Polarization Of Human Hair Follicle-derived Mesenchymal Stem Cells And Its Effects On Proliferation And Apoptosis Of B16 Cells

Background:The human hair follicle is one of appendages of the skin,consisting of at least of four type of stem cells,including: mesenchymal stem cells(h HF-MSCs),melanocytes,neural crest stem cells and keratinocytes.These cells interact synergistically in temporal and spatial manner,maintaining hair follicle self-renewal and hair cycles.Hair follicle developed pathological changes such as loss of hair and depigmentation after insulted by physical,chemical and biological factors in environment.Accumulating research show h HF-MSCs contribute to hair genesis and development as well as maintenance of hair cycles.By use of h HF-MSCs as cell sources,we resurface chronic skin loss,repigment vitiligo,and construct small diameter vascular grafts and engineer release-controlled transgenic stem cells over expressing human insulin.Although stem cell therapy is becoming spotlight in regenerative medicine,the safty,efficacy and ethic concern rais great concern in stem cell-based regenerative medicine,in particular the tumorigenesis of stem cells after implanted.Increasing evidence show MSCs increase or decrease tumor growth though paracrine by secreting immunological or inflammatory factors.However wether h HF-MSCs increase or decrease tumor growth has not been reported.By taking advantages of easily accessible and rich source of autologous stem cells,we set to explore the role of h HF-MSCs in increasing or decreasing tumor growth.Toll like receptors play an important role in the immune system and are involved in the preliminary identification of microbial pathogens and related components of pathogens.There have been many studies on the regulation of TLRs activation on the biological function of MSCs and the expression of TLRs.Toll like receptor(TLRs)mean the type I membrane protein that expressed by immune and non immune cells in the plasma membrane or intracellular(internal corpuscle).Here,immune cells refer to T and B cells,as well as inherent immune cells(include monocytes / macrophages,NK cells,T cells,neutrophils and mast cells).The difference between innate immune cells and adaptive immune cells.Non-immune cells refer to all kinds of epithelium,endothelial cells,MSCs and so on.Although TLRs is widely distributed in the immune system,the level of TLRs expression varies from cell to cell.In the research field of MSCs,the expression and application of TLRs in human BM-MSCs were discovered at first.Studies have shown that Human adipose tissue mesenchymal stem cells(h AT-MSCs)and human bone marrow mesenchymal stem cells(h BM-MSCs)express TLR1,TLR2,TLR3,TLR4,TLR5,TLR6 and TLR9.Although both BM-MSCs and AT-MSCs express TLR 4,cord derived mesenchymal stem cells(Wharton Jelly-derived stem cells)lack TLR 4 expression.It has been reported that the immunological ability of TLR3 can be mediated by activating TLR3 and TLR4 receptors in which ligand poly I:C polysaccharide becomes the specific ligand for inducing polarization.The ligand poly I / C lipopolysaccharide was used as a specific agonist to activate MSCs in a short period of time to make them polarized.By using Poly I:C to stimulate TLR3 receptor,MSCs is polarized to MSC2 phenotypic.The release of anti-inflammatory factors IL4,IL10,TGFβ1,TGFβ2,TGFβ3 had been increa sed compared with control group.It inhibits the release of pro-inflammatory factor,limits its activation to downstream effector cells,and maintains a stable tissue immune environment.In addition,the inducer LPS was used to stimulate the TLR4 receptor which polarized MSCs to the MSC1 phenotype,increased the secretion of IL6,CXCL8,IL1β,TNFα,strengthened the local inflammatory response,killed,cleared the pathogens and foreign bodies in the inflammatory site,and activated tissue repair and regeneration.This makes it possible to indirectly regulate microenvironment by activating TLRs,thus controlling the development of inflammation,promoting damage repair maintaining homeostasis of the internal environment and regulating the proliferation and apoptosis of tumor cells.Since the polarization of MSCs is related to the expression of TLRs,we explored whether the polarized h HF-MSCs could inhibit or promote the proliferation or apoptosis of melanoma cell line B16 by inducing h HF-MSCs to express TLR4 or TLR3 into h HF-MSC1 and h HF-MSC2 in vitro.Objective:To investigate the phenotypic polarization MSC1/MSC2 of hair follicle mesenchymal stem cells and its effects on proliferation and apoptosis of melanoma B16 cells.Experimental methods:1.The hair follicles were obtained by direct extraction from volunteers.h HF-MSCs were isolated by protease digestion,and the expression of specific markers on the surface of barium was detected by flow cytometry(CD44,CD73,CD90,CD90,CD105).Lipid production of h HF-MSCs was detected by oil red O and alizarin red S staining.Osteogenic differentiation ability.2.h HF-MSCs was induced to be polarized into h HF-MSC1 and h HF-MSC2.The expressions of TLR3 and TLR4 on h HF-MSC1 and h HF-MSC2 were detected by cell immunofluorescence staining combined with flow cytometry and Western blot,respectively,and the expression of TLR3 and TLR4 on h HF-MSC1 and h HF-MSC2 were detected by PCR technique.h HF-MSC1 or h HF-MSC2 were detected at m RNA level to promote the expression of inflammatory and anti-inflammatory factors.MTT and flow cytometry were used to detect the increase of h HF-MSC1 and h HF-MSC2 cells.Colonization and cycle changes were compared with h HF-MSCs.3.The supernatants of h HF-MSC1 or h HF-MSC2 were co-cultured with B16 cells.The effects of h HF-MSC1 or h HF-MSC2 supernatants on the proliferation and apoptosis of melanoma B16 were detected by MTT and cell immunofluorescence staining.Experimental results:1.Isolation and identification of h HF-MSCsHair follicles were obtained by direct extraction of hair from volunteers.h HF-MSCs were isolated by protease digestion,and the expression of specific markers(CD44,CD73,CD90,CD105)on the surface of barium was detected by flow cytometry.Lipid production of h HF-MSCs was detected by oil red O and alizarin redS staining.Osteogenic differentiation ability.2.Polarization and Identification of Mesenchymal Stem cellsAfter the study of the induced differentiation conditions,it was determined that0.1 μg / ml LPS and 10 ng / ml poly I:C excited MSCs were induced to polarization into MSC1 / MSC2.The results of flow identification showed that the positive rate of TLR4 expression was up-regulated after induction compared with that without induction.The positive rate of MSC2 expression was up-regulated,and the difference was statistically significant.The results of cellular immunofluorescence were consistent with those of flow cytometry.The positive rate after induction was higher than that of uninduced group.It has statistical significance(P<0.05)After PCR detection,MSC1 detects its expression of anti-inflammatory related factor genes: IL6,CXCL8,IL1 beta,TNF alpha,and MSC2 detects its anti-inflammatory related factor basic factor:IL10,CSF1,TGFβ1,TGFβ2,TGFβ3.3.The polarization of Mesenchymal Stem cells and the indirect effect of B16The effects of MSC1/MSC2/MSCs conditioned medium on B16 cells were studied.The results of MTT showed that the proliferation of B16 cells treated by MSC2-CM and MSC-CM was more effective than that by MSC-CM,and the difference was statistically significant(P<0.05).Conclusions :LPS or Poly I:C can induce h HF-MSCs to polarizing from h HF-MSC to h HF-MSC1 or h HF-MSC2,inhibit the proliferation of melanoma B16 cells through paracrine action,promote apoptosis or promote the proliferation of B16 cells,and inhibit their apoptosis.