Cytotechnology最新文献

Human adipose tissue-derived stem cells (hADSCs) are an attractive source for regenerative medicine. However, cryopreservation protocols-particularly with respect to optimal cell concentration-remain inadequately defined. hADSCs were isolated from adipose tissue of 12 donors (mean age: 31.8 ± 8.9 years; BMI: 22.9 ± 4.2). Second-passage cells were cryopreserved for two weeks at concentrations of 0.5 × 10⁶/mL, 1 × 10⁶/mL, 2 × 10⁶/mL, 5 × 10⁶/mL, and 10 × 10⁶/mL. Post-thaw viability, apoptosis, immunophenotype, proliferation, and tri-lineage differentiation were evaluated using standard assays. Cell viability increased significantly with higher cryopreservation concentrations, reaching 94.2 ± 2.0% at 10 × 10⁶/mL (p < 0.05 vs. 0.5 × 10⁶/mL). Early apoptosis decreased with increasing concentration, reaching its lowest level at 5 × 10⁶/mL (2.9 ± 0.5%; p < 0.05 vs. 10 × 10⁶/mL), but showed a slight increase at 10 × 10⁶/mL. Proliferation and tri-lineage differentiation into adipocytes, osteoblasts, and chondrocytes were maintained across all groups, as confirmed by histological staining and molecular analyses. Cryopreservation at 5 × 10⁶/mL offers the most favorable balance between high viability and minimal apoptosis while preserving proliferative and differentiation potential. This concentration likely represents an optimal condition for hADSC biobanking and clinical use.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00877-6.

NFIB, a pivotal transcription factor, intricately influences tumorigenesis by exerting dual roles as either an oncogenic promoter or a tumor-suppressive factor across a spectrum of tumor types. However, the specific impact of NFIB on bladder cancer remains poorly understood. This study aims to elucidate the biological function and molecular mechanism of NFIB in bladder cancer. We first found that the protein level of NFIB was downregulated in bladder cancer tissues compared to matched adjacent noncancerous tissues. Functional assays, including Wound healing and Transwell invasion assays, demonstrated that NFIB suppressed migration, invasion and epithelial-mesenchymal transition (EMT) of bladder cancer cells in vitro, wheras CCK8 assays showed NFIB had no significant effect on cell proliferation. In vivo experiments, including Xenograft and Nude mouse tail vein transfer assays, further supported these observations, indicating that NFIB inhibited lung metastasis of bladder cancer without affecting primary tumor growth. Finally, transcriptomic analysis confirmed that NFIB hindered the activation of the PI3K-AKT signaling pathway. Taken together, this study highlighted that NFIB serves as a tumor suppressor gene in bladder cancer, and suppresses cell migration, invasion and EMT through the modulation of the PI3K-AKT signaling pathway, revealing NFIB as a potential biomarker for monitoring early metastasis of bladder cancer and a target for therapy.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-026-00900-4.

Oral squamous cell carcinoma (OSCC) is a common malignant tumor of the head and neck, representing a significant public health concern. Neurexophilin-4 (NXPH4) facilitates cancer cell proliferation and invasion. E26 oncogene homolog 1 (ETS1) can influence OSCC cell migration and invasion by modulating various factors. This study aims to explore the molecular mechanism of NXPH4 and ETS1 in the progression of OSCC. Bioinformatics analysis was used to analyze the NXPH4 and ETS1 expression in OSCC tissues and explore the relationship between NXPH4 and tumor stage. The mRNA and protein levels were examined by reverse transcription-quantitative polymerase chain reaction (qRT-PCR) and western blot. Besides, the OSCC tumor models were constructed, and tumor volume and weight were detected. The immunohistochemistry (IHC) assay was used to analyze Ki-67 expression in vivo. The apoptosis and reactive oxygen species (ROS) were measured by flow cytometry. The levels of malondialdehyde (MDA) and ferrous ion (Fe²⁺) were detected using the corresponding detection kits. The cell proliferation was tested by 5‑ethynyl‑2'‑deoxyuridine (EdU) staining. Tumor Immune Estimation Resource (TIMER) 2.0 and flow cytometry were used to analyze the relationship between NXPH4 and macrophage M2 polarization. The interaction between NXPH4 and ETS1 was demonstrated by chromatin immunoprecipitation (CHIP) and a dual luciferase reporter gene assay. NXPH4 expression was significantly increased in OSCC tissues and CAL-27 and SCC-25 cell lines (P < 0.01). Silencing NXPH4 inhibited the OSCC tumor growth in vivo. In human OSCC cell lines, silencing NXPH4 promoted the apoptosis, MDA, and ROS levels and suppressed the GPX4 expression (P < 0.01), cell proliferation, and macrophage M2 polarization. Furthermore, ETS1 could bind to NXPH4 and positively regulate its expression. Knockdown of ETS1 facilitated the apoptosis, increased MDA and ROS levels, and repressed the GPX4 expression, cell proliferation, and macrophage M2 polarization. However, up-regulation of NXPH4 in vitro reversed the effect of sh-ETS1 in human OSCC cell lines. ETS1 elevates the progression of OSCC and enhances macrophage M2 polarization by contributing to NXPH4. These findings identify the ETS1/NXPH4 axis as a potential therapeutic target in OSCC.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00869-6.

Non-small cell lung cancer (NSCLC) severely impairs patients' health and quality of life, placing a substantial burden on both patients' families and society. N4-acetylcytidine (ac4C), a pivotal RNA modification among the most abundant in eukaryotes, regulates disease development. Research has shown the ac4C-related gene NAT10 promotes lung cancer progression, yet the mechanism is unclear. NAT10 expression was assessed using data from the TCGA, CPTAC, ENCORI, and TNMplot databases, as well as qRT-PCR, and Western blot analyses. Cell proliferation was evaluated using MTT and EdU assays. Apoptosis was detected using flow cytomety. Cell invasion and migration were respectively detected by transwell and wound-healing assays. Cancer stem cell properties were analyzed via tumor sphere formation assays. Moreover, dual-luciferase reporter assay, ChIP assay, and RIP assay were performed to analyze the interaction between CEBPA and NAT10. Additionally, mRNA stability was evaluated using actinomycin D assay. The animal model was established to analyze the effects of NAT10 and CEBPA on tumor growth in vivo. NAT10 showed high expression levels in NSCLC tissues and cells. Silencing NAT10 inhibited cell viability, proliferation, invasion, migration, and stemness, while promoting apoptosis in NSCLC cells. The transcription factor CEBPA could bind to NAT10, thereby promoting NAT10 expression. Notably, NAT10 mediated the ac4C modification of CEBPA mRNA, which enhanced CEBPA mRNA stability and accelerated the malignant progression of NSCLC. Finally, NAT10 upregulated CEBPA expression, which in turn promoted tumor growth in vivo. NAT10 catalyzed ac4C acetylation on CEBPA mRNA, enhancing its stability and increasing CEBPA protein expression, thereby promoting the malignant progression of NSCLC. The identification of this feedback loop provides a preclinical rationale for developing NSCLC therapeutic strategies targeting NAT10 or CEBPA.

ObjectiveTo investigate the therapeutic effects and underlying mechanisms of umbilical cord mesenchymal stem cells (UCMSCs) on chemotherapy-induced premature ovarian insufficiency (POI) in rats. Methods. A POI rat model was established using cyclophosphamide (CTX). Female Sprague Dawley rats were randomly allocated into five experimental groups and treated with varying concentrations of UCMSC transplantation via tail vein injection. Ovarian function and apoptotic activity were evaluated through comprehensive assessment including serum hormonal profiling, histopathological examination, and molecular characterization. Results Compared to controls, the CTX-treated model group demonstrated severe ovarian dysfunction characterized by reduced ovarian mass, disrupted estrous cycles, and abnormal serum hormone levels. UCMSC transplantation produced dose-dependent restoration of ovarian physiology, with the highest dose achieving near-complete functional recovery. Molecular analyses revealed that UCMSCs dose-dependently modulated apoptosis-related gene expression, characterized by upregulated BCL2 and downregulated BAX and Caspase-3 levels. Additionally, UCMSCs suppressed P53 phosphorylation while simultaneously increasing AKT phosphorylation levels, indicating activation of pro-survival signaling pathways. Conclusion UCMSC transplantation effectively mitigates chemotherapy-induced ovarian injury and improves ovarian function, likely through suppressing ovarian cell apoptosis. The therapeutic mechanism appears to involve suppression of pro-apoptotic P53 signaling coupled with enhanced PI3K-AKT pathway activation. These findings highlight UCMSCs as a promising therapeutic strategy for POI management.

Lung cancer is one of the most frequent cancers in the world and the main cause of cancer related deaths. Among them, non-small cell lung cancer (NSCLC) accounts for about 85% of lung cancer. Myristicin (1-allyl-5-methoxy-3,4-methylenedioxybenzene), an active aromatic compound, has been proved to have anti-cancer effects. However, the effects of myristicin on NSCLC are not fully illustrated. Our research aimed to elucidate the roles and explain the potential mechanism of myristicin in NSCLC. A549 and H1975 cells were exposed to 0.5, 1, 5mM myristicin for 48 h, EdU, flow cytometry, Transwell and wound healing migration assay were applied for analyzing cell proliferation, apoptosis, cycle, migration and invasion, respectively. Caspase 3 activity and cleaved-Caspase3 expression were determined by relevant kits and western blotting, respectively. Besides, the Epithelial-Mesenchymal Transition (EMT) related genes levels and Wnt/β-catenin pathway related genes expressions, including E-cadherin, N-cadherin, Wnt3a and β-catenin, were assessed by qRT-PCR, and western blot assays. The nuclear translocation of β-catenin in NSCLC cells was analyzed by immunofluorescence staining. Our data revealed that myristicin suppressed NSCLC proliferation, migration and invasion in a dose-dependent manner. Besides, myristicin led to cell apoptotic and G0/G1 arrest and enhancing Caspase3 activity in NSCLC. Moreover, myristicin inhibited NSCLC EMT and blocked Wnt/β-Catenin signaling pathway in a dose-dependent manner, as confirmed by enhanced E-cadherin expression, suppressed N-cadherin level, inhibited Wnt3a and β-catenin levels, and reduced nuclear translocation of β-catenin. Myristicin blocked the development of NSCLC via regulating cells proliferation, migration, invasion and EMT through deactivating the Wnt/β-catenin pathway, which provide a new therapeutic treatment for NSCLC in clinical.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-026-00893-0.

Glycation of low-density lipoprotein (LDL) occurs when blood glucose levels are high, as in diabetic patients. This can lead to abnormal cholesterol transport in the body. Glycated LDL (G-LDL) is harmful and triggers oxidative stress in human erythrocytes. The present study investigated the protective effect of crocin, found in saffron, against G-LDL-induced cytotoxicity, oxidative damage, and formation of reactive species in human erythrocytes. These parameters were assessed in isolated human erythrocytes exposed to 6 mg/ml G-LDL, with and without varying concentrations of crocin (0.5, 1.0, 1.5 mM). Increased hemolysis, methemoglobin, and oxoferrylHb were seen in G-LDL alone-incubated cells. A significant increase in reactive species in G-LDL-exposed erythrocytes led to enhanced oxidation of lipids, proteins, and thiols. The activities of certain key antioxidant and membrane-bound enzymes were reduced. The antioxidant capacity of cells was compromised as indicated by a diminished ability to neutralize free radicals and donate electrons. G-LDL significantly altered surface morphology, forming echinocytes and agglutinating the cells. All these characteristics were significantly restored when erythrocytes were pre-treated with crocin, before incubation with G-LDL, in a crocin concentration-dependent manner. Furthermore, erythrocytes incubated with 1.5 mM crocin alone did not show alterations in any of the above parameters, indicating that crocin was not toxic to these cells. These results clearly show that crocin is strongly cytoprotective against G-LDL-induced damage and toxicity in erythrocytes. Hence, it can be used as an efficient dietary antioxidant in various atherosclerotic cardiovascular disorders, as seen in diabetic patients.

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Sepsis, a life-threatening systemic inflammatory response syndrome mediated by dysregulated host-pathogen interactions, frequently precipitates in renal injury. Renal tubular epithelial cell (RTEC) injury is a hallmark of septic acute kidney injury (AKI). Recent studies have demonstrated the involvement of serum response factor (SRF) in septic AKI. Herein, the role and mechanism of SRF as a transcription factor in regulating RTEC dysfunction were explored. HK-2 cells (the human RTEC line) were treated with lipopolysaccharide (LPS) for the establishment of in vitro septic AKI models. HK-2 cell viability was validated using CCK-8 assay. HK-2 cell apoptosis was evaluated by flow cytometry analysis. Measurement of proinflammatory cytokine concentration was conducted using enzyme-linked immunosorbent assay kits. RT-qPCR were required for determining gene levels. Western blotting was prepared for testing the protein levels of proinflammatory cytokines, apoptosis-related markers, NF-κB p65 and NF-κB inducing kinase (NIK). The binding of SRF to NIK promoter was confirmed by chromatin immunoprecipitation and luciferase reporter assays. LPS treatment suppressed HK-2 cell viability and accelerated HK-2 cell inflammation and apoptosis, which was antagonized by SRF depletion. Mechanically, SRF as a transcription factor bound to NIK promoter. SRF silencing inhibited LPS-induced NF-κB signaling activation in HK-2 cells. In rescue assays, NIK overexpression counteracted the restrictive impact of SRF deficiency on LPS-induced HK-2 cell dysfunction. SRF aggravates LPS-elicited HK-2 cell injury via binding to NIK promoter and activating NF-κB signaling.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00854-z.

Diabetic kidney disease (DKD) is a leading cause of kidney failure. It is characterized by damage to renal tubular endothelial cells, which is exacerbated by macrophage-derived exosomes under high-glucose (HG) conditions. Triptolide (TPL), an active ingredient extracted from Tripterygium wilfordii Hook F, has been used in the treatment of DKD. In this study, we aimed to investigate whether TPL protects against renal damage induced by macrophage-derived exosomes under HG conditions and to explore the involvement of the protein kinase B/mammalian target of rapamycin (AKT/mTOR) signaling pathway in this process. Exosomes were isolated from macrophages cultured in normal glucose (NG) and HG environments and characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot. Subsequent in vitro experiments on mouse renal tubular epithelial cells (mRTECs) demonstrated that 5 ng/mL TPL enhanced cell viability, reduced apoptosis and inflammation, and promoted autophagy-effects that were reversed by the autophagy inhibitor chloroquine (CQ). Molecular docking and bioinformatic analyses suggested a stable interaction between TPL and AKT, implicating the AKT/mTOR pathway in TPL-mediated autophagy activation. Further in vivo studies using an AKT agonist supported the role of TPL in inhibiting AKT/mTOR signaling and enhancing autophagic activity, ultimately ameliorating renal injury in a DKD model. These findings provide novel insights into the mechanisms by TPL may mitigate exosome-induced renal damage, highlighting its potential as a therapeutic agent for DKD.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00883-8.

Microglial polarization plays a crucial role in Parkinson's disease (PD). This study explores how serpin family A member 1 (SERPINA1) suppresses neuroinflammation and alleviates neuronal damage in PD. Adeno-associated viruses were injected into mice to manipulate the expression of SERPINA1 or runt-related transcription factor 1 (RUNX1) in the substantia nigra, followed by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) modeling. Behavioral tests, histopathology (HE and Nissl staining), immunohistochemistry (IHC), immunofluorescence, and enzyme-linked immunosorbent assay were conducted to evaluate neuroinflammation and neuronal damage in mice. BV2 microglial cells were infected with lentiviruses overexpressing SERPINA1 and treated with 100 µM 1-methyl-4-phenyl-pyridinium (MPP)⁺. MPP⁺ increased pro-inflammatory cytokines and iNOS while decreasing anti-inflammatory cytokines and arginase-1 expression in BV2 cells. SERPINA1 and RUNX1 were upregulated in the SN of MPTP-induced mice. RUNX1 bound to the promoter region of SERPINA1 to induce its transcription. SERPINA1 or RUNX1 overexpression alleviated PD-related neuronal damage and neuroinflammation in mice and MPP⁺-induced inflammation in BV2 cells. SERPINA1 knockdown inhibited M2 polarization in the presence of RUNX1 overexpression. Taken together, RUNX1 transcriptionally activates SERPINA1, promoting microglial M2 polarization, suppressing neuroinflammation, and alleviating neuronal damage in PD.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00878-5.