in detail, which became an obstacle to understand the role of HFSC in homeostasis, wound repair and tumor initiation. In this report, we described a modified method to harvest intact hair follicles. The bulge, microdissected on the basis of morphologic feature, were cultured and passaged successfully. The biologic character, proliferating ability, specific markers expression and differentiation potential of bulge-originated cells in culture was discussed. Furthermore, the expression of markers in situ confirmed that bulge region was a putative reservoir of hair follicle stem cell.β -catenin is a cytosolic protein with dual functions, belonging to the armadillo family proteins. One is as a structural component of adherens junction, which forms the cadherin-catenin complex to mediate intercellular adhesion. The other is as a downstreameffector of wnt signaling pathway, which controls essential steps in many aspects of embryogenesis and tumorigenesis. Binding of wnt proteins to their receptors will activates the signaling pathway and causes stabilization of cytoplamic P -catenin. Accumulated P -catenin may translocate into the nucleus, where it serves as a transcriptional factor to activate various target genes. In almost all normal tissues, P -catenin has only been detected at the cell-cell junction. So far P -catenin has been detected in some malignant tumors, such as colon, ovarian, liver and oesophagus. Recently, investigator found that P -catenin was required for the initiation of hair follicle development. When P -catenin is mutated during embryogenesis, formation of the hair follicles is blocked. And P -catenin is also essential for fate decisions of skin stem cells. In order to make it clear about the relationship between P -catenin and hair follicles, this research was designed to study from four aspects. IHC and RT-PCR were used to detect the P -catenin expression in follicle cycle, hair development, wound repair and differentiation. All the data suggested that P -catenin might be an essential regulative factor in follicle formation and cycle maintenance. The main results and conclusions are as follows:1. The characterization of bulge-originated cells from hair folliclesThe bulge-originated cells from hair follicles were successfully cultured in vitro. It was the basic step for the further research. On the condition of DMEM/F12 supplemented medium, newly cells could be observed in the second day after being inoculated. Until now cells could be passaged six times and had strong ability of proliferation. The number of these cells reached the peak at the 5-6 day and maintained several days. Cultured cells had homogeneous slabstone-shape, the mitotic figure appeared and the cells had a large nuclear.cytoplasmic ratio. These characteristics suggested a primitive morphologic feature. The distribution of cell cycle showed bulge cells quickly initiated proliferation. When the intact hair follicle was cultured in vitro, a large majority of cells were emigrated from bulge region. Thus the cells with greatest proliferative potential and longest life spans in intro were occurred in the bulge region. Immunohistological staining data showed that P 1-integrin and kl9 are strongly expressed in bulge area in vivo, while klO is negative. And the cells in primary culture of bulge also indicated strong expression of P 1 and kl9, which suggested the bulge-originated cell contains early hair follicle stem cells resided in the skin.2. The expression and significance of P -catenin in hair follicleAfter the hair follicle cycles of wistar rats were definited, the P -catenin expression in different phages of hair follicle cycles were detected. The hair follicle cycle of wistar ratswere last 28d .composed of anagen (1-18)> catagen (19-22) and telogen (23-28). P -catenin was strongly expressed in anagen, while weakly in catagen and telogen. In studying the expression and significance of P -catenin in the development of hair follicle, the result display an great correlation between expression of P -catenin and the different phages. And the strong expression was concentrated in the inside of outer root sheath of adult tissues. So P -catenin might play a possible role in the formation and development of hair follicles. On the basis of a rat wound model, IHC and RT-PCR were used to detect the expression of P -catenin in wound hair follicles. It showed that the expression of P -catenin was stronger in wound hair follicle than in normal hair follicle tissue, reached a peak level after 24h and then decreased slowly, recovering normal level after 72h damage. All the data suggested that P -catenin was involved in follicle development, cycle maintenance and wound repair, which might be one of essentially regulative factor in physiological function of hair follicle. 3. The role of P -catenin in bulge cell growth and differentiation Primary cultured bulge cells were successfully induced to differentiate into sebaceous gland, which confirmed that cultured cells contained hair follicle stem cells with pluripotent differentiation potential. The expression of P -catenin and cox-2 in bulge cells was detected by ICC. Experimental data showed that P -catenin and cox-2 were correlated with bulge cells proliferation, and cox-2 might be the target genes of P -catenin signaling pathway in nucleus. On the mechanism of the effection of P -catenin, this research presumed that excessive P -catenin transflocated into the nucleus and activate cox-2 gene to promote cell proliferation. In addition, the available experimental evidence pointed toward a role of P -catenin involved in the differentiation of hair follicle stem cells. P -catenin was strongly expressed in the cell culture, but became weak after being induced.In this experimental study, the culture method of bulge cell was first established. According to their morphology, proliferating ability, specific markers expression and differentiation potential, the bulge-originated cells has been confirmed to possess hair follicle stem cells. Meanwhile, P -catenin was an essential regulative factor in cell growth, follicle development and cycle maintenance. This study has laid a solid ground and may have crucial implications for tissue engineering and cell therapy when abnormal differentiation occurred in the skin.