In Vitro Cellular & Developmental Biology. Animal最新文献

Nuclear reprogramming studies are important tools in conserving wild felids, with efficacy depending on efficient G₀/G₁ cell cycle arrest methodologies. This study evaluated different culture conditions at G₀/G₁ arrest and the quality of northern tiger cat fibroblasts. Cells from four animals were assigned to groups: 7.5 and 15 µM roscovitine (RSV) for 24 and 48 h; serum starvation (SS) for 24, 48, 72, and 96 h; and contact inhibition (CI) for 24, 48, and 72 h. Cells with 50-60% confluence were used as control. The cell quality parameters included morphology, and viability and apoptotic levels were assessed through microscopic analysis, while cell cycle phases were evaluated using flow cytometry. RSV affected the cell viable percentage and morphology with the increase of concentration and exposure time. Moreover, RSV did not improve the cells at G₀/G₁. CI did not significantly affect cell quality or increase the proportion of cells in G₀/G₁ phase. Interestingly, SS for 24 h increased the cells at G₀/G₁. However, SS affected the apoptosis levels. The SS for 24 h is the most efficient method of G₀/G₁ arrest for northern tiger cat fibroblasts. However, adjustments are still necessary to optimize cell arrest for northern tiger cat fibroblasts.

TP53TG1 is a long non-coding RNA related to the TP53 gene, which plays an important role in various biological processes such as tumorigenesis, cell cycle regulation, and DNA damage repair. In recent years, researchers have begun to explore the role of TP53TG1 in dental pulp biology, especially its potential impact on pulpitis and other pulp-related diseases. However, the role of TP53TG1 in human dental pulp stem cells (hDPSCs) remains unclear. In this study, we obtained TP53TG1 knockdown dental pulp stem cells by plasmid transfection to determine the biological role of TP53TG1 in DPSCs. We found that the expression of TP53TG1 increased significantly during odontogenic differentiation of DPSCs. SiRNA knockdown of TP53TG1 expression resulted in inhibition of proliferation of hDPSCs. During odontogenic differentiation, downregulation of TP53TG inhibited the expression of multiple differentiation-related indices, and alkaline phosphatase activity and the formation of mineralized nodules were also inhibited. In addition, Western blot found that knockdown of TP53TG1 also weakened SMAD3 and JNK1/2 signaling in DPSCs. In conclusion, our study revealed the differentiation-inducing role of TP53TG1 in DPSCs, which plays an important role in dental pulp repair and regeneration and provides new insights and approaches for the prevention and treatment of dental pulp diseases.

Osteoarthritis (OA) is a common degenerative joint disease, and cartilage dysfunction is the main cause of OA. Long non-coding RNAs (lncRNAs) have been reported to be involved in the development of OA, but the mechanism of action of lncRNA PVT1 (PVT1) in the progression of OA is still poorly understood. The purpose of this study was to explore the effect of lncRNA PVT1 on the progression of OA and the specific molecular mechanism. A rat OA model was constructed by surgery for medial meniscus instability of the right knee joint, and HC-a cells were treated with 10 μg/mL lipopolysaccharide (LPS) for 24 h to establish the OA cell model. The expression of related genes and proteins was detected by RT-qPCR and Western blot, and the damage of HC-a cells and articular cartilage tissue was evaluated by CCK-8, ELISA, flow cytometry, and HE staining. In this study, PVT1 was found to be upregulated in human or rat OA cartilage tissue, as well as in LPS-induced HC-a cells. Knockdown of PVT1 can alleviate the effect of LPS; promote the proliferation of HC-a cells; inhibit glycolysis, apoptosis, and the secretion of inflammatory cytokines TNF-α, IL-1β, and IL-6; alleviate HC-a cell damage; and alleviate the development process of OA in vivo. Mechanistically, PVT1 upregulates the expression of PKM2 by inhibiting the expression of miR-552-3p, thereby promoting the glycolysis process and cell damage, and ultimately accelerating the occurrence and development of OA. Our study suggests that inhibition of PVT1 expression may be a new target for the treatment of OA.

Multiple sclerosis (MS) is a neurodegenerative and autoimmune disease affecting the central nervous system (CNS). Recently, mesenchymal stem cells (MSCs) and their extracellular vesicles (EVs) have been extensively studied as a potential treatment for MS. In this study, we examined the impact of therapy using EVs derived from murine bone marrow MSCs (BMSC-EVs) on the proliferation of splenocytes, frequency of regulatory T cells (Tregs), and cytokine secretion in mice induced with experimental autoimmune encephalomyelitis (EAE), comparing the effects with those of their parent cells. After inducing EAE in 30 mice, the animals were divided into three groups and treated with PBS, BMSCs, or BMSC-EVs. The mice were sacrificed on day 30 post-immunization, and their splenocytes were isolated for further analysis. The proliferation of splenocytes was assessed by measuring the fluorescent intensity of CFSE dye using a FACSCalibur flow cytometer, the frequency of Treg cells was determined by flow cytometry, and cytokine levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, IL-6, IL-10, and transforming growth factor-beta (TGF-β) were measured using enzyme-linked immunosorbent assay (ELISA). The results showed that treatment with BMSC and BMSC-EV both significantly reduced splenocyte proliferation, increased Treg cell frequency, and shifted cytokine profiles toward reduced pro-inflammatory (TNF-α, IL-1β, IL-6) and increased anti-inflammatory (IL-10, TGF-β) cytokines compared to untreated EAE controls, with comparable efficacy between BMSCs and BMSC-EVs. These findings emphasize the capability of BMSC-EVs to serve as a cell-free therapy for immune response modulation in EAE.

Recurrent spontaneous abortion (RSA) represents a substantial challenge in reproductive medicine, attributed to a variety of complex factors, among which aberrations in long non-coding RNAs (lncRNAs) play a crucial role. The present study delves into the functional dynamics of the lncRNA H19 in the context of RSA, particularly focusing on its regulatory interplay with miR-29a-3p and the Suppressor of Cytokine Signaling 3 (SOCS3). A notable downregulation of H19 in villous tissues from RSA patients was observed, highlighting its potential involvement in RSA pathophysiology. Functional assays demonstrated that overexpression of H19 in HTR-8/SVneo cells enhances cellular viability while concurrently attenuating apoptotic processes, thereby indicating a pivotal role of H19 in cellular survival pathways. This study identifies miR-29a-3p as a direct regulatory target of H19, exerting significant influence on cellular viability and apoptosis. The inhibition of miR-29a-3p was observed to mitigate its pro-apoptotic effects, thereby reinforcing its critical regulatory capacity in cellular homeostasis. Moreover, SOCS3 was delineated as a downstream effector of miR-29a-3p, with its expression being inversely modulated by miR-29a-3p. Co-transfection experiments involving H19, miR-29a-3p, and SOCS3 unraveled their intricate regulatory nexus in modulating cellular survival mechanisms. Collectively, these findings elucidate that H19 orchestrates the regulation of cell viability and apoptosis in RSA through the miR-29a-3p/SOCS3 signaling axis, thereby providing valuable insights into the molecular underpinnings of RSA and unveiling novel avenues for therapeutic intervention.

Effective neovascularization is critical for tissue repair and the enhancement of cardiac function following myocardial infarction (MI). However, the hypoxic microenvironment post-MI significantly impedes neovascular formation. Although ATF4 has been implicated in heart failure and myocardial cell regeneration and repair, its role in angiogenesis remains unclear. This study utilized both in vitro and in vivo models to investigate the role of ATF4 in neovascularization after MI. In hypoxia-cultured murine endothelial cells (ECs), hypoxia was observed to inhibit EC proliferation, migration, and tube formation. In contrast, overexpression of ATF4 ameliorated these hypoxia-induced impairments. Conversely, inhibition of ATF4 further exacerbated the reduction in EC proliferation, migration, and tube formation induced by hypoxia. Notably, the beneficial effects of ATF4 were reversed by the PI3K/AKT inhibitor LY294002. Under hypoxic conditions, ATF4 overexpression significantly upregulated phosphorylated (p)-PI3K, p-AKT (T308), and p-AKT (S473) in ECs. LY294002, however, markedly reduced the expression of p-PI3K, p-AKT (T308), and p-AKT (S473) in hypoxic ECs overexpressing ATF4. In a murine MI model, ATF4 overexpression partially mitigated cardiac dysfunction and promoted neovascularization, effects that were significantly attenuated by LY294002. These findings suggest that ATF4 plays a crucial role in endothelial cell-mediated neovascularization under post-MI hypoxia by modulating the PI3K/AKT signaling pathway.