Histochemistry and Cell Biology最新文献

Cholesterol is involved in the regulation of various signaling processes, and oxysterols are essential lipid messengers. The cholesterol derivative 25-hydroxycholesterol (25-HC) is overproduced by muscle macrophages in sarcopenia, myasthenia, and neurodegenerative diseases. Herein, we examined the effects of 25-HC on Ca²⁺ signaling and contractions of the mouse diaphragm, the main respiratory muscle. We found that 25-HC increased resting levels of cytosolic Ca²⁺ in muscle fibers. This effect was dependent on estrogen receptor α (ERα) and was mediated by Ca²⁺-efflux from intracellular stores via dantrolene-insensitive and TMB-8-sensitive channels, presumably inositol trisphosphate receptors (IP₃Rs). In addition, 25-HC suppressed diaphragm contractile responses to direct stimulation of the muscle fibers. The negative effect of 25-HC on contraction force was inhibited by blockers of ERα and Ca²⁺ mobilization. Thus, 25-HC may suppress diaphragm muscle contractility due to activation of an ERα/IP₃R/Ca²⁺_in axis in muscle fibers. At the same time, 25-HC did not significantly modify the contractions elicited by phrenic nerve stimulation and respiratory activity in vivo. We discuss that the previously found enhancement of neuromuscular transmission mediated by 25-HC in the diaphragm can compensate for the reduction in the muscle contractions in the case of phrenic nerve activation.

Prostate cancer is a frequently diagnosed cancer in men, but today the prognosis of local cancer and its metastasis has improved markedly owing to multiple treatment options. In therapy, various molecular entities that induce the reversion of malignancy with the restoration of mature cells of the same histological lineage are still under development. The antler of the red deer is a large and easily available source of stem cells, rich in products with promising applications in cancer treatments. Here, for the first time, antler stem cell culture medium was used for the treatment of prostate cancer cells in vitro to determine the cellular and molecular effects. Our data revealed that antler stem cell culture medium promotes pronounced changes in the expression of the vaspin receptor GRP78, intercellular adhesion molecule 1 (ICAM-1), androgen receptor (AR), leptin, vaspin, and SRC and AKT kinase signaling pathways (connected with autophagy and cell cycle inhibition and apoptosis induction). Moreover, the inhibition of cell migration was also revealed. These results indicate useful properties of red deer antler stem cell culture medium that can control molecular and cellular mechanisms that halt carcinogenesis. All these can be considered targets for further cancer stem cell treatment or cancer diagnostic improvements.

This study aimed to quantitatively analyze subchondral vascularization during the progression of osteoarthritis (OA) in the elderly, particularly regarding the timing of initial blood vessel emergence and when their density peaks. A total of 129 independent areas from 43 human femoral heads, obtained through arthroplasty for OA or hemiarthroplasty for fractures, were analyzed. The femoral heads were grouped by Kellgren-Lawrence (KL) grades: KL 1 (6 heads), KL 2 (14 heads), KL 3 (10 heads), and KL 4 (13 heads), and the Mankin score was assessed. Quantitative measurements of blood vessel length 1 mm below the tidemark, cartilage volume and thickness, chondrocyte volume, ECM volume, subchondral bone volume, and bone marrow volume were performed using stereology and immunohistochemistry. The most substantial increase in the characteristics of blood vessels within the subchondral region began at KL 3 and peaked at KL 4. Blood vessel volume increased from 6.71 ± 5.84 mm³ in group KL 1 to 156.21 ± 138.67 mm³ in group KL 4 (p < 0.001). Blood vessel surface area showed an increase from 14.78 ± 9.89 cm² (group KL 1) to 125.20 ± 93.18 cm² (group KL 4) (p < 0.001). Likewise, blood vessel length grew from 27.53 m (IQR 13.70-65.41 m) in group KL 1 to 112.03 ± 76.07 m in group KL 4 (p = 0.001). This study offers deeper insights into the role of vascularization in OA pathophysiology, quantifying subchondral blood vessel characteristics in the femoral head across different OA stages.

Fibroblasts, the most abundant cell type in the human body, play crucial roles in biological processes such as inflammation and cancer progression. They originate from the mesoderm or neural-crest-derived ectomesenchyme. Ectomesenchyme-derived fibroblasts contribute to facial formation and do not express HOX genes during development. The expression and role of the HOX genes in adult fibroblasts is not known. We investigated whether the developmental pattern persists into adulthood and under pathological conditions, such as cancer. We collected adult fibroblasts of ectomesenchymal and mesodermal origins from distinct body parts. The isolated fibroblasts were characterised by immunocytochemistry, and their transcriptome was analysed by whole genome profiling. Significant differences were observed between normal fibroblasts from the face (ectomesenchyme) and upper limb (mesoderm), particularly in genes associated with limb development, including HOX genes, e.g., HOXA9 and HOXD9. Notably, the pattern of HOX gene expression remained consistent postnatally, even in fibroblasts from pathological tissues, including inflammatory states and cancer-associated fibroblasts from primary and metastatic tumours. Therefore, the distinctive HOX gene expression pattern can serve as an indicator of the topological origin of fibroblasts. The influence of cell position and HOX gene expression in fibroblasts on disease progression warrants further investigation.

Cutaneous wounds represent a significant health concern, and effective treatment strategies are crucial for optimal healing. This study investigates the therapeutic potential of Luetzelburgia auriculata lectin (LAA), a plant-derived protein, in accelerating wound closure. Excisional wounds were created on the backs of mice, which were subsequently treated topically with LAA solutions at two concentrations (100 µg/mL and 200 µg/mL) or saline control. Wound healing was assessed through clinical observations, including wound area measurement and inflammatory score, as well as histopathological analysis and measurement of inflammatory cytokines. LAA significantly accelerated wound closure, reduced inflammation, and promoted tissue regeneration. Histological analysis revealed enhanced re-epithelialization, increased fibroblast proliferation, and improved collagen deposition in LAA-treated wounds compared with the control group. Furthermore, LAA treatment significantly reduced the levels of proinflammatory cytokines in wound tissues (interleukin-6, tumor necrosis factor-alpha, and monocyte chemoattractant protein-1). These findings suggest that LAA exerts its beneficial effects through a multifaceted mechanism, likely involving anti-inflammatory properties and stimulation of cellular processes crucial for tissue repair. This study provides preliminary evidence for the therapeutic potential of LAA in wound healing and warrants further investigation into its underlying mechanisms and clinical applications.

A major issue facing the field of cellular imaging, immunofluorescence (IF), and immunohistochemistry (IHC) microscopy is antibody quality. One of the main methods of antibody validation is testing on positive and negative control tissues with known expression levels of a given antigen. However, this approach is reliant on availability of tissues and reliable protein expression datasets, which are not always available. In contrast, cultured cell lines often have more extensive and reproducible protein expression data available, are relatively inexpensive to maintain, and can be used to produce knockout lines for more robust and functional validation. Due to the difference in staining protocols between formalin-fixed paraffin-embedded (FFPE) tissues and cultured cell lines, an antibody that works well in cultured cells does not always produce the same results in FFPE tissues. For this reason, there is a need for methods to embed cultured cells in paraffin for antibody testing. Previous methods have been published, but many involve use of sharps, which introduces risk of cuts to the investigator, or embedded in agarose first, which results in a lower density of cells. This paper introduces a method of embedding cultured cells using custom designed silicone molds. These molds allow an easy, risk-free embedding process that results in high density cell pellet blocks which can be used for IF and IHC experiments, as well as creation of cell microarrays. Additionally, the silicone molds can be used to embed organoids for IF and IHC analysis.

The expansion of mesenchymal stem cells (MSCs) for clinical applications is often limited by replicative senescence, a growth arrest induced by various stresses during in vitro culture, yet its impact on the immunomodulatory properties of MSCs remains unclear. This study derived MSCs from the amniotic fluid (AF-MSCs) of seven first-trimester pregnancies, characterized them through flow cytometry, and evaluated their osteogenic differentiation potential before expanding the cells to compare immunoregulatory gene expression in proliferative and senescent states. Additionally, an assessment of the adipogenic differentiation potential of AF-MSCs from three samples was conducted following their recovery from approximately 9 months of cryopreservation, with results showing that these recovered cells retain the capacity for adipogenic differentiation. Molecular analysis revealed no significant differences in the expression of key immunoregulatory genes, such as TGFβ, IL-10, IDO, and VCAM-1, between proliferative and senescent cells, although senescent cells showed downregulation of FASL and upregulation of IL-6, COX1, and HLA-G. Markers of cell proliferation, including FOXM1 and B-MYB, were significantly downregulated in senescent cells, confirming the progression of replicative senescence. Despite expectations, the results indicated that some immunomodulatory markers remained stable or were even enhanced in senescent AF-MSCs. These findings highlight the resilience of AF-MSC immunomodulatory properties during prolonged in vitro expansion, supporting their potential for therapeutic applications despite the challenges posed by replicative senescence.