The Expression Of Eukaryotic VEGF-expressing Vector In HaCaT Cells And Its Biological Effects On Cultured Pig Hair Follicles In Vitro

BackgroundHair follicle (HF) is a highly sensitive mini-organ whose cyclic transformations from rapid growth (anagen), via regression (catagen) to relative quiescence (telogen) are profoundly influenced by numerous growth factors, cytokines, hormones, corresponding receptors and so on. Some common hair diseases such as androgenetic alopecia and alopecia areata not only cause cosmetic problems, but also impose much stress and pressure on patients themselves. Therefore, further investigation of detailed pathogenesis and effective therapies is one of the fundamental and imminent problems in hair research.Vascular endothelial growth factor (VEGF), also named as vascular permeability factor (VPF) or vasculotropin, is recently identified to be an important mediator of hair growth and cycling. VEGF occurs in at least five molecular forms that contain 121(VEGF₁₂₁), 145(VEGF₁₄₅), 165 (VEGF₁₆₅), 189 (VEGF₁₈₉) or 206(VEGF₂₀₆) amino acids. Among which, only VEGF₁₂₁ and VEGF₁₆₅ owing to lacking exon 6 are secreted.Substantial data have documented that VEGF was expressed in human keratinocytes, dermal papilla cells, dermal sheath cells and outer root sheath cells in anagen hair follicles. By contrast, there is only little or no VEGF expression in catagen and telogen hair follicles. Furthermore, hair follicles from androgenetic alopecia and alopecia areata often display extraordinarily lower level and even no VEGF expression, which is particularly evident in the developing lesions of alopecia.Up to now, minoxidil is the only topical drug granted by American Food and Drug Administration to cure androgenetic alopecia. Its obvious biological effects involve enhanced hair follicle size and subsequently longer and thicker hair. Minoxidil could stimulate VEGF production in cultured human hair dermal papilla cells in a dose-depedent manner. Our previous study also shows that VEGF can significantly promote the hair growth of C₅₇BL/6 mouse vibrissa follicles in vitro and its biological effects are concentration-dependent.However, VEGF is a kind of polypeptide glycoprotein, which hardly permeates into hair follicles when applied topically. Furthermore, VEGF, as a growth factor, is extremely expensive. Therefore, it is important to utilize VEGF comprehensively and effectively in clinic. ObjectivesThis study was designed to investigate the feasibility of transiently transfecting human VEGF₁₆₅ gene into HaCaT cells and to determine thebiological effects of the expressed VEGF on cultured pig hair follicles invitro.MethodsExperiment one: The amplification and identification of the eukaryotic VEGF-expressing vector pIRES2-EGFP-VEGF₁₆₅.pIRES2-EGFP-VEGF₁₆₅ was transformed into E.coli DH_5α by means of CaCl₂. After 12h culture a single clony was picked and inoculated with LB medium containing selective 30μg/mL kanamycin and then incubated overninght at 37℃. If the above medium became clouding, remove a 1mL sample to detect the sequence of target DNA. At the same time, plasmids were extracted by SDS alkaline lysis and digested with restriction endonuclease Bgl II and EcoR I. The digested products were analyzed on a 1% agarose gel to identify the palsmids. Endofree Plasmid maxi Kit was used to extract and purify plasmids on large scale. Then the concentration and purity of the plasmids were measured. The final concentration was diluted to 1μg/μL.Experiment two: The expression of the eukaryotic VEGF-expressing vector pIRES2-EGFP-VEGF₁₆₅ in HaCaT cells.pIRES2-EGFP-VEGF₁₆₅ was transiently trasfected into HaCaT cells by Lipofectamine^? 2000. HaCaT cells were transfected respectively by pIRES2-EGFP-VEGF¹⁶⁵, pIRES2-EGFP and the medium containing neither pIRES2-EGFP-VEGF¹⁶⁵ nor pIRES2-EGFP. After 36h culture, theexpression of EGFP within HaCaT cells was observed under Laser Confocal Microscopy. Then ELISA was utilized to measure the level of VEGF in the cellular supernatant.Experiment three: The biological effects of the expressed VEGF on cultured pig hair follicles in vitroPig hair follicles were cult...