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5 "Bone marrow stromal cell"
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Original Articles
Search for "Proximal Histidine" of Thyroperoxidase Using Site Directed Mutagenesis.
Won Bae Kim, Young Kee Shong
J Korean Endocr Soc. 2003;18(4):371-378.   Published online August 1, 2003
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BACKGROUND
Thyroperoxidase (TPO), a transmembrane heme containing glycoprotein, catalyzes iodide organification and thyroid hormone synthesis. It is a single peptide making a loop with more than one disulfide bond. The tertiary conformational structure is essential for its enzymatic activity and immunogenicity. The proximal histidine is thought to play a major role in enzymatic activity since it is linked to the iron center of the heme. The crystal structure of TPO has not yet been reported, but some have suggested histidine 407 be a putative proximal histidine based on comparison of a.a. sequence for TPO and that for myeloperoxidase. METHODS: The putative histidine 407 and nearby histidine 414 were mutated to arginine to verify their role as the proximal histidine. Using site directed mutagenesis of wild type, human TPO cDNA, mutants H407R and H414R were made. Mutant cDNAs were transiently transfected into COS-7 cells, and the TPO enzyme activities were measured by guaiacol assay. Four cysteine residues around the putative proximal histidines were mutated to serine and their enzymatic activities were measured to check if they were involved in the formation of intra-molecular disulfide bonds. RESULTS: TPO protein expression of H407R- and H414R- transfected cells was confirmed by Western blot, using Hashimoto's IgG as primary antibody. Both the mutants H407R and H414R showed significant peroxidase enzymatic activity, although lower than those of the wild type. None of the cysteine mutants, C375S, C389S, C598S, and C655S, were detected by Hashimoto's IgG ordisplayed any enzymatic activity. CONCLUSION: These data suggest that neither histidine 407 nor histidine 414 functions as the "proximal histidine" in human TPO. All the cysteine residues checked (375, 389, 598, 655) might be involved in the formation of disulfide bonds in TPO molecules, but this hypothesis could not be confirmed. A further search for the other putative histidine residues using the same strategy is needed to define the structure-function relationship in the human TPO molecule.
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The Effects of Aging on the Proliferation and Differentiation of Osteoblasts from Human Mesenchymal Stem Cells.
Ki Hyun Baek, Hyun Jung Tae, Ki Won Oh, Won Young Lee, Chung Kee Cho, Soon Yong Kwon, Moo Il Kang, Bong Yun Cha, Kwang Woo Lee, Ho Young Son, Sung Koo Kang, Choon Choo Kim
J Korean Endocr Soc. 2003;18(3):296-305.   Published online June 1, 2003
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BACKGROUND
Osteoblasts originate from osteoprogenitor cells in bone marrow stroma, termed mesenchymal stem cells (MSCs) or bone marrow stromal cells. Each MSC forms colonies (colony forming units-fibroblasts [CFU-Fs]) when cultured ex vivo. There are some reports about the age-related changes of the number and osteogenic potential of osteoprogenitor cells, but any relationship has not been clearly established in humans. In this study, we counted MSCs using CFU-Fs count and examined the proliferative capacity and differentiation potential of osteoprogenitor cells. Finally, we analyzed how these parameters varied with donor age. METHODS: Bone marrow was obtained from the iliac crest of young (n=6, 27.2+/-8.6 years old) and old (n=10, 57.4+/-6.7 years old) healthy donors. Mononuclear cells, including MSCs, were isolated and cultured in osteogenic medium. In primary culture, we compared the colony-forming efficiency of MSCs between the two groups and determined the matrix calcification. When primary culture showed near confluence, the cells were subcultured. Alkaline phosphatase activity, osteocalcinexpression by RT-PCR and proliferative potential by MTT assay were examined by the time course of secondary culture. RESULTS: At the 15th day of primary culture, the mean number of CFU-Fs was significantly higher in the younger donors (young: 148.3+/-28.9, old: 54.3+/-9.1, p=0.02) and the mean size of CFU-Fs was also larger in the younger donors than the older donors. However, matrix calcification was not different between the two groups (young: 103.6+/-50.6, old: 114.0+/-56.5, p=NS). In secondary culture, alkaline phosphatase activities were significantly lower in the older donors. The younger donors showed peak alkaline phosphatase activity at day 10, while the older donors didn't showed a remarkable peak (young: 935.5+/-115.0U/mg, old: 578.4+/-115.7U/mg, p<0.05). Total cell number as a proliferative index increased progressively during the secondary culture and a significantly greater cell number was noted in the younger donors. Osteocalcin expression was generally upregulated in the younger donors, but this was not statistically significant. CONCLUSION: Our study shows that the number of osteoprogenitor cells is decreased during aging and that the proliferative capacity and differentiation potential of osteoprogenitor cells seem to be reduced during aging.
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Randomized Controlled Trial
Effect of Dexamethasone and 1,25(OH)2D3 on Proliferation and Osteogenic Differentiation of Cultured Human Bone Marrow Stromal Cells.
Hye Soo Kim, Il Woo Lee, Jong Min Lee, Chang Hwan Han, Jin Hyung Sung, Min Young Park, Gil Son Khang, Hai Bang Lee
J Korean Endocr Soc. 2002;17(2):206-217.   Published online April 1, 2002
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AbstractAbstract PDF
BACKGROUND
It is crucial, in the case of regenerating bone by tissue-engineering technique, that osteoblast progenitors are proliferated and induced to differentiate to osteoblasts sequentially at the proper time. Osteoblasts can be obtained from bone itself or from osteoblast progenitors in bone marrow, even though the amount of human marrow stromal cells in marrow aspirate is usually scanty. These cells, however, have been known demonstrate the potential to easily proliferate and differentiate in osteoblasts, chondroblasts or adipocytes according to different microenvironmental factors. We evaluated the effect of dexamethasone and 1,25(OH)2D3 on the proliferation, differentiation, and mineralization of human marrow stromal cells in vitro. METHODS: We used twelve bone marrow aspirates obtained from different healthy bone marrow donors. Culture plates were randomly divided into the following four experimental groups; group 1 was cultured with control medium only, group 2 with control medium containing 1,25(OH)2D3, group 3 with control medium containing dexamethasone, and group 4 with control medium containing both 1,25(OH)2D3 and dexamethasone. 3H-thymidine uptake, protein content of cell lysates, alkaline phosphatase activities and alkaline phosphatase histochemistries were measured. Alizarin Red-S staining and quantification of dissolved dye were also performed. RESULTS: Combined stimulation of marrow stromal cells with both 1,25(OH)2D3 and dexamethasone was found to be effective to maintain stable long-term culture of the cells and to increased differentiation and mineralization of the cells. Synthesis and mineralization of matrix were highest when the cells were stimulated with 1,25(OH)2D3 alone during the early culture phase. However, 1,25(OH)2D3 shortened the lifespan of the cells. Interestingly, mineralization was higher in female donor cells than in male donor cells when stimulated with dexamethasone alone or with both dexamethasone and 1,25(OH)2D3. Neither 1,25(OH)2D3 nor dexamethasone affected cell proliferation. CONCLUSION: Our results suggest that the synergistic effect of dexamethasone and 1,25(OH)2D3 is important in maintaining long-term culture and differentiation of human marrow stromal cells. It is preferable to administer 1,25(OH)2D3 after the attachment of cultured osteoblasts to biomaterials has been established, since it could shorten cell survival despite the great increase of mineralization at the early culture phase.
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Original Articles
The Effect of Hematopoietic Stem Cell Transplantation in the Origin and the Osteoblastic Differentiation of the Human Bone Marrow Stromal Cell.
Moo Il Kang, Seong Won Cho, Eun Sook Oh, Ki Hyun Baik, Won Young Lee, Ki Won Oh, Hye Soo Kim, Je Ho Han, Kun Ho Yoon, Bong Yun Cha, Kwang Woo Lee, Ho Young Son, Sung Koo Kang, Choon Choo Kim
J Korean Endocr Soc. 2000;15(4-5):571-581.   Published online January 1, 2001
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  • 17 Download
AbstractAbstract PDF
BACKGROUND
Bone marrow transplantation is the treatment of choice for patients with certain- hematological malignancies, many of whom will survive many years thereafter. Bone disease is a potential longterm complication. But, little is known about the effects of bone marrow transplantation on bone. METHODS: In this study, bone marrow was obtained from healthy donor and transplant recipients. Then mononuclear cells including marrow stromal cells were isolated and cultured. At near confluence, bone marrow stromal cells were subcultured. Thereafter alkaline phosphatase activities of each group were measured by time course of secondary culture. We also analysed the origin of marrow stromal cells by the polymerase chain reaction using YNZ 22 minisatellite probe. RESULTS: l. Cells cultured in our system showed the characteristics of marrow stromal cells differentiated to osteoblasts. They were in fibroblast-like spindle shape and positive to alkaline pbosphatase histochemistry and Von Kossa histochemistry in secondary cultures. 2. The time required for the near confluence in the primary culture was 15 days and 22.9 days on the average in healthy donors and transplant recipients, respectively (p=0.003). 3. In secondary cultures, healthy donors and transplant recipients showed peak alkaline phosphatase activity at 10 days and 17 days, respectively (p=0.031). Alkaline phosphatase activity was lower in BMT recipients than in healthy donors during the whole period of secondary cultures. 4. In polymerase chain reaction analysis using YNZ 22 minisatellite probe, bone marrow stromal cells were of recipient origin. CONCLUSION: Recipient-derived bone marrow stromal cells may be damaged secondary to the effect of chemotherapy, glucocorticoid & total body irradiation which have given before bone marrow transplantation. So it may affect the differentiation of bone marrow stromal cells into the osteoblasts.
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Effects of Thyroid Hormone on Preduction of Interleukin-6 and Interleukin-11 in Human Bone Marrow Stromal Cells.
Chul Hee Kim, Dong Kwan Kim, Hong Kyu Kim, Young Ki Song, Ki Soo Kim
J Korean Endocr Soc. 1997;12(4):557-564.   Published online January 1, 2001
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  • 19 Download
AbstractAbstract PDF
BACKGROUND
It is well known that excessive thyroid hormone in the body is associated with bone loss. However, the mechanism by which thyroid hormone affects bone cell metabolism remains unclear. It has been shown that thyroid hormones stimulate osteoclastic bone resorption indirectly via some unknown mediators secreted by osteoblasts, This study was undertaken to determine if interleukin-6 (IL-6) or interleukin-11 (IL-l1) could be the mediator (s) of thyroid hormone-induced bone loss. METHODS: We treated primary cultured human bone rnarrow stromal cells with 3,5,3-triiodo-thyronine (T) and measured basal and interleukin-l (IL-1)-stimulated IL-6/IL-ll production. We also investigated the possible modulating effect of 17B-estradiol (17B-E2.) on thyroid hormone action. RESULTS: T3 at 10 (-12) ~ 10 (-8) M concentration, significantly increased the basal IL-6 production in a dose-dependent manner, and also potentiated the stimulatory effect of IL-1 on IL-6 production. However, T failed to elicit a detectable effect on basal or IL-1-stimulated IL-11 production. Treat#ment with l7B-E2. inhibited IL-1-stimulated IL-6 production, but the effects of T3 on IL-6 production were not affected by 17/B-E. CONCLUSION: These results suggest that thyroid hormone may increase bone resorption by increasing basal IL-6 production and potentiating IL-1-induced IL-6 production from osteoblast-lineage cells, and these effects were independent of estrogen status.
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