Effects Of EGCG On Biology Of Human Hair Follicles And Cultured Dermal Papilla Cells(DPCs) And The Expression Of HIF-1α In Human Hair Follicle And Cultured Dermal Papilla Cells

The hair follicle, a unique characteristic organ of mammals, represents a stem cell-rich, prototypic neuroectodermal-mesodermal interaction system. A unique feature of hair follicles is their cyclical behavior. Starting with anagen, the hair follicle and its shaft pass through catagen, telogen and finally exogen. The dermal papilla is necessary to both induce and maintain the hair follicle. The volume of the dermal papilla, which is determined by the number of matrix cells in the hair bulb, determines the diameter of the induced hair shaft and may also determine the duration of the anagen. On the other hand, the dermal papilla cells also play an important role in determining and maintaining the proliferation and differentiation of the matrix cells in the hair bulb, which is the key point controlling the hair growth. Epithelial-mesenchymal interactions play pivotal roles during the hair cycle.Green tea is a popular beverage around the world, while epigallocatechin-3-gallate (EGCG) is a major constituent of polyphenols from green tea. It was reported that EGCG has potential beneficial effects on cleaning up oxygen free radical, preventing ultraviolet injury, anti-cancer and anti-oxidant. However, no report has been issued to date on the effect of EGCG on human hair growth.Hypoxia inducible factor-1 (HIF-1), a DNA binding protein, was discovered by Semenza from the Hep3B cell line in 1992. It can control the expression of many genes. HIF-1αis the subunit of HIF-1 which can regulate O₂, determinate the activity of HIF-1. It can cohere to target gene and enhance its transcription, a series of hypoxia adjustment reaction is followed. It has been confirmed now that HIF can regulate erythropoiesis, iron metabolism, angiopoiesis and metabolism of glucose and so on. Meanwhile as the progression of the research on HIF-1α, it also has close relation with various kinds of cell growth mechanism.As the critical position of hair follicle growth, the hair bulb is in the deep layer of dermis, local application hardly permeates into there. Fibroblast is close below the epidermis and around the hair follicle. Our research team has successfully transfect the target gene HIF-1αto fibroblast by liposome, fibroblast around the hair follicle then can express and excrete some key factor, such as VEGF, FGF. After transfection, it can promote hair growth and achieve the purpose of healing hair diseases. As part of the subject, this experiment has successfully confirmed the expression of HIF-1αin human hair follicles and cultured dermal papilla cells, and can provide potent evidence on the theory of promotion of HIF-1αon hair growth. The thesis consists of two parts:Part one: Effects of EGCG on the growth of human hair follicles and cell proliferation of human dermal papilla cells(DPCs) in vitroIsolated hair follicles were maintained in 0.5mL Williams E medium with EGCG in ten different concentrations (0-250μg/mL). The growing speed of the hair follicles was observed and recorded on the 10th day. The human DPCs were incubated with EGCG in ten different concentrations (0-500μg/mL) for 48h, then MTT assay was used to detect the proliferating rates of the cultured DPCs. The flow cytometry was also used to analyze the cell cycle. EGCG stimulated the growth of human hair follicles in vitro at the concentrations of 0.1 and 1μg/mL (p<0.05). In the concentration ranges from 15.6 to 62.5μg/mL, EGCG can also enhanced the proliferation of cultured human DPCs (p<0.05). The increase of S%, G2M% and decrease of G1% in the human DPCs incubated in EGCG was observed at concentrations between 7.8 and 62.5μg/mL (p<0.05). We got the conclusion that EGCG can enhance the growth of human hair follicles in vitro and promote the proliferation of cultured human DPCs at certain concentrations.Part two: The expression of HIF-1αin human hair follicle and cultured dermal papilla cellsThe human intact anagen hair follicles were isolated in vitro. RT-PCR was performed to detect HIF-1αmRNA expression in human hair follicles. The cultured 3rd passage DPCs were divided into two groups, separately treated with DMEM and 200μM CoCl₂. Then RT-PCR, western blot and immunofluorescence analysis were applied to investigate the expression of HIF-1αin the cultured human hair DPCs. We found that both the human hair follicle and the cultured dermal papilla cells could express HIF-1αmRNA. HIF-1αprotein could be detected in dermal papilla cells cultured with 200μM CoCl₂. Conclusions: Human hair follicle and cultured dermal papilla cells can express HIF-1α, but HIF-1αprotein degrade immediately after its synthesis in normoxia. CoCl₂ can induce hypoxia environment, so we can detect HIF-1αprotein by western blot and immunofluorescence in DPCs cultured with CoCl₂.