In Vitro Cellular & Developmental Biology. Animal最新文献

Many strains of wild-type laboratory mice have been developed for studies in the life sciences, including skeletal muscle cell biology. Muscle regeneration capacity differs among wild-type mouse strains. However, few studies have focused on whether myogenic stem cells (satellite cells) are directly related to mouse strain-dependent myoregeneration gaps using in vitro culture models. In this study, we selected three major wild-type mouse strains, CD1 (outbred; Jcl:ICR [ICR]), C57BL/6NJcl (inbred; B6), and BALB/cAJcl (inbred; C), which are widely used in laboratory experiments. Initially, we compared myotube fusion capabilities using satellite cell-derived myoblasts. The results showed that cell cultures isolated from male ICR mice could not efficiently form myotubes owing to low expression levels of myogenic regulatory factors (e.g., MyoD, myogenin, myocyte enhancer factor [MEF] 2A, and MEF2C) compared with B6 and C mouse strains. Next, we compared the myofiber-type compositions of muscle tissues and cultured myotubes among male mice from each of the three strains. Although each muscle tissue used for satellite cell isolation similarly expressed fast-twitch myofiber markers in all mouse strains, male ICR-derived myoblasts formed abundant amounts of slow-type myotubes. By contrast, myotubes from male B6 and C mice expressed substantial levels of fast-twitch myofiber markers. We also performed a comparative experiment in female ICR, B6, and C mouse strains, similar to the male mouse experiments. The myogenic differentiation potencies of myoblasts and myofiber-type compositions of myotubes in female mouse strains were similar. Thus, male ICR-derived satellite cells (myoblasts) had low myogenic differentiation potential, which may be associated with the tendency slow-twitch myotube formation.

Skeletal muscle regeneration depends on satellite cells that maintain tissue homeostasis through self-renewal and the production of myoblasts that differentiate into mature myofibers. Dysregulation of these processes can lead to muscle degeneration, highlighting the need to elucidate their molecular mechanisms. In this study, we investigated the role of the Grb2/Sos1 signaling pathway in regulating satellite cell self-renewal and differentiation using C2C12 cells. Knockdown of either Grb2 or Sos1 significantly reduced the formation of Bcl-2-positive reserve cells and increased the proportion of differentiated myotubes. Conversely, forced expression of Grb2 increased the number of reserve cells, whereas the Grb2 P49L mutant, which disrupts its interaction with Sos1, decreased reserve cell formation and resulted in thinner myotubes. Although forced expression of Sos1 alone did not significantly increase reserve cell numbers, the chimeric protein cSos-SH2, which combines elements of Grb2 and Sos1, produced a pronounced increase of reserve cells. These results demonstrate that a precise balance between Grb2 and Sos1, along with their coordinated subcellular localization, is critical for controlling reserve cell populations. Activated by growth factor receptor tyrosine kinases and extracellular matrix/integrin interactions, the Grb2/Sos1 signaling pathway is critical for maintaining the muscle satellite cell pool, thereby playing an essential role in muscle regeneration.

Mosquito-borne diseases represent a growing global health crisis, exacerbated by climate change and insecticide resistance. RNA interference (RNAi), a natural mechanism of gene silencing, offers a promising, target-specific alternative for mosquito control. This review explores the potential of RNAi to disrupt critical physiological processes, such as reproduction and disease transmission, thereby reducing vector populations and competence. We examine the mechanisms of RNAi, its application in combatting insecticide resistance, and recent advancements in delivery systems, including nanobody- and chitosan-based nanoparticles, which enhance the stability and uptake of double-stranded RNA (dsRNA) molecules. However, significant challenges remain, such as optimizing field-effective delivery methods and assessing potential off-target effects on non-target organisms. Continued innovation in RNAi technology is pivotal for developing sustainable and environmentally sound vector control strategies. This review synthesizes current research, highlighting the molecular insights, practical applications, and future directions for integrating RNAi into modern public health initiatives.

Fish cell lines are indispensable tools for virology, biotechnology, and toxicology research. This study established a new marine fish cell line, designated EfE, from the eye tissue of the brown-marbled grouper (Epinephelus fuscoguttatus). The EfE cell line has been stably subcultured for over 70 passages in vitro for more than 300 days. It proliferated optimally in Leibovitz's L-15 medium supplemented with 15% fetal bovine serum at 28°C. Species origin was confirmed by molecular analysis of the mitochondrial CO1 gene. Chromosome analysis revealed a diploid count of 48, which is consistent with the karyotype of E. fuscoguttatus. The cell line demonstrated high transfection efficiency (25.6%) with a pEGFP-N1 plasmid, indicating its potential for genetic manipulation. In virus susceptibility tests, EfE cells were highly permissive to red-spotted grouper nervous necrosis virus (RGNNV), developing severe cytopathic effects (CPE), including extensive vacuolation, cell rounding, and detachment. Viral replication was further confirmed by semi-quantitative RT-PCR and the observation of virus particles via transmission electron microscopy (TEM). In conclusion, the novel EfE cell line provides a valuable in vitro model for virus isolation, propagation, investigation of pathogenic mechanisms, and genetic studies.

Diabetic nephropathy (DN) is a critical complication of diabetes mellitus. Icariside II, a bioactive compound from epimedium, is known for its anti-hyperglycemic properties, but its mechanism in DN remains unclear. Our study aimed to explore Icariside II's protective effects against high-glucose (HG) induced podocytes injury using an in vitro model. We assessed cell viability and proliferation using the CCK8 assay after treating cells with Icariside II. qPCR and Western blot analysis were used to measure the mRNA and protein expressions of DNMT1, α-SMA, fibronectin and collagen IV. Molecular docking studies were performed using DNMT1's 3D structure from the Protein Data Bank. DNMT1 overexpression levels were quantified via qRT-PCR and western blot. Immunofluorescence staining and ELISA assays evaluated TGF-β1, inflammatory cytokines, respectively. GSH, MDA, and intracellular Fe²⁺ were measured using biochemical assay kits and FerroOrange probes, respectively. Western blot analysis was used to measure the protein expressions of GPX4, SLC7A11, ACSL4 and TFR1. Results showed Icariside II inhibits HG induced proliferation, inflammation and extracellular matrix (ECM) accumulation in MPC-5 cells. Besides, Icariside II also reduced inflammation, ECM accumulation and ferroptosis by downregulating DNMT1. However, the intervention treatment with Ferrostatin-1 could effectively counteract this effect. Icariside II mitigated HG-induced inflammation and ECM accumulation by down-regulating DNMT1 and ferroptosis.

Improving the diagnosis and treatment of gastric cancer is a significant challenge worldwide. Circular RNAs (circRNAs), a recently identified class of endogenous non-coding RNAs with covalently closed-loop structures, have emerged as key regulators in tumorigenesis. CircFoxo3 has been studied in various cancer types, while its functional role in GC remains poorly understood. In this study, we found that circFoxo3 is significantly upregulated in GC tissues and cell lines compared to paired normal controls. Functional analyses demonstrated that knockdown of circFoxo3 markedly inhibited GC cell proliferation and migration, whereas overexpression of circFoxo3 produced the opposite effects. Mechanistically, circFoxo3 knockdown reduced forkhead box (Fox) transcription factors FOXO3 mRNA and protein levels. FOXO3a is involved in regulating cancer cell proliferation. Bioinformatic analysis revealed high expression of FOXO3 in GC tumor samples, a finding confirmed in both GC tissues and cell lines. A tumor xenograft model was used to examine the effect of circFoxo3 on tumor growth in vivo. The low circFoxo3 expression reduced the volume of the tumor and decreased its proliferation. Collectively, our findings identify circFoxo3 as an oncogenic factor in GC progression.

Non-alcoholic steatohepatitis (NASH) is a progressive form of non-alcoholic fatty liver disease (NAFLD), characterized by hepatic steatosis, inflammation, and fibrosis, which may advance to cirrhosis and hepatocellular carcinoma. Treatment options remain limited, underscoring the need to elucidate its mechanisms and develop effective therapeutics. Salidroside (Sal), a primary active compound of Rhodiola rosea, has shown potential in alleviating NASH, yet its underlying mechanisms are not fully understood. This study investigates whether Sal mitigates palmitic acid (PA)-induced hepatocyte injury by regulating the leukocyte immunoglobulin-like receptor B2 (LILRB2)-mediated autophagy pathway. In vitro NASH model were established by inducing AML-12 cells with PA. Cells were divided into control, PA, and PA + Sal groups. To validate the role of LILRB2, an LILRB2 overexpression group was included. Cell proliferation, apoptosis, inflammatory factors (TNF-α, IL-1β, IL-6), and autophagy-related proteins were detected. Autophagic flux was evaluated using mCherry-GFP-LC3B transfection. PA treatment significantly suppressed proliferation, promoted apoptosis and inflammation, and inhibited autophagy, indicated by decreased LC3B-II/Beclin-1 and accumulated p62. Sal reversed these effects. Mechanistically, Sal downregulated LILRB2 expression, which was upregulated by PA. Overexpressing LILRB2 counteracted Sal's beneficial effects. These findings reveal that Sal attenuates PA-induced injury by inhibiting LILRB2, enhancing autophagy, and reducing apoptosis and inflammation, suggesting LILRB2 as a potential therapeutic target for NASH.

Pterygium syndrome is a common eye disease that often leads to vision loss and even blindness. There is increasing evidence that miRNAs play a key role in the progression of pterygium, but the function of miR-381-3p in pterygium has not been studied. Therefore, this study aimed to investigate the effect of miR-381-3p on the progression of pterygium and to elucidate its potential molecular mechanisms. Human pterygium fibroblasts (HPFs) were isolated from clinical pterygium tissues. The expression of key genes and proteins was detected via RT-qPCR and western blotting. Cell proliferation was detected by CCK-8 and scratch assay, while cell invasion was examined by Transwell assay. Protein interactions were investigated by coimmunoprecipitation. First, we found that the expression level of miR-381-3p was significantly reduced in pterygium tissues. Second, we found that the overexpression of miR-381-3p in HPFs inhibited the proliferation, migration, and invasion abilities of HPFs while inducing cell apoptosis. In addition, in pterygium tissue, the expression of MCPIP1 was downregulated, and the expression of HACE1 and TRIP12 was upregulated. Importantly, MCPIP1 interference partially attenuated the positive effects of miR-381-3p overexpression described above, and miR-381-3p could target HACE1, while HACE1 could bind to TRIP12. Mechanistic studies revealed that miR-381-3p inhibited the binding of HACE1 to TRIP12 through the inhibition of HACE1 expression, thereby inhibiting the ubiquitination and degradation of MCPIP1 and improving the progression of pterygium. Our study highlights the powerful potential of miR-381-3p in improving the progression of pterygium, laying the foundation for the development of new intervention targets for related diseases.

Canine distemper virus (CDV), a highly contagious member of the Morbillivirus genus in the Paramyxoviridae family, continues to threaten the health of domestic and wild carnivores worldwide. However, isolation of field strains is often limited by the low susceptibility of conventional cell lines lacking essential viral entry receptors. In this study, a recombinant Vero cell line stably expressing the canine signaling lymphocyte activation molecule (SLAM/CD150) receptor was established to improve CDV isolation efficiency. The SLAM gene was amplified from canine peripheral blood mononuclear cells (PBMCs), cloned into the pTargeT mammalian expression vector, and transfected into Vero cells. Functional expression of SLAM was validated by inoculation with a field isolate (CDV34388), which induced pronounced cytopathic effects and extensive syncytium formation in Vero-dogSLAM cells, whereas no syncytia were observed in non-transfected controls. Quantitative analyses revealed a mean 2.8-fold increase in minimum effective dilution (MED) values and an average area-under-the-curve (ΔAUC) of + 98 units in SLAM-expressing cells compared with normal Vero cells (p < 0.001), indicating markedly enhanced viral replication and cytopathic activity. These findings demonstrate that canine SLAM expression significantly improves CDV detection and isolation from field samples, establishing a robust in vitro model for future diagnostic and vaccine development studies.