Cytotechnology最新文献

Inflammatory bowel disease (IBD) is a chronic, progressive, immune-mediated, gastrointestinal inflammatory disease with increasing occurrences in children. Collagen triple helix repeat containing 1 (CTHRC1), a migration-promoting protein, acts as a tumor-promoting factor in malignant tumors. However, functions and mechanisms of CTHRC1 in children with IBD remain unclear. This study aimed to determine the effects and mechanisms of CTHRC1 on dextran sodium sulfate (DSS)-treated HT-29 cells. HT-29 control cells were exposed to 2% DSS to develop an in vitro IBD model. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to assess CTHRC1 expression in serum of children with IBD and HT-29 cells. Cell viability and apoptosis were assessed using MTT and flow cytometry (FCM). Expressions of cleaved-Caspase3 and Caspase3 were determined by western blotting. The cytokine production (TNF-α, IL-1β and IL-6) in HT-29 cells was measured by ELISA assay. Activation or inactivation of NF-κB signaling pathway was confirmed by western blot assay. Results showed that CTHRC1 expression was upregulated in the IBD serum and HT-29 control cells. The level of CTHRC1 was lower in CTHRC1-siRNA transfected cells than in control siRNA-treated cells. Notably, silence of CTHRC1 markedly enhanced HT-29 cells viability, decreased apoptotic cells, suppressed cleaved-Caspase3 expression, inhibited cleaved-Caspase3/Caspase3 ratio, reduced the production of inflammatory cytokines, and blocked NF-κB signaling pathway induced by DSS. However, these effects were reversed following diprovocim treatment. Thus, that knockdown of CTHRC1 alleviated DSS-induced HT-29 cell injury by inhibiting the NF-κB signaling pathway in vitro, providing a new therapeutic target for IBD in children.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00705-x.

In a circadian cycle, the pineal gland produces and releases melatonin (MEL) into the bloodstream. By activating distinct melatonin receptors, MEL has been shown to variably change vascular endothelial dysfunction (VED) to various vascular beds. This study investigates the interaction of melatonin (MEL) and potassium ion (K⁺) on angiotensin 1-7 (Ang 1-7) vasorelaxant in streptozotocin (STZ)-induced diabetes mellitus (DM) and non-diabetes mellitus (non-DM) male albino rat aortic rings. The isometric tension of isolated aortic rings was assessed by generating a dose-response curve (DRC) for Ang 1-7 using a PowerLab data acquisition system. Accordingly, three experimental sets were carried out. In the first set the aortic rings were exposed MEL and MEL agonist ramelteon (RAM) and MEL antagonist luzindole (LUZ). In the second set, the aortic rings were exposed to various non-selective calcium activated potassium channel (K_Ca) blockers, including tetraethylammonium (TEA), a small and large-conductance calcium-activated K⁺ [(SK_Ca) and (BK_Ca)] channels blocker charybdotoxin (ChTx) and intermediate calcium-activated K⁺ channel (IK_Ca) blocker clotrimazole (CLT). In the third set, the aortic rings were exposed to various selective K⁺ channels blockers, including the selective blocker of K_ATP channel, glibenclamide (Glib), 4-aminopyridine (4-AP), a selective blocker of K_v channels and BaCl₂, delayed inward rectifier K⁺ channels (K_ir) blocker. The results highlight the significant role of MEL in modulating vascular reactivity, particularly in the DM aorta. By enhancing the vasorelaxant effects of Ang 1-7 through mechanisms involving its receptors and antioxidant activities, MEL demonstrates its potential to counteract oxidative stress and VED associated with diabetes. These findings advance the understanding of vascular reactivity in diabetes and suggest MEL as a promising therapeutic agent for improving vascular health in diabetic conditions.

Molecular profiles of mesenchymal stem cells (MSCs) are needed to standardize the composition and effectiveness of MSC therapeutics. This study employs RNA sequencing to identify genes to be used in concert with CD264 as a molecular profile of aging MSCs at a clinically relevant culture passage. CD264^- and CD264⁺ populations were isolated by fluorescence-activated cell sorting from passage 4 MSC cultures. CD264⁺ MSCs exhibited an aging phenotype relative to their CD264^- counterpart. Donor-matched CD264^-/+ mRNA samples from 5 donors were subjected to pair-ended, next-generation sequencing. An independent set of 5 donor MSCs was used to validate differential expression of select genes with quantitative reverse transcription PCR. Pairwise differential expression analysis identified 2,322 downregulated genes and 2,695 upregulated genes in CD264⁺ MSCs relative to donor-matched CD264^- MSCs with a Benjamini-Hochberg adjusted p-value (BH p _adj ) < 0.1. Nearly 25% of these genes were unique to CD264^-/+ MSCs and not differentially expressed at a significance level of BH p _adj  < 0.1 in previous RNA sequencing studies of early- vs. late-passage MSCs. Least Absolute Shrinkage and Selection Operator regression identified microtubule-associated protein 1A (MAP1A) and pituitary tumor-transforming gene 1 (PTTG1) as predictive genes of CD264⁺ MSCs. Combined MAP1A and PTTG1 expression correctly classified CD264 status of MSC samples with an accuracy of 100%. Differential expression and predictive ability of MAP1A and PTTG1 compared favorably with that of existing senescence markers expressed in early passage CD264^-/+ MSCs. This study provides the first linkage of MAP1A to CD264, aging and senescence. Our findings have application as quality metrics to standardize the composition of MSC therapies and as molecular targets to slow/reverse cellular aging.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00724-8.

Status epilepticus (SE) is a serious neurological emergency that brings significant risks to health and life. microRNAs (miRNAs) and their targets show involvement in the pathophysiology of SE. We identified plasma exosomal miRNAs and analyzed the miRNA-messenger RNA (mRNA) regulatory pathways in SE mice. Mice were subjected to SE induction by kainic acid injection, and plasma exosome (Exo) extraction. Exo morphology, particle size distribution, and Exo-positive marker proteins were evaluated. Differentially-expressed miRNAs in Exos of SE mice were analyzed and verified by sequencing and RT-qPCR. Functional enrichment analysis on target genes and protein-protein interaction (PPI) network were performed. Hippocampal neuron cells HT-22 were cultured in vitro, and the targeted binding association between Exos-derived miR-205-5p and target genes was invalidated. There were 64 differentially-expressed miRNAs in plasma Exos of SE mice from healthy mice (32 up-regulated, 32 down-regulated). Among the top 10 differentially-expressed miRNAs, 5 were up-regulated, and 5 were down-regulated. The PPI network of collective target genes was developed, including 11 edges and 9 nodes. The genes related to nerve injury were phosphatase and tensin homolog (Pten), glycogen synthase kinase 3 beta (Gsk3b), and leucine-rich repeat kinase 2 (Lrrk2). SE mouse plasma Exos targeted Gsk3b, Lrrk2 and Pten in neuronal cells and reduced cell viability. Plasma exosomal miRNAs of SE mice were differentially expressed, and their target genes participated in the regulation of multiple pathways, mainly related to nervous system development. miR-205-5p could target Gsk3b, Lrrk2 and Pten, and suppress neuronal viability.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00708-8.

In conventional Chinese hamster ovary (CHO) cell line development, static culture is used for early-stage screening, whereas suspension culture is generally used for the manufacturing process. Shaken flask (SF) fed-batch culture allows evaluation with culture mode, which is closer to the final process. However, due to its laborious and low-throughput characteristics, only a limited number of clones can be evaluated. To attain high-throughput fed-batch culture evaluation, a shaken 24-deep-well plate (24 DWP) culture process was developed. 24 DWP culture allows multiple plates to be run in parallel, and is therefore suitable for early-stage screening. One challenge of well plate culture is the nonuniform evaporation rate among wells, which may result in unnecessary bias on clone evaluation. The 'sandwich lid system' introduced here provides a uniform evaporation rate, and showed no significant difference in cell culture performance by well location. 192 antibody-producing CHO clones were evaluated by 24 DWP fed-batch culture, and 30 clones were selected. On comparison of clone sets selected by 24 DWP fed-batch culture and the conventional scheme, average antibody concentration in SF fed-batch culture was 3.6 g/L and 2.9 g/L, respectively. 24 DWP fed-batch culture process showed a high correlation ratio with SF fed-batch culture in antibody productivity and similar cell culture characteristics. These characteristics-high-throughput and sufficient culture volume to support cell culture performance monitoring-indicate that 24 DWP fed-batch culture can be applied in the clone selection stage in place of SF, and will shorten the time required for cell line development.

Pyroptosis and inflammation play crucial roles in the development of chronic obstructive pulmonary disease (COPD), and Zinc deficiency is commonly observed in COPD patients. In this study, we aimed to explore the impact of Zinc supplementation on pyroptosis and inflammation in a cigarette smoke (CS)-induced COPD mouse model, as well as the underlying mechanisms. The COPD mouse model was established through CS exposure, and mouse pulmonary epithelial cells (MLE-12) were exposed to cigarette smoke extract (CSE) to further validate the effects of Zinc supplementation. CS exposure resulted in significant alveolar wall damage, increased thickening of the alveolar walls, and elevated levels of interleukin-1β (IL-1β), IL-6, IL-18, and tumor necrosis factor-α (TNF-α) in the lung tissues of COPD mice. However, treatment with dexamethasone (a positive control) or Zinc supplementation alleviated these damages. Furthermore, the expressions of pyroptosis markers, including NLRP3, cleaved-Caspase-1, and GSDMD-N proteins, were upregulated in the lung tissues after CS exposure. Zinc supplementation, however, reversed these changes. Additionally, Zinc supplementation upregulated the protein expressions of nuclear factor erythroid 2-related factor 2 (Nrf2), hemeoxygenase-1 (HO-1), and quinone oxidoreductase-1 (NQO-1), and promoted the ubiquitination of Kelch-like ECH-associated protein 1 (Keap1) mediated by tripartite motif 25 (TRIM25) in the lung tissues of CS-induced mice. Importantly, the Nrf2 signaling inhibitor ML385 abolished the beneficial effects of Zinc in CS-exposed mice. Similar results were observed in MLE-12 lung epithelial cells exposed to CSE. In summary, Zinc supplementation inhibits pyroptosis and attenuates inflammation in COPD mice by activating the Nrf2 pathway.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00725-7.

The long-term treatment of malignancies, particularly brain tumors, is challenged by abnormal protein expression and drug resistance. In terms of potency, selectivity, and overcoming drug resistance, Proteolysis Targeting Chimeras (PROTACs), a cutting-edge method used to selectively degrade target proteins, beats traditional inhibitors. This review summarizes recent research on using PROTACs as a therapeutic strategy for brain tumors, focusing on their mechanism, benefits, limitations, and the need for optimization. The review draws from a comprehensive search of peer-reviewed literature, scientific databases, and clinical trial databases. Articles published up to the knowledge cutoff date up to 14 April 2023 were included. Inclusion criteria covered PROTAC-based brain tumor therapies, including preclinical and early clinical studies, with no restrictions on design or publication type. We included studies using in vitro, in vivo brain tumor models, and human subjects. Eligible treatments involved PROTACs targeting proteins linked to brain tumor progression. We evaluated the selected studies for methodology, including design, sample size, and data analysis techniques. A narrative synthesis summarized key outcomes and trends in PROTAC-based brain tumor therapy. Recent research shows PROTACs selectively degrade brain tumor-related proteins with minimal off-target effects. They offer enhanced potency, selectivity, and the ability to combat resistance compared to traditional inhibitors. PROTACs hold promise for brain tumor treatment offering advantages over traditional inhibitors, but more research is needed to refine their mechanisms, efficacy, and safety. Larger-scale trials and translational studies are essential for assessing their clinical utility.

Intervertebral disc degeneration (IVDD) represents a major cause of lower back pain, whose prevalence rises with age. This study probed into the mechanism of microRNA (miR)-124-3p regulating function of nucleus pulposus cells (NPCs) by targeting calpain-1 (CAPN1). Rat IVD NPCs were cultured in vitro and transfected with miR-124-3p mimics, miR-124-3p inhibitor, oe-CAPN1 and their negative controls. The mRNA levels of miR-124-3p and CAPN1 were assessed by RT-qPCR. Cell proliferation, apoptosis and migration were evaluated by CCK-8, flow cytometry and Transwell assays. Levels of CAPN1 protein, apoptosis-related proteins (BAX, Cleaved-Caspase3, BCL-2) and extracellular matrix (ECM) proteins (Collagen II, Aggrecan, Fibronectin, Collagen I, matrix metalloproteinase [MMP]-13) were determined by Western blot. The target binding relationship between miR-124-3p and CAPN1 was verified by dual-luciferase assay. miR-124-3p overexpression facilitated NPC function and the maintenance of ECM homeostasis, as evidenced by increased NPC proliferation and migration, decreased apoptosis, elevated apoptosis-related protein BCL-2 level, diminished BAX and Cleaved-Caspase3 levels, reduced levels of ECM homeostasis-associated factors Collagen I and MMP-13 proteins, as well as raised levels of Collagen II, Aggrecan and Fibronectin proteins. Conversely, miR-124-3p knockdown brought about the opposite results. miR-124-3p targeted CAPN1. Furthermore, overexpression of CAPN1 partially reversed the regulatory effects of miR-124-3p on the ECM homeostasis, proliferation and migration in NPCs, and promoted apoptosis. miR-124-3p contributed to proliferation and migration of IVD NPCs, and reduced their apoptosis by inhibiting CAPN1 expression, thereby modulating ECM homeostasis and maintaining the function of IVD NPCs.

The study was undertaken to determine the mechanism of miR-372-3p activating hepatic stellate cell (HSC). Transforming growth factor-β1 (TGF-β1) induced LX-2 cells were transfected with miR-372-3p mimics and/or RhoC overexpression vector (oe-RhoC), after which the miR-372-3 and RhoC expressions were detected and the biological functions of transfected cells were assessed. The relation between miR-372-3p and RhoC predicted online was validated using the dual-luciferase assay. Protein level of Collagen I (COL I), α-smooth muscle actin (α-SMA), and key proteins in the RhoC/ROCK pathway were determined using western blot. Activated LX-2 cells had decreased miR-372-3p and increased RhoC expression. Overexpression of miR-372-3p led to inhibited LX-2 cell proliferation, accelerated apoptosis, and decreased protein level of COL I and α-SMA, while such an expression pattern can be partially reversed by RhoC overexpression. miR-372-3p can bind and target RhoC expression. miR-372-3p inhibited RhoC expression to block the activation of the Rho/ROCK pathway and thus mediate LX-2 cell proliferation and apoptosis. miR-372-3p mediated RhoC/ROCK pathway to inhibit HSC activation.

Fermented plant product (FPP) is a dietary supplement made by fermentation and aging of a variety of plants, including fruits, vegetables, and grains. A previous study has shown that oral FPP supplementation prevents the development of allergic rhinitis-like nasal symptoms in a murine model of Japanese cedar pollinosis without affecting systemic immune response. However,　the mode of action by which FPP exerts an anti-allergic effect remains to be elucidated. Here, we show that FPP acts on mast cells to suppress immediate hypersensitivity reactions in vitro as well as in vivo. We found that stimulation with FPP potently suppressed IgE antibody-mediated degranulation of RBL-2H3 rat basophilic leukemia cells. We also found that oral feeding with FPP significantly suppressed passive cutaneous anaphylaxis (PCA), an in vivo model of IgE- and mast cell-mediated hypersensitivity reactions. Mechanistic analysis revealed that FPP extensively suppressed the high-affinity IgE receptor (FcεRI) signaling pathway, in which FPP not only inhibited intracellular Ca²⁺ influx upon FcεRI ligation but also negatively regulated another Ca²⁺-independent FcεRI signaling pathway leading to granule translocation through microtubule formation. These results suggest that FPP fulfills its anti-allergic activity by acting on the IgE-mast cell axis to suppress immediate hypersensitivity reactions.