• Journal of Veterinary Science
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• v.24 no.1
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• pp.4.1-4.16
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• 2023

Background: In vitro culture of preantral follicles is a promising technology for fertility preservation. Objectives: This study aims to investigate an optimized three-dimensional (3D) fetal bovine serum (FBS)-free preantral follicle culture system having a simple and easy operation. Methods: The isolated follicles from mouse ovaries were randomly divided in an ultra-low attachment 96-well plates supplement with FBS or bovine serum albumin (BSA) culture or encapsulated with an alginate supplement with FBS or BSA culture. Meanwhile, estradiol (E₂) concentration was assessed through enzyme-linked immunosorbent assay of culture supernatants. The diameter of follicular growth was measured, and the lumen of the follicle was photographed. Spindle microtubules of oocytes were detected via immunofluorescence. The ability of oocytes to fertilize was assessed using in vitro fertilization. Results: The diameters were larger for the growing secondary follicles cultured in ultra-low attachment 96-well plates than in the alginate gel on days 6, 8, and 10 (p < 0.05). Meanwhile, the E2 concentration in the BSA-supplemented medium was significantly higher in the alginate gel than in the other three groups on days 6 and 8 (p < 0.05), and the oocytes in the FBS-free system could complete meiosis and fertilization in vitro. Conclusions: The present study furnishes insights into the mature oocytes obtained from the 3D culture of the preantral follicle by using ultra-low attachment 96-well plate with an FBS-free system in vitro and supports the clinical practices to achieve competent, mature oocytes for in vitro fertilization.

• Journal of Ginseng Research
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• v.47 no.2
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• pp.199-209
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• 2023

Background: Due to the interrupted blood supply in cerebral ischemic stroke (CIS), ischemic and hypoxia results in neuronal depolarization, insufficient NAD+, excessive levels of ROS, mitochondrial damages, and energy metabolism disorders, which triggers the ischemic cascades. Currently, improvement of mitochondrial functions and energy metabolism is as a vital therapeutic target and clinical strategy. Hence, it is greatly crucial to look for neuroprotective natural agents with mitochondria protection actions and explore the mediated targets for treating CIS. In the previous study, notoginseng leaf triterpenes (PNGL) from Panax notoginseng stems and leaves was demonstrated to have neuroprotective effects against cerebral ischemia/reperfusion injury. However, the potential mechanisms have been not completely elaborate. Methods: The model of middle cerebral artery occlusion and reperfusion (MCAO/R) was adopted to verify the neuroprotective effects and potential pharmacology mechanisms of PNGL in vivo. Antioxidant markers were evaluated by kit detection. Mitochondrial function was evaluated by ATP content measurement, ATPase, NAD and NADH kits. And the transmission electron microscopy (TEM) and pathological staining (H&E and Nissl) were used to detect cerebral morphological changes and mitochondrial structural damages. Western blotting, ELISA and immunofluorescence assay were utilized to explore the mitochondrial protection effects and its related mechanisms in vivo. Results: In vivo, treatment with PNGL markedly reduced excessive oxidative stress, inhibited mitochondrial injury, alleviated energy metabolism dysfunction, decreased neuronal loss and apoptosis, and thus notedly raised neuronal survival under ischemia and hypoxia. Meanwhile, PNGL significantly increased the expression of nicotinamide phosphoribosyltransferase (NAMPT) in the ischemic regions, and regulated its related downstream SIRT1/2/3-MnSOD/PGC-1α pathways. Conclusion: The study finds that the mitochondrial protective effects of PNGL are associated with the NAMPT-SIRT1/2/3-MnSOD/PGC-1α signal pathways. PNGL, as a novel candidate drug, has great application prospects for preventing and treating ischemic stroke.

• Journal of Veterinary Science
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• v.23 no.2
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• pp.40.1-40.13
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• 2022

Background: Somatic cell nuclear transfer (SCNT) is used widely in cloning, stem cell research, and regenerative medicine. The type of donor cells is a key factor affecting the SCNT efficiency. Objectives: This study examined whether urine-derived somatic cells could be used as donors for SCNT in pigs. Methods: The viability of cells isolated from urine was assessed using trypan blue and propidium iodide staining. The H3K9me3/H3K27me3 level of the cells was analyzed by immunofluorescence. The in vitro developmental ability of SCNT embryos was evaluated by the blastocyst rate and the expression levels of the core pluripotency factor. Blastocyst cell apoptosis was examined using a terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. The in vivo developmental ability of SCNT embryos was evaluated after embryo transfer. Results: Most sow urine-derived cells were viable and could be cultured and propagated easily. On the other hand, most of the somatic cells isolated from the boar urine exhibited poor cellular activity. The in vitro development efficiency between the embryos produced by SCNT using porcine embryonic fibroblasts (PEFs) and urine-derived cells were similar. Moreover, The H3K9me3 in SCNT embryos produced from sow urine-derived cells and PEFs at the four-cell stage showed similar intensity. The levels of Oct4, Nanog, and Sox2 expression in blastocysts were similar in the two groups. Furthermore, there is a similar apoptotic level of cloned embryos produced by the two types of cells. Finally, the full-term development ability of the cloned embryos was evaluated, and the cloned fetuses from the urine-derived cells showed absorption. Conclusions: Sow urine-derived cells could be used to produce SCNT embryos.

• Applied Chemistry for Engineering
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• v.33 no.2
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• pp.173-178
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• 2022

A lateral flow immunoassay (LFIA) method using carbon nanodot@silica as a signaling material was developed for analyzing the concentration of retinol-binding protein 4 (RBP4), one of the lung cancer biomarkers. Instead of antibodies mainly used as bioreceptors in nitrocellulose membranes in LFIA for protein detection, aptamers that are more economical, easy to store for a long time, and have strong affinities toward specific target proteins were used. A 5' terminal of biotin-modified aptamer specific to RBP4 was first reacted with neutravidin followed by spraying the mixture on the membrane in order to immobilize the aptamer in a porous membrane by the strong binding affinity between biotin and neutravidin. Carbon nanodot@silica nanoparticles with blue fluorescent signal covalently conjugated to the RBP4 antibody, and RBP4 were injected in a lateral flow manner on to the surface bound aptamer to form a sandwich complex. Surfactant concentrations, ionic strength, and additional blocking reagents were added to the running buffer solution to optimize the fluorescent signal off from the sandwich complex which was correlated to the concentration of RBP4. A 10 mM Tris (pH 7.4) running buffer containing 150 mM NaCl and 0.05% Tween-20 with 0.6 M ethanolamine as a blocking agent showed the optimum assay condition for carbon nanodot@silica-based LFIA. The results indicate that an aptamer, more economical and easier to store for a long time can be used as an alternative immobilizing probe for antibody in a LFIA device which can be used as a point-of-care diagnosis kit for lung cancer diseases.

• Clinical and Experimental Pediatrics
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• v.49 no.9
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• pp.977-982
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• 2006

Purpose : There has been an increasing amount of literature concerning the association between Mycoplasma pneumoniae and asthma pathogenesis. Interleukin(IL)-6 stimulates the differentiation of monocytes, and can promote Th2 differentiation and simultaneously inhibit Th1 polarization. IL-8 is a potent chemoattractant and, it has been suggested, has a role in asthma pathogenesis. Nitric oxide (NO) synthesized by airway epithelium may be important in the regulation of airway inflammation and reactivity. Vascular endothelial growth factor(VEGF) has been reported to be a mediator of airway remodeling in asthma. We investigated the effects of M. pneumoniae on IL-6, IL-8, NO and VEGF production in human respiratory epithelial cells. Methods : A549 cells were cultured and inoculated with M. pneumoniae at a dose of 20 cfu/cell. After infection, the presence of M. pneumoniae in epithelial cell cultures was monitored by immunofluorescence and confirmed by polymerase chain reaction(PCR) detection. IL-6, IL-8 and VEGF were determined by an enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction. NO was measured using the standard Griess reaction. Results : In A549 cells, M. pneumoniaeinduced IL-6, IL-8, NO and VEGF release in time-dependent manners. It also induced mRNA expression of IL-6, IL-8 and VEGF in similar manners. Conclusion : These observations suggest that M. pneumoniae might have a role in the pathogenesis of the allergic inflammation of bronchial asthma.

• Pediatric Infection and Vaccine
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• v.23 no.1
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• pp.31-39
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• 2016

Purpose: The aim of this study was to investigate the prognostic factors for Pneumocystis jirovecii pneumonia (PCP) and to evaluate the influence of PCP prophylaxis in pediatric patients. Methods: From January 2002 to April 2015, patients aged <18 years with a diagnosis of confirmed PCP at our institute were reviewed retrospectively. Clinical characteristics and outcomes were compared according to the groups with or without PCP prophylaxis. Risk factors associated with PCP-related death were analyzed by logistic regression analysis. Results: During study period, a total of 24 patients were diagnosed with PCP by immunofluorescence assay and/or PCR. The median age of the patients was 5 years (range, 3 months-18 years) and 23 (96%) had immunocompromised conditions including hematologic disorders with or without hematopoietic stem cell transplantation (n=15), solid organ transplantation (n=4), and primary immune deficiency (n=4). Most common presenting symptoms were tachypnea and cough (92%, each). At the time of diagnosis, 79% (19/24) and 25% (6/24) suffered from respiratory failure and multi-organ dysfunction syndrome (MODS), respectively. Mechanical ventilation was required in 8 (33%) patients and 5 (21%) patients died of PCP. Multivariate analysis showed that MODS at initial presentation was an indicator of poor prognosis (OR, 17.1 [95% CI 1.13-257.67]; P=0.04). Compared to the patients without PCP prophylaxis, the frequency of MODS at diagnosis, need for mechanical ventilation and length of hospital days were significantly less common in the children who received PCP prophylaxis. Conclusions: MODS at presentation was a significant predictor for poor outcome and PCP prophylaxis could alleviate the clinical courses of pediatric PCP. Prospective study will be mandatory to determine the risk factors for development and deterioration of PCP in children.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.18
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• pp.7849-7855
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• 2014

Purpose: To investigate the effect of deacetylase inhibitory trichostatin A (TSA) on anti HepG2 liver carcinoma cells and explore the underlying mechanisms. Materials and Methods: HepG2 cells exposed to different concentrations of TSA for 24, 48, or 72h were examined for cell growth inhibition using CCK8, changes in cell cycle distribution with flow cytometry, cell apoptosis with annexin V-FTIC/PI double staining, and cell morphology changes under an inverted microscope. Expression of ${\beta}$-catenin, HDAC1, HDAC3, H3K9, CyclinD1 and Bax proteins was tested by Western blotting. Gene expression for ${\beta}$-catenin, HDAC1and HDAC3 was tested by q-PCR. ${\beta}$-catenin and H3K9 proteins were also tested by immunofluorescence. Activity of Renilla luciferase (pTCF/LEF-luc) was assessed using the Luciferase Reporter Assay system reagent. The activity of total HDACs was detected with a HDACs colorimetric kit. Results: Exposure to TSA caused significant dose-and time-dependent inhibition of HepG2 cell proliferation (p<0.05) and resulted in increased cell percentages in G0/G1 and G2/M phases and decrease in the S phase. The apoptotic index in the control group was $6.22{\pm}0.25%$, which increased to $7.17{\pm}0.20%$ and $18.1{\pm}0.42%$ in the treatment group. Exposure to 250 and 500nmol/L TSA also caused cell morphology changes with numerous floating cells. Expression of ${\beta}$-catenin, H3K9and Bax proteins was significantly increased, expression levels of CyclinD1, HDAC1, HDAC3 were decreased. Expression of ${\beta}$-catenin at the genetic level was significantly increased, with no significant difference in HDAC1and HDAC3 genes. In the cytoplasm, expression of ${\beta}$-catenin fluorescence protein was not obvious changed and in the nucleus, small amounts of green fluorescence were observed. H3K9 fluorescence protein were increased. Expression levels of the transcription factor TCF werealso increased in HepG2 cells following induction by TSA, whikle the activity of total HDACs was decreased. Conclusions: TSA inhibits HDAC activity, promotes histone acetylation, and activates Wnt/${\beta}$-catenin signaling to inhibit proliferation of HepG2 cell, arrest cell cycling and induce apoptosis.

• Tuberculosis and Respiratory Diseases
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• v.58 no.1
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• pp.43-53
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• 2005

Emphysema is characterized by air space enlargement and alveolar destruction. The mechanism responsible for the development of emphysema was thought to be protease/antiprotease imbalance and oxidative stress. A very recent study shows that alveolar cell apoptosis causes lung destruction and emphysematous changes. Thus, this study was performed to support the evidence for the role of apoptosis in the development of emphysema by characterizing cigarette smoke extract (CSE)-induced apoptosis in A549 (type II pneumocyte) lung epithelial cells. CSE induced apoptosis at low concentration (10% or less) and both apoptosis and necrosis at high concentration (20%). Apoptosis was demonstrated by DNA fragmentation using FACScan for subG1 fraction. Discrimination between apoptosis and necrosis was done by morphologic analysis using fluorescent microscopy with Hoecst 33342/propium iodide double staing and electron microscopy. Cytochrome c release was confirmed by using immunofluorescence with monoclonal anti-cytochrome c antibody. However, CSE-induced cell death did not show the activation of caspase 3 and was not blocked by caspase inhibitors. This suggests that CSE-induced apoptosis might be caspase-independent apoptosis. CSE-induced cell death was near completely blocked by N-acetylcystein and bcl-2 overexpression protected CSE-induced cell death. This results suggests that CSE might induce apoptosis through intracellular oxidative stress. CSE also activated p53 and functional knock-out of p53 using stable overexpression of HPV-E6 protein inhibited CSE-induced cell death. The characterization of CSE-induced cell death in lung epithelial cells could support the role of lung cell apoptosis in the pathogenesis of emphysema.

• Journal of Life Science
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• v.31 no.8
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• pp.710-718
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• 2021

Placenta-derived mesenchymal stem cells (PD-MSCs) are promising candidates for cell-based therapy in regenerative medicine. The migration and homing potential of PD-MSCs to injured sites is a critical property of MSC engraftment. MicroRNAs (miRNAs) have recently been shown to regulate the critical functions of MSCs, such as proliferation, survival, and migration. The objective of the present study was to identify the miRNA and target genes involved in PD-MSCs homing in a bile duct ligation (BDL) rat model. We selected candidate miRNAs targeting genes for PD-MSCs homing based on microarray analysis. PD-MSC engraftment in BDL-injured rat liver was identified by immunofluorescence assay and human-specific Alu gene expression by quantitative real-time polymerase chain reaction (qRT-PCR) one week after transplantation. Compared with migrated naïve PD-MSCs under hypoxic and normoxic conditions (Hyp/Nor), the transplanted group with PD-MSCs (Tx) showed distinct differences in miRNA expressions in BDL-injured rat liver. We also validated the miRNAs and their target genes for PD-MSCs homing. The expressions of integrin α4 (ITGA4) and integrin α5 (ITGA5) target genes for miR-199a-5p and miR-148a-3p were significantly upregulated in the Tx group (p<0.05). In addition, integrin β1 (ITGB1) and integrin β8 (ITGB8) were upregulated by suppressing miR-183-5p and miR-145-5p, respectively. These results demonstrated that PD-MSCs regulate miRNA expression related to the integrin family for their homing effects on the BDL-injured rat liver. The findings further suggest that miRNA-mediated regulation of the integrin family contributes to the therapeutic efficacy of PD-MSCs in the rat hepatic fibrosis model by BDL.