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OBJECTIVE@#To determine the expression level of Sonic hedgehog (Shh) in the passage of hair follicle stem cells (HFSCs), analyze the effect of Shh overexpression on the proliferation activity of HFSCs, and explore the survival of HFSCs after Shh overexpression and its effect on hair follicle regeneration.@*METHODS@#Hair follicles from the normal area (H1 group) and alopecia area (H2 group) of the scalp donated by 20 female alopecia patients aged 40-50 years old were taken, and the middle part of the hair follicle was cut under the microscope to culture, and the primary HFSCs were obtained and passaged; the positive markers (CD29, CD71) and negative marker (CD34) on the surface of the fourth generation HFSCs were identified by flow cytometry. The two groups of HFSCs were transfected with Shh-overexpressed lentivirus. Flow cytometry and cell counting kit 8 assay were used to detect the cell cycle changes and cell proliferation of HFSCs before and after transfection, respectively. Then the HFSCs transfected with Shh lentivirus were transplanted subcutaneously into the back of nude mice as the experimental group, and the same amount of saline was injected as the control group. At 5 weeks after cell transplantation, the expression of Shh protein in the back skin tissue of nude mice was detected by Western blot. HE staining and immunofluorescence staining were used to compare the number of hair follicles and the survival of HFSCs between groups.@*RESULTS@#The isolated and cultured cells were fusiform and firmly attached to the wall; flow cytometry showed that CD29 and CD71 were highly expressed on the surface of the cells, while CD34 was lowly expressed, suggesting that the cultured cells were HFSCs. The results of real-time fluorescence quantitative PCR and Western blot showed that the expression levels of Shh protein and gene in the 4th, 7th, and 10th passages of cells in H1 and H2 groups decreased gradually with the prolongation of culture time in vitro. After overexpression of Shh, the proliferation activity of HFSCs in the two groups was significantly higher than that in the blank group (not transfected with lentivirus) and the negative control group (transfected with negative control lentivirus), and the proliferation activity of HFSCs in H1 group was significantly higher than that in H2 group before and after transfection, showing significant differences ( P<0.05). At 5 weeks after cell transplantation, Shh protein was stably expressed in the dorsal skin of each experimental group; the number of hair follicles and the expression levels of HFSCs markers (CD71, cytokeratin 15) in each experimental group were significantly higher than those in the control group, and the number of hair follicles and the expression levels of HFSCs markers in H1 group were significantly higher than those in H2 group, and the differences were significant ( P<0.05).@*CONCLUSION@#Lentivirus-mediated Shh can be successfully transfected into HFSCs, the proliferation activity of HFSCs significantly increase after overexpression of Shh, which can secrete and express Shh continuously and stably, and promote hair follicle regeneration by combining the advantages of stem cells and Shh.
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Animais , Feminino , Camundongos , Alopecia/cirurgia , Folículo Piloso , Proteínas Hedgehog/genética , Camundongos Nus , Regeneração , Células-TroncoRESUMO
Objective:To investigate the effect of the transcriptional coactivator Mediator 1 (Med1) on mouse hair regeneration, and to explore potential mechanisms.Methods:Med1 flox/flox C57BL/6J mice were mated with K14-Cre mice, and the mice with epidermis-specific knockout of Med1 gene, namely K14-Cre-expressing Med1 flox/flox mice (knockout group) , were obtained by using the Cre-Loxp system, while Med1 flox/flox mice without K14-Cre expression served as control group. Mice in the two groups (3 mice in each group) were raised together for 8 weeks followed by dorsal hair removal. Hair regeneration was observed for 12 consecutive days after hair removal. After 12 days, all mice in the two groups were sacrificed, their depilated and non-depilated dorsal skin tissues were resected, and total RNA was extracted from the tissues. Real-time quantitative PCR was performed to determine the mRNA expression of hair keratin genes, vitamin D receptor/β-catenin pathway-related genes, and genes associated with maintenance of hair follicle stem cell proliferation and quiescence. Paraffin-embedded sections of depilated and non-depilated mouse skin tissues were prepared, and immunofluorescence staining was conducted to determine the number of stem cells in the hair follicle bulge. Two-independent-sample t test was used for comparisons between two groups. Results:From days 0 to 12 after depilation, hair regeneration was delayed in the depilated skin area in the knockout group compared with the control group. Real-time quantitative PCR showed significantly decreased mRNA relative expression levels of hair keratin genes Ha1 and Krt2-16, vitamin D receptor/β-catenin pathway-related genes S100a3, Dlx3 and Tubb3, and genes associated with maintenance of hair follicle stem cell proliferation and quiescence including Lhx2, Sox9 and Nfatc1 in the depilated skin tissues in the knockout group (22.09 ± 12.32, 2.07 ± 0.20, 0.02 ± 0.01, 12.36 ± 2.12, 1.75 ± 0.46, 0.39 ± 0.02, 4.42 ± 0.76, 0.44 ± 0.07, respectively) compared with the control group (70.53 ± 9.46, 7.76 ± 0.49, 0.05 ± 0.01, 26.16 ± 2.96, 2.60 ± 0.14, 0.71 ± 0.09, 11.93 ± 0.42, 0.75 ± 0.04, respectively; t = 5.40, 18.64, 3.89, 6.57, 3.04, 6.10, 15.03, 6.18, respectively, all P < 0.05) . Immunofluorescence staining showed that the number of CD34 +K15 + hair follicle stem cells in the hair follicle bulge in both depilated and non-depilated skin tissues was significantly lower in the knockout group than in the control group. Conclusion:Med1 gene knockout may down-regulate the expression of downstream genes of the vitamin D receptor/β-catenin pathway and genes associated with maintenance of hair follicle stem cell proliferation and quiescence (Sox9, Nfatc1 and Lhx2) , and reduce the number of hair follicle stem cells, leading to hair follicle differentiation disorder and hair regeneration delay.
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Adult body harbors powerful reservoir of stem cells that maintains homeostasis by tissue regeneration and in response to disease and injury. Hair follicle is a dynamic mini organ supporting important biological functions of the body in maintaining homeostasis and skin tissue self-renewal. This study was carried out with the objective of finding the adult stem cells in canine hair follicular tissue. To conduct this study, adult canine skin samples (n=12) irrespective of breed and sex were collected. To characterize the hair follicle stem cells, paraffin sections of canine hair follicles were immunostained with positive hair follicle stem cell markers like Anti- cytokeratin 15 (CK15) and Anti-cytokeratin 19 (CK19) and FITC conjugated and HRP conjugated secondary antibodies were used. Immunoreactivities for CK15 and CK19 were observed in the bulge/isthmus region of hair follicles in between the infundibulum and suprabulbar regions and occupied most part of the peripheral layer of outer root sheath cell. Immunophenotyping of canine Hair Follicle Stem Cells (cHFSCs) in the bulge region of hair follicle helps in confirmation of in vitro culture of cHFSCs from the bulge region which will be further used for translational research
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Background & objectives: Skin is an established tissue source for cell based therapy. The hair follicle has been introduced later as a tissue source for cell based therapy. The ease of tissue harvest and multipotent nature of the resident stem cells in skin and hair follicle has promoted basic and clinical research in this area. This study was conducted to evaluate skin stem cells (SSCs) and hair follicle stem cells (HFSCs) as candidate cells appropriate for neuronal and melanocyte lineage differentiation. Methods: In this study, SSCs and hair follicle stem cells (HFSCs) were expanded in vitro by explant culture method and were compared in terms of proliferative potential and stemness; differentiation potential into melanocytes and neuronal lineage. Results: SSCs were found to be more proliferative in comparison to HFSCs, however, telomerase activity was more in HFSCs in comparison to SSCs. Capacity to differentiate into two lineages of ectoderm origin (neuronal and melanocyte) was found to be different. HFSCs cells showed more propensities towards melanocyte lineage, whereas SSCs were more inclined towards neuronal lineage. Interpretation & conclusions: The study showed that SSCs had differential advantage over the HFSCs for neuronal cell differentiation, whereas, the HFSCs were better source for melanocytic differentiation.
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Objective Hair follicle stem cells (HFSCs) can be cultured in vti ro to differentiate into chondrocytes, osteoblasts and adipocytes .The aim of the article was to investigate the culture method of HFSCs and their adipogenic and osteogenic differentiation potentials. Methods Microdissection and two-step enzyme digestion were applied to obtain HFSCs , and HFSCs are purified by differ-ential adhesion method.The cells were cultured in keratinocyte serum-free medium obtaining 10% fetal bovine serum.Flow cytometry was used to detect CD34、β1-integrin and CK15.The third-generation HFSCs were cultured by osteogenic inductor and adipogenic induc-tor respectively . Results Cultured HFSCs were homogeneous in shape , bearing the property of great refraction and typical cobble-stone-morph.By flow cytometry, the expression rates of CD34,β1-integrin and CK15 were 59.6%, 97.0%and 61.1%.Stem cells were in good growing condition from the third-generation cell growth curve.21 days after being induced by osteogenic inductor, calcified nodules appeared after alizarin red staining, while 14 days after being induced by adipogenic inductor, intracellular lipid droplets could be observed after oil red O staining. Conclusion Cultured HFSCs are chaterized by strong growth capacity and multiple differentiation potential .
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Objective To establish a simple , practical , highly-effective and stable method for the isolation and cultivation of rat hair follicle cells. Methods Under sterile condition, single hair follicle was taken out after the skin around the barbel of SD neonatal rats was sheared off. And then the hair follicles were digested with two-step method with Type Ⅰcollagenase and trypsin. The obtained cell suspension was planted into the culture plate which was covered with extracellular matrix, and then was cultivated with K-SFM culture medium containing fetal bovine serum with volume fraction of 1%. On the next day, K-SFM culture medium was replaced with serum-free culture medium. The remaining tissues were cut into pieces and spread out in the culture flask, and then were cultivated with HG-DMEM culture medium containing serum. Two kinds of cells were harvested and then were identified by immunofluorescence. The hair follicle epithelial cells were tested by flow cytometer. Results The hair follicle epithelial cells obtained through the above methods showed rapid adherence, and were round or polygon-like , with typical cobblestone-like morphology. The long spindle-shaped cells were seen around the tissues cultivated, having many protrusions on the surface of the cells, and they were interconnected into reticular structure. The expression of cytokeratin 15, cytokeratin 19 and β1 integrin in epithelial cells were positive. Most of the epithelial cells were in the G1 phase, accounting for 75.6%. The expression of laminin ( LN), fibronectin ( FN) and vimentin in the connective tissue sheath cells were also positive. Conclusion The cells harvested by modified two-step enzyme digestion method have confirmed as hair follicle cells and fibroblasts, and the obtained cells are of rapid adherence, good homogeneity, and active proliferation.
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Objective To compare the phenotypes and biological characteristics between the rabbit hair follicle stem cells and urethral mucosa stem cells, and to explore the feasibility of using hair follicle stem cells as seed cells for tissue engineering urethra. Methods Rabbit hair follicle stem cells and urethral mucosa stem cells were isolated and cultured in vitro, and the cells with diameter <10 μm and integrin-α6+ /CD71- were sorted using flow cytometry. The maximum amplification factor and clone forming ability of the two kinds of cells were calculated. The positive rates of K19, p63 and β1-integrin in the two kinds of cells were detected by flow cytometry. The enriched stem cells were implanted in the urethral stent to construct tissue engineering urethra. Histology and fluorescent staining were used to observe the tissue-engineered urethra. Results The maximum amplification multiples of rabbit hair follicle stem cells and urethral mucosa stem cells were (6. 1±0. 8)×104 and (7. 1±1. 1)×103, respectively; and the clone formation rates were (10. 1±1. 3)% and (4. 3±1. 1)%, respectively. The positive rates of K19, p63, and β1-integrin in hair follicle stem cells were (90. 53±6. 77) %, (93.31±5.57)%, and (91. 17±6.98)%, respectively; and those in urethral mucosa stem cells were (88. 50±4. 95)%, (91. 63±5. 86)%, and (93. 35±6. 28) %, respectively. Both hair follicle stem cells and urethral mucosa stem cells formed complex layer epithelioid structure on the stents, with positive staining for AE1/AE3. Conclusion Rabbit hair follicle stem cells and urethral mucosa stem cells can both serve as seed cells for constructing tissue engineering urethra. As for the availability of seed cells, hair follicle stem cells are more superior to the urethral mucosa stem cells.
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Objective To construct the lentivirus carrying human ?-catenin-EGFP(enhanced green fluorescent protein)and observe its expression in human follicle stem cells.Methods The ?-catenin gene sequence was amplified by RT-PCR from extraction of total RNA of human vascular endothelial cells.TA cloning technique was utilized to acquire gene subcloned pUCm-T-?-catenin.After transformation reaction,candidate clone was further analyzed by PCR and gene sequencing.Then the plasmid was transfected into FT293 cells.After identification by Western blotting,the plasmid was transfected into FT293 cells again for packaging.Infection titer was monitored by green EGFP expression.The expression of ?-catenin-lentivirus in human follicle stem cells were observed under inverted fluorescence microscope.Results The ?-catenin gene was cloned into the lentivirus successfully.The high expression of green fluorescence protein in FT293 cell line was found under fluorescent microscope.Viral titer checked by real-time PCR was about 2.0?108 TU/mL.When the multiplicity of infection(MOI)was 10,the infection efficiency of ?-catenin-lentivirus in human follicle stem cells was nearly 80% after infection 48 h around.After 3 weeks of continuous observation,we found the infection efficiency still keeping in the range of 80%-90%.Conclusion The lentivirus expression vector for ?-catenin was successfully constructed.It can steadily infect human follicle stem cells and the infection efficiency is considerable high.
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Objective To investigate the expression and significance of androgen receptor(AR) of rat vibrissa follicle stem cells in culture and vibrissa follicle stem cells in vivo at three different phases of a hair follicle cycle,including anagen,catagen and telogen.Methods AR was detected using immunohistochemistry.The expression of AR mRNA was examined by total RNA extraction and RT-PCR.Results In vivo the vibrissa follicle stem cells expressed AR in the whole hair follicle cycle.AR mRNA was strongly expressed in anagen,weakly in catagen and telogen.AR expressed in the cultured vibrissa follicle stem cells.Conclusion The expression of AR mRNA was altered regularly in rat vibrissa follicle stem cells in vivo throughout the hair follicle cycle,which suggested that the interaction between androgen and AR may facilitate the differentiation of vibrissa follicle stem cells.