Effect study of Sonic hedgehog overexpressed hair follicle stem cells in hair follicle regeneration / 中国修复重建外科杂志

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.

Primary cilia support cartilage regeneration after injury / 国际口腔科学杂志·英文版

In growing children, growth plate cartilage has limited self-repair ability upon fracture injury always leading to limb growth arrest. Interestingly, one type of fracture injuries within the growth plate achieve amazing self-healing, however, the mechanism is unclear. Using this type of fracture mouse model, we discovered the activation of Hedgehog (Hh) signaling in the injured growth plate, which could activate chondrocytes in growth plate and promote cartilage repair. Primary cilia are the central transduction mediator of Hh signaling. Notably, ciliary Hh-Smo-Gli signaling pathways were enriched in the growth plate during development. Moreover, chondrocytes in resting and proliferating zone were dynamically ciliated during growth plate repair. Furthermore, conditional deletion of the ciliary core gene Ift140 in cartilage disrupted cilia-mediated Hh signaling in growth plate. More importantly, activating ciliary Hh signaling by Smoothened agonist (SAG) significantly accelerated growth plate repair after injury. In sum, primary cilia mediate Hh signaling induced the activation of stem/progenitor chondrocytes and growth plate repair after fracture injury.

Mechanism of ursolic acid in regulating colorectal cancer cell HCT116 autophagy through hedgehog signaling pathway / 中国中药杂志

To prove that ursolic acid(UA)could activate the autophagy of colorectal cancer HCT116 cells by inhibiting hedgehog signaling pathway. The effect of UA on the viability of HCT116 cells was determined by MTT assay. The effect of UA on the proliferation and migration of HCT116 cells was detected by crystal violet staining and scratch test. In the study on autophagy, the time points were screened out first: the autophagy fluorescence intensity of UA acting on HCT116 at different time points were detected by Cell Meter~(TM) Autophagy Assay Kit; Western blot was used to detect the expression of autophagy protein P62 at different time points. Then, Cell Meter~(TM) Autophagy Assay Kit was used to detect the effect of UA on autophagy fluorescence intensity of HCT116 cells. The effect of different doses of UA on the expressions of LC3Ⅱ and P62 proteins in HCT116 cells were detected by Western blot. Further, AdPlus-mCherry-GFP-LC3 B adenovirus transfection was used to detect the effects of UA on autophagy flux of HCT116 cells; UA combined with autophagy inhibitor chloroquine(CQ) was used to detect the expression of LC3Ⅱ by Western blot. In terms of mechanism, the effect of UA on hedgehog signaling pathway-related proteins in HCT116 cells was detected by Western blot. The results showed that UA inhibited the activity, proliferation and migration of HCT116 cells. UA enhanced the fluorescence intensity of autophagy in HCT116 cells, while promoting the expression of LC3Ⅱ and inhibiting the expression of P62, in a time and dose dependent manner. UA activated the autophagy in HCT116 cells, which manifested that UA resulted in the accumulation of fluorescence spots and strengthened the fluorescence intensity of autophagosomes; compared with UA alone, UA combined with autophagy inhibitor CQ promoted the expression of LC3Ⅱ. UA reduced the expressions of PTCH1, GLI1, SMO, SHH and c-Myc in hedgehog signaling pathway, while increased the expression of Sufu. In conclusion, our study showed that UA activated autophagy in colorectal cancer HCT116 cells, which was related to the mechanism in inhibiting hedgehog signaling pathway activity.

Effect of dietary nutrition on tail fat deposition and evaluation of tail-related genes in fat-tailed sheep

BACKGROUND: The effects of dietary nutrition on tail fat deposition and the correlation between production performance and the Hh signaling pathway and OXCT1 were investigated in fat-tailed sheep. Tan sheep were fed different nutritional diets and the variances in tail length, width, thickness and tail weight as well as the mRNA expression of fat-related genes (C/EBPα, FAS, LPL, and HSL) were determined in the tail fat of sheep at three different growth stages based on their body weight. Furthermore, the correlations between tail phenotypes and the Hedgehog (Hh) signaling pathway components (IHH, PTCH1, SMO, and GLI1) and OXCT1 were investigated. RESULTS: C/EBPα, FAS, LPL, and HSL were expressed with differences in tail fat of sheep fed different nutritional diets at three different growth stages. The results of the two-way ANOVA showed the significant effect of nutrition, stage, and interaction on gene expression, except the between C/EBPα and growth stage. C/EBPα, FAS, and LPL were considerably correlated with the tail phenotypes. Furthermore, the results of the correlation analysis demonstrated a close relationship between the tail phenotypes and Hh signaling pathway and OXCT1. CONCLUSIONS: The present study demonstrated the gene-level role of dietary nutrition in promoting tail fat deposition and related tail fat-related genes. It provides a molecular basis by which nutritional balance and tail fat formation can be investigated and additional genes can be identified. The findings of the present study may help improve the production efficiency of fat-tailed sheep and identify crucial genes associated with tail fat deposition.

Exploring parent-of-origin effects for non-syndromic cleft lip with or without cleft palate on PTCH1, PTCH2, SHH, SMO genes in Chinese case-parent trios / 北京大学学报(医学版)

OBJECTIVE@#Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a common birth defect, affecting 1.4 per 1 000 live births, and multiple genetic and environmental risk factors influencing its risk. All the known genetic risk factors accounted for a small proportion of the heritability. Several authors have suggested parent-of-origin effects (PoO) may play an important role in the etiology of this complex and heterogeneous malformation. To clarify the genetic association between PTCH1, PTCH2, SHH and SMO in hedgehog (HH) pathway and NSCL/P, as well as testing for potential PoO effects in Chinese case-parent trios.@*METHODS@#We tested for transmission disequilibrium tests (TDT) and PoO effects using 83 common single nucleotide polymorphic (SNP) markers of HH pathway genes from 806 NSCL/P case-parent trios. These trios were drawn from an international consortium established for a genome-wide association studies (GWAS) of non-syndromic oral clefts of multiple ethnicities. DNA samples were collected from each trio. Single marker and haplotype based analysis were performed both in TDT tests and PoO effects. SNPs were excluded if they (ⅰ) had a call rate of < 95%, (ⅱ) had a minor allele frequency (MAF) of < 0.05, (ⅲ) had Mendelian errors over all trios of >5%, (ⅳ) had a genotype distribution in the parents that deviated from the Hardy-Weinberg equilibrium (HWE) (<i>P</i> < 0.000 1). The process was done using Plink (version 1.07, <a href="http://pngu.mgh.harvard.edu/~purcell/plink/data.shtml" target="_blank">http://pngu.mgh.harvard.edu/~purcell/plink/data.shtml</a>). TDT test was performed in Plink v1.07. A log-linear model was used to explore PoO effects using Haplin v6.2.1 as implemented in R package v3.4.2. Significance level was assessed using the Bonferroni correction.@*RESULTS@#A total of 18 SNPs were dropped due to low MAF, thus leaving 65 SNPs available for the analysis. Thus the Bonferroni threshold was 7.7×10-4 (0.05/65). Nominal significant association with NSCL/P was found at a SNP (rs4448343 in PTCH1, P=0.023) and six haplotypes (rs10512249-rs4448343, rs1461208-rs7786445, rs10512249-rs4448343, rs16909865-rs10512249-rs4448343, rs1461208-rs7786445-rs12698335, and rs288756-rs288758-rs1151790, P < 0.05). A total of six haplotypes (rs288765-rs1233563, rs12537550-rs11765352, rs872723-rs288765-rs1233563, rs288765-rs1233563-rs288756, rs6459952-rs12537550-rs11765352, and rs12537550-rs11765352-rs6971211) showed PoO effect (P < 0.05). None of the results remained significant after the Bonferroni correction (P>7.7×10-4).@*CONCLUSION@#Neither significant association between SNPs within HH pathway and the risk of NSCL/P nor PoO effects was seen in this study.

Muscarinic acetylcholine receptor M1 mediates prostate cancer cell migration and invasion through hedgehog signaling / 亚洲男科学杂志(英文版)

The autonomic nervous system contributes to prostate cancer proliferation and metastasis. However, the exact molecular mechanism remains unclear. In this study, muscarinic acetylcholine receptor M1 (CHRM1) expression was measured via immunohistochemical analysis in human prostate cancer tissue array slides. PC-3, LNCaP, and A549 cells were treated with pirenzepine or carbachol, and the cell migration and invasion abilities were evaluated. Western blotting and quantitative real-time PCR were performed to measure GLI family zinc finger 1 (GLI1), patched 1 (PTCH1), and sonic hedgehog (SHH) expression levels. High expression of CHRM1 was found in early-stage human prostate cancer tissues. In addition, the selective CHRM1 antagonist pirenzepine inhibited PC-3, LNCaP, and A549 cell migration and invasion, but the agonist carbachol promoted the migration and invasion of these three cell lines. Muscarinic signaling can be relayed by hedgehog signaling. These data show that CHRM1 is involved in the regulation of prostate cancer migration and invasion through the hedgehog signaling pathway.

Molecular basis of basal cell carcinoma

Abstract: Basal cell carcinoma is the most common cancer, presenting low mortality but high morbidity, and it has as risk factor exposure to sunlight, especially UVB spectrum. The most important constitutional risk factors for basal cell carcinoma development are clear phototypes (I and II, Fitzpatrick classification), family history of basal cell carcinoma (30-60%), freckles in childhood, eyes and light hair. The environmental risk factor better established is exposure to ultraviolet radiation. However, different solar exposure scenarios probably are independent risk factors for certain clinical and histological types, topographies and prognosis of this tumor, and focus of controversy among researchers. Studies confirm that changes in cellular genes Hedgehog signaling pathway are associated with the development of basal cell carcinoma. The cellular Hedgehog signaling pathway is activated in organogenesis, but is altered in various types of tumors.

Identification of p.Glu131Lys Mutation in the IHH Gene in a Korean Patient With Brachydactyly Type A1

No abstract available.

Localization of Bmp-4, Shh and Wnt-5a transcripts during early mice tooth development by in situ hybridization

A comparative nonisotopic in situ hybridization (ISH) analysis was carried out for the detection of Bmp-4, Shh and Wnt-5a transcripts during mice odontogenesis from initiation to cap stage. Bmp-4 was expressed early in the epithelium and then in the underlying mesenchyme. Shh expression was seen in the odontogenic epithelial lining thickening, being stronger in the enamel knot area, during the cap stage. Wnt-5a transcripts were expressed only in the mesenchyme during the initiation, bud and cap stages, with strong expression in the dental mesenchyme during the bud stage. The present results showed that Bmp-4, Shh and Wnt-5a are expressed since the very early stages of tooth development, and they suggest that the Wnt-5a gene is expressed in different cell populations than Bmp-4 and Shh.

No presente trabalho, realizou-se uma análise comparativa não isotópica por hibridização in situ a fim de se detectar a presença de transcritos de Bmp-4, Shh e Wnt-5a durante as fases iniciais da odontogênese em camundongos, desde a iniciação até o estágio de capuz. No estágio de iniciação, observou-se expressão precoce de Bmp-4 no epitélio e no mesênquima subjacente, enquanto que a expressão de Shh ocorreu durante o estágio de capuz, na região de espessamento do revestimento epitelial odontogênico, tornando-se mais intensa na área de nó do esmalte. Os transcritos de Wnt-5a foram expressos somente no mesênquima durante os estágios de iniciação, botão e capuz, com intenso sinal na região no mesênquima na fase de botão. Estes resultados mostraram que Bmp-4, Shh e Wnt-5a são expressos desde os estágios mais precoces do desenvolvimento dentário, sugerindo que o gene Wnt-5a seja expresso em populações celulares distintas daquelas que expressam Bmp-4 e Shh.