摘要: Objectives Myoblast transfer therapy (MTT) is a technique to replace muscle satellite cells with genetically repaired or healthy myoblasts, to treat muscular dystrophies. However, clinical trials with human myoblasts were ineffective, showing almost no benefit with MTT. One important obstacle is the rapid senescence of human myoblasts. The main purpose of our study was to compare the various methods for scalable generation of proliferative human myoblasts. Methods We compared the immortalization of primary myoblasts with hTERT, cyclin D1 and CDK4(R24C), two chemically defined methods for deriving myoblasts from pluripotent human embryonic stem cells (hESCs), and introduction of viral MyoD into hESC-myoblasts. Results Our results show that, while all the strategies above are suboptimal at generating bona fide human myoblasts that can both proliferate and differentiate robustly, chemically defined hESC-monolayer-myoblasts show the most promise in differentiation potential. Conclusions Further efforts to optimize the chemically defined differentiation of hESC-monolayer-myoblasts would be the most promising strategy for the scalable generation of human myoblasts, for applications in MTT and high-throughput drug screening.

摘要: Objectives Interleukin-34 (IL-34) is associated with hepatitis B virus (HBV) infection and hepatocellular carcinoma (HCC). However, the role and associated mechanisms of IL-34 in HBV-related HCC remain unclear. In this study, the expression, biological function and associated mechanisms of IL-34 in HBV-related HCC cells were investigated. Methods IL-34 expression induced by HBV and HBV X (HBX) gene was measured in hepatoma cells. The role of CCAAT/enhancer-binding protein alpha (CEBP/alpha) in HBX-induced IL-34 expression was examined. The signal pathways involved in the expression of CEBP/alpha and IL-34 induced by HBX were assessed. The role of IL-34 in the proliferation and migration of HCC cells, and related mechanisms were explored. Results Dependent on HBX, HBV increased IL-34 expression in hepatoma cells, and HBX upregulated and interacted with CEBP/alpha to enhance the activity of IL-34 promoters. CEBP/alpha mediated by HBX was associated with the activation of PI3-K and NF-kappa B pathways to promote IL-34 expression. Via CSF1-R and CD138, IL-34 promoted the proliferation and migration of hepatoma cells, and contributed to the activation of ERK and STAT3 pathways and the upregulation of Bcl-xl and c-Myc mediated by HBX. Conclusion We demonstrate that IL-34 contributes to HBX-mediated functional abnormality of HCC cells and provides a novel insight into the molecular mechanism of carcinogenesis mediated by HBX.

摘要: Objectives To investigate the role of hypoxia in vasculogenic mimicry (VM) of salivary adenoid cystic carcinoma (SACC) and the underlying mechanism involved. Materials and methods Firstly, wound healing, transwell invasion, immunofluorescence and tube formation assays were performed to measure the effect of hypoxia on migration, invasion, EMT and VM of SACC cells, respectively. Then, immunofluorescence and RT-PCR were used to detect the effect of hypoxia on VE-cadherin and VEGFA expression. And pro-vasculogenic mimicry effect of VEGFA was investigated by confocal laser scanning microscopy and Western blot. Moreover, the levels of E-cadherin, N-cadherin, Vimentin, CD44 and ALDH1 were determined by Western blot and immunofluorescence in SACC cells treated by exogenous VEGFA or bevacizumab. Finally, CD31/ PAS staining was performed to observe VM and immunohistochemistry was used to determine the levels of VEGFA and HIF-1 alpha in 95 SACC patients. The relationships between VM and clinicopathological variables, VEGFA or HIF-1 alpha level were analysed. Results Hypoxia promoted cell migration, invasion, EMT and VM formation, and enhanced VE-cadherin and VEGFA expression in SACC cells. Further, exogenous VEGFA markedly increased the levels of N-cadherin, Vimentin, CD44 and ALDH1, and inhibited the expression of E-cadherin, while the VEGFA inhibitor reversed these changes. In addition, VM channels existed in 25 of 95 SACC samples, and there was a strong positive correlation between VM and clinic stage, distant metastases, VEGFA and HIF-1 alpha expression. Conclusions VEGFA played an important role in hypoxia-induced VM through regulating EMT and stemness, which may eventually fuel the migration and invasion of SACC.

摘要: Objectives Chondrocyte proliferation and differentiation are crucial for endochondral ossification, but their regulatory mechanism remains unclear. The present study aimed to determine the physiological function of TGF beta 1 signalling in the proliferation and differentiation of antler chondrocytes and explore its relationship with Notch, Shh signalling and Foxa. Materials and methods Immunofluorescence, Western blot, MTS assay, flow cytometry, RNA interference and real-time PCR were used to analyse the function and regulatory mechanisms of TGF beta 1 signalling in antler chondrocyte proliferation and differentiation. Results TGF beta 1, TGFBR1 and TGFBR2 were highly expressed in antler cartilage. TGF beta 1 promoted chondrocyte proliferation, increased the proportion of S-phase cells and induced the expression of hypertrophic chondrocyte markers Col X, Runx2 and Alpl. However, this induction was weakened by TGF beta receptor inhibitor SB431542 and Smad3 inhibitor SIS3. Simultaneously, TGF beta 1 activated Notch and Shh signalling whose blockage attenuated the above effects of rTGF beta 1, whereas addition of rShh rescued the defects in chondrocyte proliferation and differentiation elicited by SB431542 and SIS3. Further analysis revealed that inhibition of Notch signalling impeded TGF beta 1 activation of the Shh pathway. Knockdown of Foxa1, Foxa2 and Foxa3 abrogated the effects of TGF beta 1 on chondrocyte differentiation. Notch and Shh signalling mediated the regulation of Foxa transcription factors by TGF beta 1. Conclusions TGF beta 1 signalling could induce the proliferation and differentiation of antler chondrocytes through Notch-Shh-Foxa pathway.

摘要: Objectives Glioblastoma is the most common malignant glioma of all brain tumours. It is difficult to treat because of its poor response to chemotherapy and radiotherapy and high recurrence rate after treatment. The aetiology of glioblastoma is a result of disorders of multiple factors. Depending on cell signal transduction, these glioblastoma-associated factors lead to cell proliferation, differentiation and apoptosis. Therefore, investigation of the potential factors which involved in the development of glioblastoma could provide a new target for the treatment of glioblastoma. Materials and methods We analysed the transcript expression of CLEC5A in glioblastoma by accessing The Cancer Genome Atlas (TCGA). qRT-PCR was performed to detect the RNA expression of genes in cells and tissues, and Western blot was used to measure the protein levels (Cyclin D1, Bcl-2, BAX, PCNA, MMP2, MMP9, Akt and Akt phosphorylation) in tissues and cells. Cell proliferation, migration, invasion, cycle and apoptosis were measured by CCK-8, transwell and flow cytometry assays, respectively. Ki67 level and lung metastasis were determined by immunochemistry and H&E staining. Results In this study, we found that CLEC5A was highly upregulated in glioblastoma compared to normal brain tissues, which had an opposite relation with the overall patient survival. Downregulation of CLEC5A could inhibit cell proliferation, migration and invasion via promoting apoptosis and G1 arrest. In contrast, overexpression of CLEC5A stimulated cell proliferation, migration and invasion. In addition, we found that CLEC5A level was positively correlated with Akt phosphorylation level. Akt inhibitor or agonist could reverse the modulation effects of CLEC5A in glioblastoma. Moreover, In vivo results suggested that inhibition of CLEC5A significantly reduced tumour size, weight, cell proliferation ability and lung metastasis via inhibition of phosphorylation Akt. Conclusion Both in vitro and in vivo evidences supported that CLEC5A was involved in glioblastoma pathogenesis via regulation of PI3K/Akt pathway. Thus, CLEC5A might serve as a potential therapeutic target in the treatment of glioblastoma in the future.