| 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 DPC 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 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. In order to investigate the function of a new gene, it is necessary to obtain its recombinant protein. Based on the above cognition, in this study, the open reading frame of HSPC016 genewas cloned into Escherichia coli and Pichia pastoris. Then the recombinant protein was inducted to be expressed. This work has made a foundation for the function investigation of the HSPC016 gene. The results of our work revealed that the ORF of HSPC016 gene was successfully amplified by PCR and the squence of 195bp was identical with Genbank (accession No:AY508979). The gene was subcloned into the plasmid pMD-18T and then cut out. After purified, the HSPC016 gene was inserted into the prokaryotic expression vector pET-28a(+)and the prokaryotic expression vector pPIC9K respectively. The pET-28a(+)/HSPC016 was then transformed into E.coli BL21 and recombinant protein was expressed after induced by isopropyl-1-thio-β-D-galactoside. SDS-PAGE analysis showed that the molecular weight of the expressed product was 7.2kD which was identical to value of theory expects, and the expression rate was about 20%. The result of the N-terminal amino acid residual sequencing is identical to that of the molecular design. After primary purification, the target protein of HSPC016 was used to immunize two rabbits, and a high titer antiserum against HSPC016 has been obtained (320,000 EU detected by ELISA). The pPIC9K/HSPC016 was then transformed into P. pastoris GS115 strain after electroporation and recombinant protein was expressed after inducted by methanol, and the expression rate was about 11.7%. The N-terminal amino acid residual sequencing of the protein was made after SDS-PAGE analysis. The results of SDS-PAGE showed that the molecular weight of the expressed product was 7.2kD, and the result of the N-terminal amino acid residual sequencing is identical to that of the molecular design. The recombinant protein has shown a reasonable immuno-reactivity by Western blot assay. In conclusion, the results suggested that HSPC016 gene was successfully constructed and well expressed in eukaryotic and prokaryotic expression system. What I do is a fundamental part of the whole project carried out by our department, and I hope that my work will be significant.
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