• Animal Bioscience
• /
• v.37 no.4
• /
• pp.609-621
• /
• 2024

Objective: Hair follicle stem cells (HFSCs) differentiation is a critical physiological progress in skin hair follicle (HF) formation. Goat HFSCs differentiation is one of the essential processes of superior-quality brush hair (SQBH) synthesis. However, knowledge regarding the functions and roles of miR-133a-3p and miR-145-5p in differentiated goat HFSCs is limited. Methods: To examine the significance of chi-miR-133a-3p and chi-miR-145-5p in differentiated HFSCs, overexpression and knockdown experiments of miR-133a-3p and miR-145-5p (Mimics and Inhibitors) separately or combined were performed. NANOG, SOX9, and stem cell differentiated markers (β-catenin, C-myc, Keratin 6 [KRT6]) expression levels were detected and analyzed by using real-time quantitative polymerase chain reaction, western blotting, and immunofluorescence assays in differentiated goat HFSCs. Results: miR-133a-3p and miR-145-5p inhibit NANOG (a gene recognized in keeping and maintaining the totipotency of embryonic stem cells) expression and promote SOX9 (an important stem cell transcription factor) expression in differentiated stem cells. Functional studies showed that miR-133a-3p and miR-145-5p individually or together overexpression can facilitate goat HFSCs differentiation, whereas suppressing miR-133a-3p and miR-145-5p or both inhibiting can inhibit goat HFSCs differentiation. Conclusion: These findings could more completely explain the modulatory function of miR-133a-3p and miR-145-5p in goat HFSCs growth, which also provide more understandings for further investigating goat hair follicle development.

• International Journal of Stem Cells
• /
• v.16 no.3
• /
• pp.326-341
• /
• 2023

Background and Objectives: Osteoarthritis (OA) is a degenerative disease that leads to the progressive destruction of articular cartilage. Current clinical therapeutic strategies are moderately effective at relieving OA-associated pain but cannot induce chondrocyte differentiation or achieve cartilage regeneration. We investigated the ability of wedelolactone, a biologically active natural product that occurs in Eclipta alba (false daisy), to promote chondrogenic differentiation. Methods and Results: Real-time reverse transcription-polymerase chain reaction, immunohistochemical staining, and immunofluorescence staining assays were used to evaluate the effects of wedelolactone on the chondrogenic differentiation of mesenchymal stem cells (MSCs). RNA sequencing, microRNA (miRNA) sequencing, and isobaric tags for relative and absolute quantitation analyses were performed to explore the mechanism by which wedelolactone promotes the chondrogenic differentiation of MSCs. We found that wedelolactone facilitates the chondrogenic differentiation of human induced pluripotent stem cell-derived MSCs and rat bone-marrow MSCs. Moreover, the forkhead box O (FOXO) signaling pathway was upregulated by wedelolactone during chondrogenic differentiation, and a FOXO1 inhibitor attenuated the effect of wedelolactone on chondrocyte differentiation. We determined that wedelolactone reduces enhancer of zeste homolog 2 (EZH2)-mediated histone H3 lysine 27 trimethylation of the promoter region of FOXO1 to upregulate its transcription. Additionally, we found that wedelolactone represses miR-1271-5p expression, and that miR-1271-5p post-transcriptionally suppresses the expression of FOXO1 that is dependent on the binding of miR-1271-5p to the FOXO1 3'-untranscribed region. Conclusions: These results indicate that wedelolactone suppresses the activity of EZH2 to facilitate the chondrogenic differentiation of MSCs by activating the FOXO1 signaling pathway. Wedelolactone may therefore improve cartilage regeneration in diseases characterized by inflammatory tissue destruction, such as OA.

• Journal of the korean academy of Pediatric Dentistry
• /
• v.45 no.3
• /
• pp.271-279
• /
• 2018

Previous studies to elucidate the etiology of cyclosporine(Cs)-induced gingival overgrowth in children have not completely excluded all factors that may cause differences among individuals. This study examined the effect of cyclosporine on the metabolism of type 1 collagen(CoL-I) in experimental models that controlled the effects of biological variations on individuals. Five 5-week-old male Sprague-Dawley rats were administered Cs by gastric feeding for 6 weeks. Gingival specimens were harvested from the mandibular posterior area before beginning Cs administration and at 2, 4, and 6 weeks thereafter. Gingival fibroblasts were cultured from all the 20 biopsies collected from the gingiva. Half of the fibroblasts collected prior to the Cs administration were designated as Control. The other half of the fibroblasts were treated with Cs in vitro and called in vitro test group(Tt). The fibroblasts collected 2, 4, and 6 weeks after the Cs administration were called in vivo test groups : T2, T4, T6, respectively. Immunofluorescence microscopy was used to detect CoL-I in all the fibroblasts. CoL-I was analyzed at both the gene and protein expression levels by real-time polymerase chain reaction and western blotting. Changes in CoL-I before and after Cs treatment were evaluated from the gingiva of each rat. There was no significant difference in gene expression of CoL-I in the control and test groups. CoL-I protein expression levels of fibroblasts increased in in vitro Cs treatment for each individual, and also increased in in vivo Cs treatment. In this study, the experimental method that control biological variations that can occur due to differences among individuals was useful. Subsequent studies on other factors besides CoL-I and in-depth studies in humans are needed.

• Pediatric Infection and Vaccine
• /
• v.5 no.1
• /
• pp.104-114
• /
• 1998

Purpose : Enteroviruses are the most common cause of aseptic meningitis. The epidemics of aseptic meningitis in 1993 and 1996 were mostly caused by echovirus type 9. Identification of the causative virus of aseptic meningitis in epidemics, is very important not only for diagnosis but also for epidemiologic purpose. The purpose of this study was to identify the causative virus and investigate the relationship between aseptic meningitis, prevailed in Masan and surrounding areas in Kyoungsangnamdo in 1997, and its clinical manifestations. Methods : One hundred twenty eight cerebrospinal fluid(CSF) and 239 stool specimens were obtained from 239 patients(213 children and 26 adult patients) with aseptic meningitis were admitted to Masan Fatima Hospitals from March to October 1997. Viral isolation and serotype identification was performed by cell culture and immunofluorescent test. Enteroviruses not typed by immunofluorescent test was confirmed by reverse transcription-polymerase chain reaction(RT-PCR). Results : 1) The peak incidence was noted in June. 2) The age of 239 patients(pediatrics-213 cases, internal medicine-26 cases) that were diagnosed ranged from neonate to 35 years, the age of the patients of pediatrics ranged from neonate to 15years(mean 4.9 years), the age of the patients of internal medicine (above 16 years) ranged from 16 years to 35 years(mean 24.2 years). 3) Fifty-three(41.4%) of 128 CSF specimens were positive for enteroviruses, and 163(68.2%) of 239 stool specimens were positive for enteroviruses respectively. 4) Serotypes of 53 enteroviruses isolated from CSF were 16(30.2%) of echovirus type 30, 6(11.3%) of echovirus type 6, 1 of echovirus type 4, 4 of untyped echovirus, 1 of coxsackievirus type B5, and 24 isolates of untyped enteroviruses. Of 163 enterovirus isolated from stool were 72(44.2%) of echovirus type 30, 21(12.9%) of echovirus type 6, 1 of echovirus type 4, 17(10.4%) of undetermined subtyped echovirus, 1 of coxsackievirus type B5, 2 of A24, 3 of undetermined subtyped coxsackievirus type B, and 46 isolates of untyped enterovirus. Conclusion : There were epidemics of aseptic meningitis in the central areas of Kyoungsangnamdo from March to October 1997. The main causative organism was thought to be the echovirus type 30, and echovirus type 4, 6, coxsackievirus B5 and A24 were also thought to contribute to the epidemics.

• Korean Journal of Veterinary Research
• /
• v.15 no.1
• /
• pp.57-67
• /
• 1975

The poxvirus group is considered to be a typical cytoplasmic inclusion forming virus. Every poxvirus has been reported to produce only one kind of inclusion in the infected tissues. A vague concept that inclusions of poxviruses are eosinophilic or acidophilic has prevailed. Although many papers and theories about the nature of the inclusion have been presented, most of them are not quite convincing on the point of the relations with virus multiplication, and an analysis of papers published showed that there seem to be many discrepancies in the descriptions of the nature of the poxvirus inclusions. Comparative studies on host-virus interaction with cowpox, orf, swinepox and fowlpox viruses which selected from each Group (I-IV) of poxviruses were performed from the morphological and virological standpoints. At first, in cowpox virus-FL cell system, as a comparative model, cytoplasmic inclusion, nucleic acid metabolism by autoradiography and detection of viral antigen by immunofluorescence were studied and obtained the results as follows: 1. The focus-like cytopathic effect (CPE) at early stage developed to entire culture at terminal stage of infection, and also the developing status of CPE was correlated to viral doses for inoculation. Two kinds of cytoplasmic inclusions which named A and B type were easily observed by Giemsa, hematoxylin-eosin (H & E) and May-Greenwald Giemsa (MGG) stainings in the infected cells. The B type inclusions were formed at early stage of infection and the A type inclusions were produced subsequently the B type formation. The B type which common type inclusion in poxviruses was a small compact or aggregate at early stage and developed to a large diffuse body at terminal stage of infection. On the other hand, the A type inclusion which depend upon the kind of virus was appeared as round and discrete shape, and its size and number was increased gradually during the culture period. It was characteristic to form distinct halos around the both types of inclusions in acid fixed, H & E stained preparations of infected cultures. The B type inclusion was always positive in Feulgen reaction and showed as DNA containing body but the A type inclusion was not. 2. In the relationship between inclusion and DNA metabolism of infected cells by the qualitative autoradiography using 3H-thymidine, the appearance of silver grains was coincided with B type inclusion but not with A type inclusion. This showed that the DNA synthesis was proceeded in all B type inclusions except those in the terminal stage with a diffuse form. This suggested that the B type inclusions are only sites of DNA synthesis and this was proceeded after the cell infection independently. The activity of DNA synthesis of the inclusions was nearly the same as that of the nucleic of normal cells and non-inclusion bearing cells. and non-inclusion bearing cells. Regardless of the size of the degree of DNA synthesis of the B type inclusion, inclusion bearing cells all showed remarkable suppression of nuclear DNA synthesis. 3. By the direct fluorescent antibody technique viral antigen in infected cells was detected. The B type inclusions have been proved to contain a great deal of viral antigen, whereas the basic substance of A type inclusion did not show antigenicity except the round edge. It was suggested that the round edge fluorescence might be caused by the glare of cytoplasmic viral antigen which pushed out and concentrated by the A type inclusion development. 4. Hemorrhagic red pock formations on chorioallantoic membrane of embryonated chicken egg had proved the characteristic of used viral strain. 5. By the above studies on the nature of two types of inclusions and the role they play in virus multiplication, it was concluded that the B type inclusion must be the site of the synthesis of viral DNA and protein as well as the site of the virus.

• Childhood Kidney Diseases
• /
• v.15 no.2
• /
• pp.138-145
• /
• 2011

Purpose : To test whether the expression of ${\beta}$-catenin, a component of podocyte as a filtration molecule, would be altered by puromycin aminonucleoside (PAN) in the cultured podocyte in vitro. Methods : We cultured rat glomerular epithelial cells (GEpC) with various concentrations of PAN and examined the distribution of ${\beta}$-catenin by confocal microscope and measured the change of ${\beta}$-catenin expression by Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). Results :We found that ${\beta}$-catenin relocalized from peripheral cytoplasm to inner cytoplasm, therefore, intercellular separations were seen in confluently cultured cells by high concentrations of PAN in immunofluorescence views. In Western blotting of GEpC, PAN ($50{\mu}g/mL$) decreased ${\beta}$-catenin expression by 34.9% at 24 hrs and 34.3% at 48 hrs, compared to those in without PAN condition (P<0.05). In RT-PCR, high concentrations ($50{\mu}g/mL$) of PAN also decreased ${\beta}$-catenin mRNA expression similar to protein suppression by 25.4% at 24 hrs and 51.8% at 48 hrs (P<0.05). Conclusion : Exposure of podocytes to PAN in vitro relocates ${\beta}$-catenin internally and reduces ${\beta}$-catenin mRNA and protein expression, which could explain the development of proteinuria in experimental PAN-induced nephropathy.

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