The Expression And Promoter Activity Analysis Of CCDC72 In Human Dermal Papilla Cells

Hair follicle,the main body of hair,is a typical regenerative system.Once hair follicle formed in uteri,it starts to cycle continuously over the total lifetime of a mammal including human.Hair has many useful biologic functions and is psychosocially important in our society,patients with hair loss such as alopecia or excessive hair growtn often suffer tremendously.Depression,low self-confidence,and humiliation are oberved frequently in the patients of all ages.Not surprisingly,understanding the morphogenetic mechanism,growth cycle of hair,changes in gene expression within anagen follicle,and developing new drugs to treat hair disease is the key subject in the hair biological research area.The papilla is an inductive structure that sends and receives signals to affect the development of hair follicle and hair cycling. The dermal papilla cells(DPCs) 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 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 growth associated gene of the aggregating dermal papilla cells in the folliculomorphogenesis and mature hair follicle cycling.DPCs in the state of aggregative growth pattern in vitro still sustained the potency to induce hair follicles formation from afollicular skin.In order to know which gene expressed in DPCs at the state of aggregative growth pattern,Song et al set up the human DPCs subtractive library and screened out some up-regulated genes and down-regulated genes successfully. CCDC72 gene is one of the differential expression gene in DPCs. It has been firstly identified in the CD34+ hematopoietic stem/progenitor cells,but its function is not clear. Dr.Wang JW et al made CCDC72 gene expression decreased or negative in DPCs by RNA interfering technology.The DPCs koncked down with CCDC72 grew much slower than normal DPCs.They lost aggregative properties and the shape of the cell also changed significantly. Dr.Xia RS et al discovered that recombined CCDC72 protein promoted the proliferation and DNA synthesis of high-passage DPCs.These results indicated that CCDC72 gene played a critical role in the control of DPCs proliferation and differentiation.For these reasons,this study invesgated the gene expression, protein distribution and promoter activity of CCDC72 gene in dermal papilla cells.1. Two step enzyme method is simple, quick and economic method to isolate and culture dermal papilla cells. The human adult dermal papilla cells were firstly isolated and cultured from scalp hair follicle, and these cultured dermal papilla cells exhibited an aggregative behavior. The aggregative ability would be losing along with the dermal papilla cells'cultivation, and the aggregative characteristics of dermal papilla cells were correlated with their biologic functions.2. Immunofluorescence,mitochondrion-selective stain and laser scanning confocal microscopy were used to find that the CCDC72 gene-encoded protein located in the cytoplasm,probably in the mitochondria of dermal papilla cells. We found the expression of CCDC72 gene at the level of mRNA was decreasing along with the dermal papilla cells'cultivation through fluorescence semiquantitative real-time PCR assay.The expression levels of CCDC72 gene in passage 1～3 DPCs were much higher than in passage 4～8 DPCs, compared with the 8 passage, the higher expression in 4-6 passage was also significant.3. Sequence analyzing by Cister online indicated that there are many cis-acting elements in the regions of -200～-1bp and -1000～-600bp from up steam start codon ATG.To evaluate promoter activity of the CCDC72 gene,the 1924bp of genomic DNA immediately 5'to the start codon ATG(-1920～+4) were obstained and four luciferase report gene vectors with promoter segments with different lengths were constructed successfully. The four different reconstruct luciferase plasmids were transfected into passage 3 DPCs and passage 8 DPCs,and the report activities were measured by using dual luciferase reporter assay. The results showed incresed luciferase expression of the four different reconstruct plasmids in passage 3 DPCs,the -600～+4 bp fragment was significant greater than those of the other three fragments.The luciferase activitiy decreased significantly in passage 8 DPCs,the -200～+4 bp fragment induced a large increase in basal luciferase activity,which may contain the activity of minimal core promoter,was essential for basal transcription. The -600～+4 bp fragment in passage 3 DPCs was the highest activity of the four luciferase,but it had decreased obviously in passage 8 DPCs.This implied there are cis-acting elements in the regions of -600～-200bp which regulated CCDC72 gene transcription in the passage 8 DPCs.