Experiment Of Human Gingival Fibroblasts Induction Into Nerve-Like Cells

Objective: Mainly consisting of nerve tissue,nerve system plays a key role in regulating the physiological activity in human body.Nerve system is mainly composed of nerve cell and neuroglia cells.It is the nerve cell that constitutes the structure and functional unit of the nerve system.Not only there are numbers nerve cells,but also they possess the capacity to receive stimulation,impulse conduction and integrate information.Moreover,nerve cells also have endocrine function.Nevertheless,the nerve cells are so difficult to regenerate after the trauma of nervous system.The treatment of the nervous system disease has become a difficult point in clinical treatment.As one of the periodontal support tissues,gingiva is shallow in position and easy to obtain.Some researches prove that human gingival fibroblasts have the potential to differentiate into other different kinds of tissues cells,such as epithelial-like cell and muscle-like cells.Furthermore,it has an excellent ability of self-repairing.In this study,human gingival fibroblasts were cultured and induced into nerve-like cells in order to investigate the possibility of human gingival fibroblasts induction into nerve-like cells and provide a new and reliable source of seed cells for nerve tissue engineering.Methods:1.The isolation and culture of the human gingival fibroblasts(HGFs)The normal human gingiva was collected from a patient with an extraction of impacted tooth in Department of oral Maxillofacial Surgery,Hospital of Stomatology,Hebei Medical University.The primary culture of human gingival fibroblasts were with the method of tissue piece method in low-glucose DMEM containing 15% fetal bovine serum.The cells were digested and passaged by 2.5% tripsin.2.The identification of the human gingival fibroblastsThe fourth generation gingival fibroblasts were identified by immunocytochemical stains with mouse-anti-human vimentin,GFAP and MAP-2 respectively.3.The induction of the human gingival fibroblastsThe fourth generation gingival fibroblasts were induced with the cell inducing medium,which concludes 1mmol/L ?-ME,20μmol/L RA,2%DMSO and induced for 12 days.4.The identification of the nerve-like cellsThe induced nerve-like cells were examined by immunocytochemical stains with three antibodies against human vimentin,GFAP and MAP-2respectively.Furthermore,the nerve-like cells were also identified with immunoflurescent stains by GFAP and MAP-2.5.Enzyme-linked immunosorbent assay(ELISA)of neurotransmitterThe medium of the HGFs and nerve-like cells induced for 12 days were collected respectively.The former was recorded as control group,and the latter was recorded as experimental group.ELISA was used to test the neurotransmitter secreted by the nerve-like cells.Meanwhile,the experimental data of the two groups were collected and analyzed by t-test,besides,the bar chart were made,and the P was calculated.Results1.The cell culture of the human gingival fibroblastsAfter cultured for 7-9days,spindle cells were observed by the inverted microscope around the gingival tissue.The elongation cells with round or ellipse nucleus and 2-3 nucleolus grows vortex in shape.2.The identification of the human gingival fibroblastsImmunocytochemical stains showed that vimentin was positive strongly in the gingival fibroblasts,and GFAP and MAP2 were negative in the gingival fibroblasts.3.The induction of the human gingival fibroblastsThe fourth generation gingival fibroblasts in logarithmic phase were induced in low-glucose DMEM containing 15%FBS,1mmol/L ?-ME,20μmol/L RA,2% DMSO and induced for 12 days.Most of the fibroblasts were changed into triangular or polygonal in shape with single or several protrusions which were long and thin,separated or connected into a network observed by inverted microscope.4.The identification of the nerve-like cellsImmunocytochemical stains indicated that the nerve-like cells showed positive for human vimentin,GFAP and MAP-2,so did the results of the immunoflurescent stains with MAP-2 and GFAP.5.Enzyme-linked immunosorbent assay(ELISA)of neurotransmitterDopamine and Acetylcholine could be detected by the enzyme-linked immunosorbent assay kit.Furthermore,the concentration of both two kinds of neurotransmitter between control group and experimental group had statistical differences(P < 0.05).At the same time,the amount of the Dopamine was approximately 56.25 pg /ml,and the acetylcholine was about 75 pg /ml.Conclussion1.Human gingival fibroblasts have the capability of being induced into nerve-like cells.2.The nerve-like cells can produce the neurotransmitter dopamine and acetylcholine.