Expression Of Hypoxia Inducible Factor -1α In Cells And Its Biological Effects On Hair Follicle And Cells

BackgroundHair follicle (HF) is a highly sensitive mini-organ whose cyclictransformations from rapid growth (anagen), via regression (catagen) torelative quiescence (telogen) are profoundly influenced by numerousgrowth factors, cytokines, hormones, corresponding receptors and so on.Some common hair diseases such as androgenetic alopecia and alopeciaareata not only cause cosmetic problems, but also impose much stress andpressure on patients themselves. Therefore, further investigation ofdetailed pathogenesis and effective therapies is one of the fundamentaland imminent problems in hair research.Hypoxia inducible factor-1(HIF-1), a DNA binding protein, isdiscovered by Semenza in 1992, can control the expression of manygenes. HIF-1αis the subunit of HIF-1 which can regulate O₂, determinatethe activity of HIF-1. It can cohere to target gene and enhance itstranscription, a series of hypoxia adjustment reaction is followed. It isconfirmed now HIF can regulate erythropoiesis, iron metabolism,angiopoiesis and metabolism of glucose and so on. Meanwhile as theprogression of the research on HIF-1α, it also has close relation with various kinds of mechanisms of cell growth.However, as the critical position of hair follicle growth, the hair bulbis in the deep layer of dermis, local application hardly permeates intothere. Fibroblast is close below the epidermis and around the hair follicle.It is fairly feasible to transfect the target gene to fibroblast by liposome,fibroblast around the hair follicle can express and excrete some key factorafter transfection, thus it can promote hair growth, achieve the purpose ofhealing hair diseases and supply a new treatment for hair diseases.ObjectivesThis study was designed to investigate the feasibility of transfectinghuman HIF-1 a gene into fibroblast and to determine the biological effectsof the expressed HIF-1 on cultured human hair follicles and cells in vitro,and detect the expression of downstream cytokine.Methods1. The amplification and identification of the eukaryoticpcDNA3.0-HIF-1α.2. pcDNA3.0-HIF-1αwas transiently trasfected into fibroblast byLipofectamine^TM 2000.3. Expression of HIF-1αwithin fibroblast was observed byimmunohistochemistry.4. Then ELISA was utilized to measure the level of VEGF in the cellularsupematant. MTT was utilized to detect the activity of cells cultured with the cellular supernatant.5. The biological effects of the expressed VEGF on cultured human hairfollicles in vitro.Human hair follicles were cultured with Williams E medium plus thesupernatant collected from the cell culture transfected respectively bypcDNA3.0-HIF-1α(group A), pcDNA3.0 (group B) and only transfectedmedium (group C). Hair growth and morphological changes wereobserved under microscope.6. pcDNA3.0-HIF-1αwas stably trasfected into fibroblast byLipofectamine^TM 2000.7. Expression of HIF-1αwithin fibroblast was observed by Westernblotting.8. Then ELISA was utilized to measure the level of VEGF in the cellularsupernatant. RT-PCR was utilized to measure the level of bFGF in thecells.9. MTT was utilized to detect the activity of cells (fibroblast and dermalsheath cells) cultured with the cellular supernatant.Results1. DNA sequence confirmed that the target DNA was pcDNA3.0-HIF-1α.2. 36h after transfection HIF-1αwas found in fibroblast transfected bypcDNA3.0-HIF-1α. 3. ELISA tests showed that VEGF level was significantly higher insupernatant transfected by pcDNA3.0-HIF-1αthan that transfected bypcDNA3.0 or only transfected medium (P＜0.01).4. MTT showed the activity of fibroblast was significantly higher insupernatant transfected by pcDNA3.0-HIF-1αthan that transfected bypcDNA3.0 or only transfected medium (P＜0.01).5. On day 8 of culture, hair growth was significantly longer in the groupA than that in the group B and the group C (P＜0.01). However, nosignificant difference was detected between the group B and the group C(P＞0.05). The morphological study showed that hair follicles in thegroup A still maintained in anagen, in contrast, which representedcatagen/telogen stages in both of the group B and the gruoup C.6. When stable transfection is established, HIF-1αwas found in fibroblasttransfected by pcDNA3.0-HIF-1α.7. ELISA tests showed that VEGF level was significantly higher insupernatant transfected by pcDNA3.0-HIF-1αthan the control group(P＜0.01).8. RT-PCR showed that bFGF level was significantly higher in fibroblasttransfected by pcDNA3.0-HIF-1αthan the control group (P＜0.01).9. MTT showed the activity of fibroblast and dermal sheath cells wassignificantly higher in supematant transfected by pcDNA3.0-HIF-1αthanthe control group (P＜0.05). ConclusionsHuman HIF-1αgene could be successfully transfected into fibroblastby Lipofectamine ^TM2000 with the high level of HIF expression. Theexpressed HIF significantly stimulate hair growth of cultured human hairfollicles and enhance the activity of fibroblast and dermal sheath cells byimproving the expression of VEGF and bFGF.