In Vitro Cellular & Developmental Biology. Animal最新文献

Atherosclerosis (AS) is a primary contributor to cardiovascular disease (CVD), resulting in high mortality. Ferroptosis, triggered by lipid peroxidation, contribute to AS development. This study aimed to explore the regulatory relationships of Trim28, ALDH4A1, P53, and ferroptosis in the pathogenesis of AS. The AS cell model was constructed by treating HUVECs with oxidized low-density lipoprotein (ox-LDL). The roles of Trim28 overexpression in regulating AS development, P53 ubiquitination, and ferroptosis of vascular endothelial cells were investigated. Moreover, the interaction between Trim28 and ALDH4A1 was explored, followed by analyzing the effect of ALDH4A1 knockdown on P53 ubiquitination. Additionally, the impact of ALDH4A1 knockdown and P53 overexpression on AS development and ferroptosis of vascular endothelial cells was explored. Reduced Trim28 expression and increased ALDH4A1 and P53 expression were observed in HUVECs after treatment with ox-LDL. Overexpression of Trim28 mitigated AS development, promoted P53 ubiquitination, and suppressed ferroptosis of vascular endothelial cells. Additionally, ALDH4A1 could interact with Trim28, and ALDH4A1 knockdown enhanced P53 ubiquitination. Moreover, P53 overexpression reversed the inhibitory effects of ALDH4A1 knockdown on AS development and ferroptosis of vascular endothelial cells. Our findings indicate that Trim28, ALDH4A1, and P53 may be key regulators in AS development. Silencing of ALDH4A1 may alleviate AS development through regulating Trim28-mediated P53 ubiquitination to inhibit ferroptosis of vascular endothelial cells. These molecules may by promising therapeutic targets for AS and related CVD.

Anoikis resistance, the evasion of programmed cell death when cells detach from the extracellular matrix (ECM), is a critical feature of glioblastoma (GBM) malignancy, contributing to tumor survival, spread, and resistance to therapy. We focused on the role of growth factor receptors, particularly platelet-derived growth factor receptor-α (PDGFRα), and integrin expression patterns in mediating this resistance. We first cultured cells under non-adherent conditions using polyHEMA-treated plates to induce anoikis resistance. We performed assays like cell survival, migration, and sphere formation. To delineate the role of PDGFRα signalling in anoikis resistance, we further employed pharmacological inhibitors of key signalling molecules such as AG1295 (PDGFRα blocker), HS173 (PI3K inhibitor), U0126 (ERK inhibitor), and AG490 (JAK-STAT inhibitor) which led to a decrease in cell survival, proliferation, and migration. These findings highlight the critical role of PDGFRα and associated signalling pathways in mediating anoikis resistance in GBM, offering potential therapeutic targets for intervention.

Differentiation of the human monocytic leukemia cell line THP-1 is widely used to analyze the function of monocyte/macrophage-like cells in vitro. Although chemotaxis, a critical function of monocytes/macrophages enabling tissue accumulation, has been extensively studied, methods to evaluate sustained, long-distance chemotaxis remain underexplored. Therefore, we aimed to evaluate macrophage-like cells in vitro by differentiating THP-1 cells into monocyte/macrophage-like cells exhibiting sustained, strong chemotaxis over long distances (up to 260 μm). Using various reagents, we identified the combination of vitamin D, panobinostat, and granulocyte-macrophage-colony-stimulating factor as optimal for achieving high directionality and velocity in cell migration, as analyzed using the TAXIScan cell dynamics assay device. The differentiated cells matured into M1 macrophage-like cells and displayed reduced migratory capacity post-maturation, along with enhanced phagocytosis and reactive oxygen species production. Collectively, our differentiation and analysis methods provide a reliable platform for basic research into cellular maturation processes and drug development targeting the regulation of monocyte/macrophage dynamics.

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.