Park, Se-Ah;Kang, Hyun-Mi;Heo, Jin-Yeong;Yoon, Jin-Ah;Kim, Hae-Kwon
Clinical and Experimental Reproductive Medicine
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v.36
no.1
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pp.23-34
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2009
Objectives: Mesenchymal stem cells (MSC) comprise a promising tool for cellular therapy. It is known that long-term in vitro culture of human bone marrow and adipose tissue derived-MSCs lead to a reduction of life span and a change of stem-like characters. The aim of our study was to examine whether stem cell properties of human umbilical cord-derived stem cells (HUC) could be affected by in vitro expansion. Methods: HUC were isolated from human umbilical cord and cultured for 10 passages in vitro. Morphology and population doubling time (PDT) were investigated, and changes of stem cell properties were examined using RT-PCR and immunocytochemistry during serial subcultures. Results: Morphology and PDT of HUC began to change slightly from the 7th passage (p7). Expression level of nestin and vimentin mRNAs increased along with the culture period from p4 until p10. In contrast, expression level of SCF mRNA decreased during the same culture period. Expression level of Oct-4 and HNF-4${\alpha}$ mRNAs was not significantly changed throughout the culture period until p10. Expression level of BMP-4, FGF-5, NCAM and HLA-ABC mRNAs appeared to increase as the culture continued, however, the difference was not significant. Immunocytochemical studies showed that HUC at p3, p6 and p9 positively were stained with antibodies against SSEA-3 and SSEA-4 proteins. Interestingly, staining intensity of HUC for ICAM-1 and HLA-ABC gradually increased throughout the culture period. Intensity against thy-1 and fibronectin antibodies increased at p9 while that against TRA-1-60 and VCAM-1 antibodies began to decrease at p6 until p9. Conclusions: These results suggest that HUC change some of their stem cell characteristics during in vitro culture. Development of culture system might be needed for the maintenance of characteristics.
Tak, Min-Jin;Jung, Il-Kook;Kim, Dae-Keun;Jung, Han-Sol;Lee, Chang-Hyun
Journal of Physiology & Pathology in Korean Medicine
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v.21
no.1
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pp.136-144
/
2007
Biotae Orientalis has been widely used for treatment of relaxion of smooth muscle, gastrointestinal hemorrhage and alopecia in Oriental Medicine. This experiment examined the effect of an extracts, obtained from the acetone and MeOH extracts of dried or fresh Biotae Orientalis, on hair growing activity of the C57BL/6N mice after topical application to skin. First, We examined on hair growth activity of extracts of Biotae Orientalis compare to control and 1% minoxidil groups. Second, We investigated on the number of hair follicle and mast cells after topical application of extracts of the Biotae Orientalis to skin for 16 day. Third, We investigated immunoreactive density of vascular endothelial growth factor(VEGF), protein kinase C-${\alpha}$(PKC-${\alpha}$) and stem(mast) cell factor(SCF) in skin of C57BL/6N mice by immunohistochemical methods. I fourth investigated changes of subpopulation of splenocytes and thymocytes in C57BL/6N mice for 16day using laser flow cytometry. The results were as follows : Hair growing effect of acetone and MeOH extracts of dried and fresh Biotae Orientalis was observed in 70%, 90% and 60% in hair removed skin area in 16 day respectively. Immunoreactive density of VEGF and PKC-${\alpha}$ in skin of experimental groups was weakly stained compare to control group in 10 day. Immunoreactive density of stem cell factor in skin of experimental group was heavily stained compare to control group in 10 day. Splenic TH/TC Iymphocytes of lived MeOH extracts group significantly increased compare to control group. TH cells in thymic T lymphocytes were increased compare to control group. These experiment suggest that acetone and MeOH extracts of Biotae Orientalis may be used for topical treatment of alopecia areata.
Aim of the study: An alternative source of adult stem cells that could be obtained in large quantities, under local anesthesia, with minimal discomfort would be advantageous. Adipose tissue could be processed to obtain a fibroblast-like population of cells or adipose tissue-derived stromal cells (ATSCs). This study was performed to confirm the availability of ATSCs in bone tissue engineering. Materials amp; Methods: In this study, adipose tissue-derived mesenchymal stem cell was extracted from the liposuctioned abdominal fat of 24-old human and cultivated, and the stem cell surface markers of CD 105 and SCF-R were confirmed by immunofluorescent staining. The proliferation of bone marrow mesenchymal stem cell and ATSCs were compared, and evaluated the osteogenic differentiation of ATSCs in a specific osteogenic induction medium. Osteogenic differentiation was assessed by von Kossa and alkaline phosphatase staining. Expression of osteocyte specific BMP-2, ALP, Cbfa-1, Osteopontin and osteocalcin were confirmed by RT-PCR. With differentiation of ATSCs, calcium concentration was assayed, and osteocalcin was evaluated by ELISA (Enzyme-linked immunosorbant assay). The bone formation by 5-week implantation of HA/TCP block loaded with bone marrow mesenchymal stem cells and ATSCs in the subcutaneous pocket of nude mouse was evaluated by histologic analysis. Results: ATSCs incubated in the osteogenic medium were stained positively for von Kossa and alkaline phosphatase staining. Expression of osteocyte specific genes was also detected. ATSCs could be easily identified through fluorescence microscopy, and bone formation in vivo was confirmed by using ATSC-loaded HA/TCP scaffold. Conclusions: The present results show that ATSCs have an ability to differentiate into osteoblasts and formed bone in vitro and in vivo. So ATSCs may be an ideal source for further experiments on stem cell biology and bone tissue engineering.
Stable Compression Fractures(SCF hereafter) are most often caused by trauma such as traffic accidents. These SCFs usually occur in the thoracic and lumbar regions of the spinal cord. Human life spans have increased as a result of medical advances, which in turn has led to an increase in the elderly population. SCFs are divided into the categories of stable and unstable. The categories are determined through X-rays and P/Ex tests. The D.I.T.I. is then used to diagnose the severity and prognosis of the fracture injuries, and it allows an objective evaluation of the symptoms. This author researched 40 patients who were treated at Wonkwang University Oriental Medical Center from October 1995 to December 1996. The patients were diagnosed by X-ray as having SCFs and treated primarily with acupuncture. Both the changes in D.I.T.I. and patients' self-diagnoses of their conditions before and after treatment yielded the meaningful results which have been presented here. 1. 75% of those who suffer from Stable Compression Fractures are 60 years of older. Of those, the ratio of male to female is 1 to 4. This is due in part to the fact that many elderly women seek Oriental medicine treatment for conditions associated with advanced age. 2. 53.3% of these injuries occurred between T11 and L2. 3. 65% of patients were hospitalized for 10 to 29 days, and then able to be treated on an outpatiens basis. 4. The D.I.T.I. results showed 50% below $0.2^{\circ}C$, 30% between 0.3 and $0.5^{\circ}C$, 10% between 0.5 and $0.7^{\circ}C$, 10% between 0.7 and $0.9^{\circ}C$, and 0% over $0.9^{\circ}C$. 5. The results of treatment using Modified Evaluation System in Thoracolumbar Vertebral Fractures Method showed that 55% of patients achieved excellent recovery and 40% achived good recovery. After comparing the D.I.T.I. results before and after treatment, we found 50% of patients showed excellent recovery $({\Delta}T{\leq}0.2)$ and 40% showed good recovery$(0.2<{\Delta}T{\leq}0.5)$.
Lee, C.K.;Moore, K.;Scales, N.;Westhusin, M.;Newton, G.;Im, K.S.;Piedrahita, J.A.
Asian-Australasian Journal of Animal Sciences
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v.13
no.5
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pp.587-594
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2000
At present embryonic stem (ES) cells with confirmed pluripotential properties are only available in the mouse. Recently, we were able to isolate, culture and genetically transform primordial germ cell (PGC)-derived cells from pig embryos and demonstrate their ability to contribute to chimera development in the pig. In order to determine whether the system we developed could be used to isolate embryonic germ (EG) cells from other mammalian species, we placed isolated PGCs from cattle, goats, rabbits and rats in culture. Briefly, PGCs were isolated from fetuses of cow (day 30-50), goat (day 25), rabbit (day 15-18) and rat (day 11-12), and plated on STO feeder cells in Dulbecco's modified Eagle's medium (DMEM): Ham's F10 medium (1:1) supplemented with 0.01 mM nonessential amino acids, 2 mM L-glutamine, 0.1 mM $\beta$ - mercaptoethnol, soluble recombinant human stem cell factor (SCF; 40ng/ml), human basic fibroblast growth factor (bFGF; 20ng/ml) and human leukemia inhibitory factor (LIF; 20ng/ml). For maintenance of the cells, colonies were passed to fresh feeders every 7-10 days. In all species tested, we were able to obtain and maintain colonies with ES-like morphology. Their developmental potential was tested by alkaline phosphatase (AP) staining and in vitro differentiation assay. For genetic transformation, cells were electroporated with a construct containing the green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter. GFP-expressing colonies were detected in cattle, rabbits and rats. These results suggest that PGC-derived cells from cattle, goats, rabbits and rats can be isolated, cultured, and genetically transformed, and provide the basis for analyzing their developmental potential and their possible use for the precise genetic modification of these species.
Proceedings of the Korean Society of Crop Science Conference
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2017.06a
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pp.141-141
/
2017
The ubiquitin-proteasome pathway is the major regulatory mechanism in a number of cellular processes for selective degradation of proteins and involves three steps: (1) ATP dependent activation of ubiquitin by E1 enzyme, (2) transfer of activated ubiquitin to E2 and (3) transfer of ubiquitin to the protein to be degraded by E3 complex. F-box proteins are subunit of SCF complex and involved in specificity for a target substrate to be degraded. F-box proteins regulate many important biological processes such as embryogenesis, floral development, plant growth and development, biotic and abiotic stress, hormonal responses and senescence. However, little is known about the F-box genes in wheat. The draft genome sequence of wheat (IWGSC Reference Sequence v1.0 assembly) used to analysis a genome-wide survey of the F-box gene family in wheat. The Hidden Markov Model (HMM) profiles of F-box (PF00646), F-box-like (PF12937), F-box-like 2 (PF13013), FBA (PF04300), FBA_1 (PF07734), FBA_2 (PF07735), FBA_3 (PF08268) and FBD (PF08387) domains were downloaded from Pfam database were searched against IWGSC Reference Sequence v1.0 assembly. RNA-seq paired-end libraries from different stages of wheat, such as stages of seedling, tillering, booting, day after flowering (DAF) 1, DAF 10, DAF 20, and DAF 30 were conducted and sequenced by Illumina HiSeq2000 for expression analysis of F-box protein genes. Basic analysis including Hisat, HTseq, DEseq, gene ontology analysis and KEGG mapping were conducted for differentially expressed gene analysis and their annotation mappings of DEGs from various stages. About 950 F-box domain proteins identified by Pfam were mapped to wheat reference genome sequence by blastX (e-value < 0.05). Among them, more than 140 putative F-box protein genes were selected by fold changes cut-offs of > 2, significance p-value < 0.01, and FDR<0.01. Expression profiling of selected F-box protein genes were shown by heatmap analysis, and average linkage and squared Euclidean distance of putative 144 F-box protein genes by expression patterns were calculated for clustering analysis. This work may provide valuable and basic information for further investigation of protein degradation mechanism by ubiquitin proteasome system using F-box proteins during wheat development stages.
Kim, Myoung-Joo;Shon, Hye-Jin;Baek, So-Young;Lee, Kang-Eun;Lee, Young-Joon;Lee, Hyun-Ah
IMMUNE NETWORK
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v.6
no.3
/
pp.154-162
/
2006
Background: Dendritic cell (DC)-based cancer immunotherapy is studied for several years. However, it is mainly derived from autologous PBMC or leukapheresis from patient, which has limitations about yield and ability of DC production according to individual status. In order to solve these problems, inquiries about allogeneic DCs are performed but there are no preclinical trial answers for effect or toxicity of allogeneic DC to use for clinical trial. In this study, we compared the anti-tumor effect of allogeneic and autologous DCs from mouse bone marrow stem cells in mouse metastatic melanoma model. Methods: B16F10 melanoma cells ($5{\times}10^4$/mouse) were injected intravenously into the C57BL/6 mouse. Therapeutic DCs were differentiated from autologous (C57BL/6: CDC) or allogeneic (B6C3F1: BDC) bone marrow stem cells with GM-CSF, SCF and IL-4 for 13days and pulsed with B16F10 tumor cell lysate (Blys) for 18hrs. DC intra-peritoneal injections began on the 8th day after the tumor cell injection by twice with one week interval. Results: Anti-tumor response was observed by DC treatment without any toxicity especially in allogeneic DC treated mice (tumor burden score: $2.667{\pm}0.184,\;2.500{\pm}0.463,\;2.000{\pm}0.286,\;1.500{\pm}0.286,\;1.667 {\pm}0.297$ for saline, CDC/unpulsed-DC: U-DC, CDC/Blys-DC, BDC/U-DC and BDC/Blys-DC, respectively). IFN-${\gamma}$ secretion was significantly increased in allogeneic DC group stimulated with B16F10 cell lysate ($2,643.3{\pm}5,89.7,\;8,561.5{\pm}2,204.9.\;6,901.2{\pm}141.1pg/1{\times}10^6$ cells for saline, BDC/U-DC and BDC/Blys-DC, respectively) with increased NK cell activity. Conclusion: Conclusively, promising data was obtained that allogeneic DC can be used for DC-based cancer immunotherapy.
One of the most extensively studied populations of multipotent adult stem cells are mesenchymal stem cells (MSCs). MSCs derived from the human umbilical cord vein (HUC-MSCs) are morphologically and immunophenotypically similar to MSCs isolated from bone marrow. HUC-MSCs are multipotent stem cells, differ from hematopoietic stem cells and can be differentiated into neural cells. Since neural tissue has limited intrinsic capacity of repair after injury, the identification of alternate sources of neural stem cells has broad clinical potential. We isolated mesenchymal-like stem cells from the human umbilical cord vein, and studied transdifferentiation-promoting conditions in neural cells. Dopaminergic neuronal differentiation of HUC-MSCs was also studied. Neural differentiation was induced by adding bFGF, EGF, dimethyl sulfoxide (DMSO) and butylated hydroxyanisole (BHA) in N2 medium and N2 supplement. The immunoreactive cells for $\beta$-tubulin III, a neuron-specific marker, GFAP, an astrocyte marker, or Gal-C, an oligodendrocyte marker, were found. HUC-MSCs treated with bFGF, SHH and FGF8 were differentiated into dopaminergic neurons that were immunopositive for tyrosine hydroxylase (TH) antibody. HUC-MSCs treated with DMSO and BHA rapidly showed the morphology of multipolar neurons. Both immunocytochemistry and RT-PCR analysis indicated that the expression of a number of neural markers including NeuroD1, $\beta$-tubulin III, GFAP and nestin was markedly elevated during this acute differentiation. While the stem cell markers such as SCF, C-kit, and Stat-3 were not expressed after neural differentiation, we confirmed the differentiation of dopaminergic neurons by TH/$\beta$-tubulin III positive cells. In conclusion, HUC-MSCs can be differentiated into dopaminergic neurons and these findings suggest that HUC-MSCs are alternative cell source of therapeutic treatment for neurodegenerative diseases.
In the development of enteral foods for the patients with diarrhea, soybean hull, by-products of soybean processing, was used to prepare crude dietary fiber extracts (soybean hull fiber, SHF). Total dietary fiber content of SHF is 85% and their composition are 86.1% cellulose, 8.1% hemi-cellulose, and 4.7% lignin. The effects of SHF on the prevention of diarrhea were studied in animal. Spraque-Dawley (SD) rats were fed AIN93G diets containing 5% dietary fiber for 3days simultaneously inducing diarrhea with the phenolphthalein Mg citrate solution. On day 4, feces were collected at different time point. Dietary fibers used for the animal study were SHF, soybean cotyledon fiber (SCF), psyllium husk fiber (PHF), and chicory fiber (CF). ${\alpha}$-cellulose was used as a control. Body weight gain, calorie consumed and food efficiency ratio among the experimental groups were not different. However, water content in the feces of SHF group was significantly lower by 10%, compared with other groups at 24hrs. time point. This effect was even greater in the feces collected later than 24 hrs. time point. SHF seems to have a greater effects on slow the symptom of diarrhea. Based on the previous results, enteral food enriched with SHF were prepared and its effect was compared with other commercially available products from domestic or imported ones. Weight changes among experimental groups were not different, but the moisture content of feces consumed SHF enriched products were lower than that of other products. Approximately 10% decrease in water content was observed from feces collected at 24 hr time point. According to the sensory evaluation, overall acceptability of the enteral food enriched with SHF was 3.24 out of 5 indicating that taste of this product is acceptable.
Osteoporosis is one of the major health problem affecting postmenopausal women. Estrogen deficiency results in an increase in bone turnover, lead to bone resorption and an increase risk of fracture. The aim of this study was to evaluate the effects of Capsosiphon fulvecense extract (SCF) on the collagen content of the connective tissues and alkaline phosphatase activity of serum in ovariectomized estrogen-deficient rats. Three groups were surgically ovariectomized (OVX). The fourth group was sham operated. Sprague-Dawley female rats were randomly assigned to the following groups : sham-operated rats (Sham), ovariectomized control rats (OVX-control), ovariectomized rats supplemented with CsF at 50mg/kg body wt (OVX-CSF50) and ovariectomized rats supplemented with CsF at 200mg/kg body wt (OVX-CSF200). The Capsosiphon fulvecense extracts were orally administrated at 1mL per day. The ovariectomy caused a decreasing in the levels of collagen content in bone, cartilage, skin and lung tissues. However CSF groups, supplementation with Capsosiphon fulvecense extract, were increased the level of collagen content in bone, cartilage, skin and lung tissues than OVX-control group. Alkaline phosphatase activity also were increased and calcium levels were decreased than OVX-control on serum. These results suggest that Capsosiphon fulvecense supplementation prevents postmenopausal bone loss, thus it may be used possibly to improve the quality of life in menopausal women.
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