Biotechnic & Histochemistry最新文献

Endothelial progenitor cells (EPCs) play a crucial role in neovascularization and tissue repair, with significant therapeutic potential in ischemic diseases, tumor therapy, and as gene carriers. However, the current methods for isolating and culturing EPCs are not standardized, leading to inconsistencies in cell numbers and functionality. This study aimed to optimize the in vitro culture conditions for EPCs using an orthogonal design, focusing on four main factors: cell density, culture medium, fetal bovine serum (FBS) concentration, and vascular endothelial growth factor (VEGF) concentration. The most effective conditions among those tested were a cell density of 1 × 10⁶/cm², EGM-2 medium, 10% FBS, and 20 ng/mL VEGF. Under these conditions, EPCs exhibited significantly enhanced proliferation, migration, and pro-angiogenic (paracrine) capacity. Immunohistochemistry and fluorescent staining confirmed high expression of EPC-specific markers, such as CD133 and KDR, and the ability to uptake DiI-ac-LDL and bind FITC-UEA-1. Angiogenesis assays showed that most effective conditions among those tested significantly increased the number of vessel-like structures. Additionally, the migration rate and proliferative activity of EPCs were significantly higher under the most effective conditions among those tested compared to conventional conditions. These findings provide a robust foundation for further refining in vitro EPC culture and pave the way for more effective clinical applications. Future studies should validate these optimized conditions in in vivo models to fully realize the therapeutic potential of EPCs.

Clinical and experimental studies have shown that sitagliptin regulates blood glucose levels. This study was designed because it is thought that sitagliptin may reduce diabetes-induced apoptosis in testes by affecting blood glucose levels and may have a beneficial effect on spermatogenesis by regulating hormonal activity. Thirty-four male Sprague-Dawley rats were divided into three groups: Control group (n = 10), given citrate buffer only; Diabetes group (n = 12), after 2 weeks of the high-fat diet, given a single dose of 35 mg/kg streptozotocin (STZ, dissolved in citrate buffer, intraperitoneally); Diabetes + Sitagliptin group (n = 12), after 2 weeks of the high-fat diet, diabetes was induced with STZ and 10 mg/kg sitagliptin (intragastric) was administered daily for 6 weeks. At the end of the experiment, blood glucose levels measured in the sitagliptin-treated group were found to be significantly lower than in the diabetes group. Serum testosterone, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels, seminiferous tubule diameter, Johnsen score, and proliferation indices were significantly lower in the diabetic groups compared to the control group, while no significant difference was found between the diabetes and sitagliptin groups. Basement membrane thickness, apoptotic cell and apoptotic tubul indexes, Fas, FasL, and caspase8 immunoreactivities were higher in diabetic groups compared to the control group, while no difference was found between the diabetic and sitagliptin groups. In conclusion, although 10 mg/kg sitagliptin reduced blood glucose levels in diabetes-induced hyperglycemia, it did not alter serum testosterone, FSH and LH levels, and did not appear to have a beneficial effect on diabetes-induced apoptosis and proliferation in the testes.

Beta-glucans (βTGs) are a class of dietary fibers and biologically active polysaccharides derived from natural sources, known for their diverse bioactive properties. Their documented effects include anti-tumor, anti-inflammatory, prebiotic, anti-obesity, anti-allergic, anti-microbial, antiviral, anti-osteoporotic, and immunomodulating activities. Despite these well-established benefits, the role of βTG in dehydroepiandrosterone (DHEA)-induced polycystic ovary syndrome (PCOS) remains largely unexplored. This study investigated the protective effects of βTG treatment on PCOS and its potential to reverse PCOS-induced changes. Female Sprague-Dawley (SD) rats were randomly divided into four groups (n = 8 each): control, PCOS, PCOS+βTG, and βTG. We assessed biochemical markers related to oxidative stress, antioxidant status, inflammation, cytokines, and hormone levels. Additional analyses included immunohistochemistry and histopathology. Membrane array analysis was used to profile growth factors, cytokines, and chemokines. However, βTG normalized deviations in the estrous cycle caused by PCOS and positively affected the reproductive system (p < 0.05). It also reduced the inflammatory response in PCOS rats by decreasing inflammatory cytokines (p < 0.05). Furthermore, oxidative stress was significantly reduced, and antioxidant enzyme activities were markedly elevated in the βTG group (p < 0.05). Histopathological alterations were prevented by βTG, which also induced the expression of essential proteins such as beta-nerve growth factor (bNGF), tissue inhibitor of metalloproteinase-1 (TIMP-1), Agrin, cytokine-induced neutrophil chemoattractant-1 (CINC-1), brain-derived neurotrophic factor (BDNF), and basic fibroblast growth factor (FGF-2/bFGF) (p < 0.05). In conclusion, βTG treatment effectively protects against oxidative stress, inflammation, hormone imbalance, and histopathological damage in ovarian tissue caused by PCOS.

Groundwater pollution with lead, fluoride, and nitrate presents a growing environmental and health challenge. This study aimed to evaluate the nephrotoxic effects of these pollutants in male albino rats and assess the potential ameliorative effects of curcumin and ascorbic acid in counteracting their toxicity for 135 days. A total of ten treatment groups were established viz. control, lead + fluoride + nitrate (BIS), lead + nitrate, lead + nitrate + curcumin + ascorbic acid, lead + fluoride, lead + fluoride + curcumin + ascorbic acid, fluoride + nitrate, fluoride + nitrate + curcumin + ascorbic acid, lead + fluoride + nitrate, lead + fluoride + nitrate + curcumin + ascorbic acid. Exposure to lead, fluoride, and nitrate resulted in a significant decrease in the activity of oxidative stress enzymes viz. superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase, and a notable increase in the lipid peroxidation levels. Further, significantly increased urea and creatinine levels in plasma and renal damage including glomerular shrinkage, widened Bowman's space, and tubular degeneration were also observed. The greatest damage was recorded in the lead + fluoride + nitrate group followed by lead + fluoride, lead + nitrate, and fluoride + nitrate. Co-treatment with curcumin and ascorbic acid demonstrated remarkable protective effects, with improvements in oxidative stress markers, plasma urea, and creatinine levels along with a significant restoration of glomerular structure and normalization of Bowman's space reflecting improved renal function. This research highlights the kidneys' susceptibility to environmental toxicants and the combined efficacy of curcumin and ascorbic acid in mitigating nephrotoxicity.

Ulcerative colitis is a chronic inflammatory condition of the gastrointestinal tract that can predispose patients to colonic neoplasms. Various natural compounds have been explored for their therapeutic potential. Indole-3-carbinol (I3C), a natural compound derived from cruciferous vegetables, is recognized for its tissue-protective and regenerative properties. This study aimed to investigate the effects of I3C on experimental ulcerative colitis in rats. Thirty-two Wistar rats were randomly assigned to four groups: a control group receiving isotonic saline, a TNBS group administered trinitrobenzene sulfonic acid (TNBS) intrarectally, an I3C group receiving I3C via gastric gavage, and a TNBS+I3C group treated with I3C following TNBS induction. After 7 days, all animals were euthanized under anesthesia, and pathological, histochemical, and immunohistochemical evaluations were conducted. The results revealed that I3C mitigated the severity of TNBS-induced colonic lesions and facilitated tissue repair. The I3C-treated group exhibited reduced tissue damage and enhanced mucosal regeneration. Additionally, vessel count, collagen, and myofibroblastic activity were markedly increased following I3C treatment. In conclusion, I3C exhibits both protective and reparative effects in experimental ulcerative colitis, potentially through anti-inflammatory mechanisms and the activation of tissue repair pathways.

Cultivating cells in 3D is considered a significant advancement in cell culture models, as it better reflects natural cellular environments compared to 2D cultures. However, analytical methods like standard light microscopy are less effective for 3D cultures. In this study, 3D cell cultures were generated using the liquid overlay technique with 10,000, 50,000, 100,000 and 200,000 Normal Human Dermal Fibroblasts, analyzed on days 1, 2, and 3 post-seeding. We quantified the influence of fixation with paraformaldehyde or glutardialdehyde/dehydration on their morphology compared to living 3D cell cultures. They were analyzed by light microscopy, scanning electron microscopy as well as by digital light microscopy (height profile measurement). Over time, the cultures decreased in size, likely due to cell shrinkage and structural reorganization. The size reduction could be mathematically described by an exponential decay function. The proportion of round spheroids versus indented aggregates depended on cell number, culture age, and fixation method. On day 1, cultures seeded with 10,000 cells formed nearly 100% round spheroids, regardless of fixation. Higher cell numbers led to fewer round spheroids, and fixation further reduced their number. This suggests that large cell quantities sediment in layers due to steric hindrance, forming indentations. Since aldehydes are responsible for cross-linking proteins, we hypothesize that this chemical reaction, combined with low stability of the 3D cell cultures, leads to the increased formation of the indented 3D cell aggregates. This is consistent with an overall increase in the number of round spheroids and a decrease of the negative influence of fixation over time. In summary, it is important to consider the number of seeded cells, the incubation time, as well as the possible fixation effects when generating stable spheroids using the liquid overlay technique for down-stream experiments.

In this study, we aimed to investigate the specific role of the acetyl-CoA carboxylase (ACACA) gene in oral squamous cell carcinoma (OSCC). We constructed the human tongue carcinoma cell line SAS with low ACACA expression and evaluated changes in cell proliferation, apoptosis, and ferroptosis. Then, the effect of combined treatment with cisplatin and ferroptosis inducer erastin was measured. To assess the impact of ACACA expression on tumor growth in vivo, we established xenograft models with varying ACACA levels in twelve male BALB/c nude mice. ACACA knockdown significantly reduced the proliferation ability of SAS cells, and increased the number of apoptotic cells. ACACA knockdown also induces ferroptosis, and this effect was enhanced by combined treatment with cisplatin and erastin. In vivo experiments demonstrated lower tumor volume and weight in the ACACA knockdown group than those in the control group. Exploring the combined effect of ACACA knockdown and cisplatin treatment revealed a promising synergistic effect against ferroptosis signaling and downstream signaling pathways in SAS cells and in vivo. These findings suggest that targeting the ACACA gene has the potential to be a novel therapeutic strategy for oral cancer treatment.

Dogs represent the most common domesticated animal outside of the research arena for whom tissue for pathological diagnosis is requested. Currently, there is a paucity of literature describing the cross-reactivity of anti-human antibodies on canine tissue for oncological diagnosis. This study aimed to evaluate the cross-reactivity of commonly used anti-human diagnostic antibodies in veterinary histopathology. One hundred seventy-one various samples from 153 canine tissues were processed to paraffin and tested with 76 fully validated anti-human antibody clones frequently employed in hospital pathology laboratories using immunohistochemistry methods to determine which ones exhibited comparable cross-reactivity and therefore potential use in veterinary pathology laboratories. Some of the anti-human antibodies were excluded from the study due to a lack of comparable canine tissues on which to test. However, almost half of the antibodies demonstrated results similar to those in human tissues and about one quarter showed weaker or less consistent labeling. The antibodies that demonstrated weaker or inconsistent labeling suggest areas where further development and modification could improve their employment on canine specimens. Fully one-third of antibodies tested failed to show labeling congruent with human tissues. The outcomes from this study could facilitate the adoption of established anti-human antibody markers in veterinary histopathology practices.