Intrauterine adhesion (IUA) causing infertility and recurrent miscarriage of reproductive female mammals usually results from endometrium injury. Nevertheless, there is no efficient therapeutic method to avoid IUA. Bone marrow derived mesenchymal stem cells (BMSCs) are an important cell source for tissue regeneration. This study designs and explores the ability of BMSC‐loaded elastic poly(glycerol sebacate) (PGS) scaffold to prevent IUA and compares the effect of PGS with poly(lactic‐co‐glycolic acid) (PLGA) and collagen scaffolds in resumption of damaged rat uteruses. The 3D architecture provided by PGS scaffolds favors the attachment and growth of rat BMSCs. In vivo bioluminescence imaging shows that compared with direct BMSC intrauterine injection, PLGA, and collagen scaffolds, the PGS scaffold significantly prolongs the retention time of BMSCs in a wounded rat uterus model. More importantly, BMSCs can directly differentiate into endometrial stromal cells after transplantation of PGS/BMSCs constructs, but not PLGA/BMSCs and collagen/BMSCs. It is found that the level of transforming growth factor β1 (TGF‐β1), basic fibroblast growth factor (bFGF), vascular endothelial growth factor, and insulin‐like growth factors in the injured endometrium adjacent to PGS/BMSCs constructs is higher than those of rats receiving PLGA/BMSCs, collagen/BMSCs, or BMSCs intrauterine transplantation. Besides, transplantation of PGS/BMSCs leads to better morphology recovery of the damaged uterus than PLGA/BMSCs and collagen/BMSCs. The receptive fertility of PGS/BMSCs is 72.2 ± 6.4%, similar to the one of collagen/BMSCs, but significantly higher than 42.3 ± 3.9% in PLGA/BMSCs. Taken together, PGS/BMSCs may be a promising candidate for preventing IUA.
Long noncoding RNA (lncRNA) has been suggested to play an important role in a variety of diseases over the past decade. In a previous study, we identified a novel lncRNA, termed HOXA11‐AS, which was significantly up‐regulated in calcium oxalate (CaOx) nephrolithiasis. However, the biological function of HOXA11‐AS in CaOx nephrolithiasis remains poorly defined. Here, we demonstrated that HOXA11‐AS was significantly up‐regulated in CaOx nephrolithiasis both in vivo and in vitro. Gain‐/loss‐of‐function studies revealed that HOXA11‐AS inhibited proliferation, promoted apoptosis and aggravated cellular damage in HK‐2 cells exposed to calcium oxalate monohydrate (COM). Further investigations showed that HOXA11‐AS regulated monocyte chemotactic protein 1 (MCP‐1) expression in HK‐2 cell model of CaOx nephrolithiasis. In addition, online bioinformatics analysis and dual‐luciferase reporter assay results showed that miR‐124‐3p directly bound to HOXA11‐AS and the 3'UTR of MCP‐1. Furthermore, rescue experiment results revealed that HOXA11‐AS functioned as a competing endogenous RNA to regulate MCP‐1 expression through sponging miR‐124‐3p and that overexpression of miR‐124‐3p restored the inhibitory effect of proliferation, promotion effects of apoptosis and cell damage induced by HOXA11‐AS overexpression. Taken together, HOXA11‐AS mediated CaOx crystal–induced renal inflammation via the miR‐124‐3p/MCP‐1 axis, and this outcome may provide a good potential therapeutic target for nephrolithiasis.
Active DNA demethylation occurs after a sperm enters an egg. However, the mechanisms for the active DNA demethylation remain poorly understood. Ten-eleven translocation enzymes were recently shown to catalyze the conversion of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). Thus, we decided to investigate the role of 5hmC in active demethylation. We analyzed the methylation and hydroxymethylation status in metaphase II oocytes as well as 1-cell stage and cleavage stage embryos. In zygotes, 5hmC was mainly detected in the paternal pronucleus and it increased from the pronuclear-2 (PN2) to PN5 stages, an indication that 5hmC was involved in paternal genomic DNA demethylation. Bisulfite-sequencing PCR and qGluMS-PCR (DNA glucosylation and digestion before quantitative PCR) results showed that a large reduction of methylcytosine and hydroxymethylcytosine in LINE1 (long interspersed nuclear element 1) occurred between the 4- and 8-cell stages, which indicates that demethylation potentially occurred after the 4-cell stage. We then microinjected mouse zygote with plasmids that were methylated in vitro by SssI methylase and analyzed for the hydroxymethylation status of the plasmids promoter region. We found that the rapid onset of expression of the unmethylated plasmids in mouse embryos happened in <12 h, but the expression of methylated plasmids was delayed until 50 h when most embryos were at the 8-cell stage. Quantitative GluMS-PCR results suggested that 5hmC was present in the plasmid's promoter region at the MspI site where the active demethylation occurred. Our results demonstrate that 5hmC is involved in active demethylation in mice.
MSCs (mesenchymal stem cells) are a stem cell source that can be easily obtained from bone marrow. Despite the increasing importance of the pig as a large animal model, little is known about foetal pMSCs (porcine MSCs). In this study, we observed the gene expression of pluripotent markers in foetal pMSCs and the capacity of pMSCs to differentiate into adipocytes, osteocytes and neural-like cells using quantitative RT–PCR (reverse transcription–PCR), normal histological staining and immunohistochemistry. Foetal pMSCs have either a spindle or a flattened shape, and flow cytometry revealed the expression of the MSC-related proteins CD44 and CD105 (endoglin) but not CD34 and CD45. pMSCs express pluripotent markers such as Oct4 (octamer-binding transcription factor 4) and Nanog at the protein and mRNA levels. qRT-PCR (quantitative real-time PCR) analyses revealed that pMSCs expressed nestin [for NSCs (neural stem cells)]. Immunocytochemical and RT–PCR data showed that 29% and 23% of pMSCs expressed MAP2 (microtubule-associated protein 2) for neurons and β-tubulin III (Tuj1) for immature neurons, respectively, after induction of neural differentiation. These findings demonstrate the plasticity of pMSCs and their potential for use in cellular replacement therapy for neural diseases.
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