The Expression And Biological Significance Of HSPC016 In Human Dermal Papilla Cells

Hair has many useful biological functions, such as to protect the organism from heat loss and afford the underlying epidermis a"first line of defense"from abrasion and penetration of noxious chemical agents, etc. As for humankinds, the most important function of hair is to form varied size of body hairs, such as eyelashes, eyebrows, scalp hairs and so on, and these body hairs act as physical medium of social communication; In fact, scalp, facial, and body hairs are essentially the only body part by which an individual can shape to influence social relationship. Patients with hair loss such as alopecia or excessive hair growth often suffer tremendously. Depression, low self-confidence, and humiliation are observed frequently in the patients of all ages.Hair follicle, the main body of hair, is a regenerative system. Once hair follicle formed in uteri, it starts to cycle continuously over the total lifetime of a mammal including human. Follicle cycling includes anagen, catagen and telogen. If anagen stopped ahead and telogen prolonged abnormally, alopecia will occur. Normal development and cycling of hair follicle depend on the interaction of the follicular epithelium with the adjacent mesenchymal follicle papillae. The dermal papilla cell (DPC) is the unique cell within follicle papillae which induces hair-follicle formation from the overlying epithelium during fetal development, and at the onset of each new follicular cycle in adults DPCs play important roles in the control of follicle cycling through interactions with follicle epithelia. The dermal papilla cells originate from condensed mesenchymal cells that lie beneath the epithelial hair germ cells (placode) in embryonic skin.Dermal papilla cells have now been cultured from a great variety of follicles, including rat whisker, rat pellage, sheep, and human skin. Cultured dermal papilla cells resemble fibroblast in appearance, but generally exhibit an aggregative behavior in culture, forming small clumps of cells overlapping each other with other areas of the dish remaining clear , unlike the parallel array of confluent dermal fibroblast. The character of aggregation would be losing along with the dermal papilla cells'cultivation, those after sixth passage would fail to coagulate.The tendency of aggregation is one of the significant characters of the dermal papilla cells, and the aggregative behavior of the dermal papilla cells is associated with their biologic function and differentiating state. However little is known about the molecular mechanisms of the aggregative behavior of dermal papilla cells, and about the importance of the specific gene(s) expression of the aggregating dermal papilla cells in the folliculomorphogenesis and mature hair follicle cycling.The papilla is an inductive strcture that sends and receives signals to affect the development of hair follicle and hair cycling. Although most of our current knowledge of the substances that modulate hair growth in humans is derived from clinical observations, studies in mice/rat models have identified some of the molecular events associated with hair follicle cycling, such as fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs), sonic hedgehog (Shh), neurotrophins, platelet derived growth factor (PDGF), and so on. Most of these factors are derived from the epithelial cells, and act as a cytokine network controlling follicle developing and hair cycling. At present we couldn't identify any key molecules in this cytokine network. So the specific mechanisms of the follicular development of and the hair cycling couldn't be clearly interpreted from the studies of these factors, and little is known about target genes in the dermal papilla cells that have a direct role in the dermal condensation process and hair follicle cycling. It is said that the biologic significance of the specific gene being expressed in the dermal papilla cells would be an important content in the present hair follicle study.Before this study, the gene of HSPC016 has been firstly identified by a scholar from Fudan University in the hematopoietic stem/progenitor cell (HSPC), and our colleagues Dr. Song ZQ and Prof.HAO F have established a cDNA library of DPCs different expression and discovered HSPC016's up-regulated expression in the status of agglomerating growth for the first time. At the same time, they have demonstrated that the gene of HSPC016 is accurately expressed in the aggregated adult and fetal human dermal papilla cells, but not in the non-aggregated dermal papilla cells and the dermal fibroblast. This result shows that the gene of HSPC016 is specially expressed in the aggregated dermal papilla cells, and the expression of HSPC016 is possibly concerned with their biologic specialities. But the specific function and regulate mechanism of HSPC016 are not sure yet. For these reasons, this study investigated the biological significance of HSPC016 expressed in human dermal papilla cells of different growth status.1. Gene cloning, expression, purification of HSPC016The primer was designed and synthesized to amplify the HSPC016 by PCR . The target gene was cloned into expression vector plasmid through restriction sites after T-A cloning . Then the recombinant prokaryotic expression plasmid pET-22b(+)/HSPC016 was transformed into E.coli BL21(DE3) and the target protein was induced by IPTG. The expressed product with His-tag was purified by Ni-NTA and identified by SDS-PAGE, ELISA and Western blot. The recombinant proteins of HSPC016 was used to immunize rabbit . The HSPC016 gene of dermal papilla cell was successfully expressed in prokaryotic expression system. Purity of HSPC016 was all>95% and a high titer antiserum against HSPC016 has been gotten. The double immunodiffusion titer of the antiserum against HSPC016 was about 1:16.The ELISA titer of the antiserum against HSPC016 was about 1:320 000.Western blot analysis showed that the antiserum could bind to the expressed HSPC016 specifically.2. Preparation and identification biological activity of anti- HSPC016 mcAb BALB/c mice were immunized with purified mitochondrial HSPC016. Spleen cells were fused with SP2/0 cells using polyethylene glycol and hybridoma cells were selected by HAT medium and screened with ELISA.The hybridoma cell lines that can secrete anti-HSPC016 mcAb with high affinity and specificity were established by hybridoma technique. Single cell line which stably produce antibody against HSPC016 was picked out and identified by enzyme linked immunosorbent assay(ELISA). We injected them to abdominal cavity of Balb/c mice for ascites, and we obtained a mount of monoclonal antibodies from ascites. After mcAb was purified, we identified their purity by SDS-PAGE and the titer degree of ascites by ELISA. The specific of mcAbs was identified by Western blotting. The affinity of mcAbs was examined by non-competitive enzyme immuno-assay. We obtained one hybridoma cell line named A7B7D7, which produce mcAbs specificity against HSPC016. It own 103 chromosomes. It could steadly secrete antibodies after the cell line had been continuous cultivated three months and repeatedly been frozen under -196℃in liquid nitrogen. The purities of IgG in ascites was over 78.3%. The isotypes of A7B7D7 monoclonal antibodies was IgG12a and the affinity constant was 4.313×1010 (mol/L)-1, It was evaluated by Western blotting that the antibodies can bind to HSPC016 specifically and was made of only single protein strap.3. Party of bioactivity analysis of HSPC016The HSPC016 was labeled with horseradish peroxidase(HRP)and was applied to detect serum HSPC016 of patients with hair loss and healthy people. Dermal papillae were isolated from scalp tissues by the two-step enzyme method. DPCs were cultured and serially subcultivated to passage 10. To digest the passage 3 and 9 DPCs in log growth phase and to adjust the concentration to 3×105cells/mL and incubate the DPCs to 24 hole cell culture plank. The 0.1mg/mL recombined HSPC016 protein was added to DPCs in log growth phase. The cells were observed by phase-contrast light electron microscopy. One group of cells were measured by flow cytometer, the others were supplemented with 3H-TdR and incubated for 6 hours. The cells were digested and the proliferative capacity was measured. Use the solid phase enzyme immunoassay to test roughly the distributes condition of HSPC016 in serum from healthy people and Patients with hair loss such as alopecia or excessive hair growth. The results show that the HSPC016 in serum of patients with hair loss is remarkably lower than healthy people control(p<0.01). The DPCs treated with recombined HSPC016 protein proliferated actively than control. The results of flow cytometer and 3H-TdR showed that recombined HSPC016 protein promoted the proliferation and DNA synthesis of passage 9 DPCs, however, HSPC016 protein had no effect obviously on passage 3 DPCs.In this study, HSPC016 protein was highly expressed by procaryotic expression vector in E.coli and was purified. HSPC016 monoclonal antibody was prepared by lymphocytic hybridoma technique and could be used for specifically detection of HSPC016 in cells and tissues by Western blotting, et al. The quantitatively determining HSPC016 in human serua of patients and control were tested roughly by SPEIA(solid phase enzyme immunoassay,SPEIA)and the result showed that there was a lot of contrast between healthy people and patients with hair loss such as alopecia or excessive hair growth.Dermal papillae were isolated from scalp tissues by the two-step enzyme method. The results of flow cytometer and 3H-TdR showed that recombined HSPC016 protein had the function of promoting the proliferation of high-passage DPCs.