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마우스 골수세포 배양시 Prostaglandin E_2가 파골세포-유사세포 생성에 미치는 영향에 관한 연구
고성희,김관식,정동균 대한구강생물학회 1990 International Journal of Oral Biology Vol.14 No.2
Osteoclasts are primary cells responsible for bone resorption. It has been reported that osteoclasts are formed by fusion of mononuclear precursors derived from hematopoietic progenitor cells. Prostaglandin E_2(PGE_2) is known to be a potent bone resorbing agent, but its mechanisms are not known. This experiment was performed to study the effect of PGE_2 and calcitonin on the generation of osteoclasts from their precursor cells. The bone marrow cells were prepared from 7 to 9 week-old male mice. The femur and tibia were dissected aseptically and the marrow cavity was flushed with 1ml α-minimum essential medium by slow injection. The collected marrow cells were cultured at 1.5∼2.0×10^6 cells/well in 24-well plate for 5, 8 and 11 days. In experimental group, PGE_2(10^-5,10^-6,10^-7,10^-8M) and calcitonin(5, 30, 90ng/ml) were added. After cultures, staining for tartrate-resistant acid phosphatase(TRACP)-marker enzyme of osteoclast-was performed according to the modified method of Burstone. The TRACP-positive multinucleated cells, which have 3 or more nuclei were counted, The observed results were as follows. 1. In control group, TRACP-positive mononuclear cells were present, but no TRACP-positive multinucleated cells appeared. 2. In 8 days of culture, PGE_2 increased significantly the number of osteoclast-like cells in a dose-dependent manner in the mouse marrow cell cultures. 3. In 5, 8 and 11 days of culture with PGE_2(10^-6M), the number of osteoclast-like cells was increased up to 8 day and decreased thereafter. 4. In 8 days of culture, calcitonin decreased significantly the number of osteoclast-like cells induced by PGE_2(10^-6M) in a dose-dependent manner.
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고추수침엑스가 Streptococcus mutans B-13의 세포외 다당류 합성에 미치는 영향에 관한 전자현미경적 연구
고재승,정태영,이종흔,정동균,김각균 대한구강생물학회 1980 International Journal of Oral Biology Vol.4 No.1
Streptococcus mutans B-13 was cultured in sucrose broth containing water-extract of capsicum and the effect of the extract was observed with the aid of electron microscope, in an effort to elucidate the anti-cariogenic effect of capsicum in white rats observed during the previous experiment. Growth curve was also obtained in the presence or in the absence of the extract. The results were as follows : 1. S. mutans B-13 produce no extracellular polysaccharide in glucose broth. 2. In sucrose broth, extracellular polysaccharides with two components were produced ; that is, electron-dense, globular and relatively homogenous material and widespread fibrillar component. 3. In sucrose broth containing water-extract of capsicum, production of extracellular polysaccharide was almost completely suppressed. Growth was also influenced by the extract. 4. It seems that certain material which is capable of suppressing the production of extracellular polysaccharide and of influencing growth is present in water-extract of capsicum and that anti-cariogenic effect of capsicum is probably related to this unidentified material.
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골흡수 기전에 관한 연구 : 파골세포의 활성화 기전 MECHANISM OF OSTEOCLAST ACTIVATION
정동균,고재승,김관식,김각균,민병무,김세원 대한구강생물학회 1989 International Journal of Oral Biology Vol.13 No.1
Although the osteoclast has long been recognized to be the cell responsible for bone resorption, little is known of the mechanisms by which its activity is controlled. Recently, it has been suggested that osteoblasts ─ the bone-forming cells ─ seem to be the target cells of PTH, the bone-resorbing hormone, and mediate osteoclastic bone resorption by producing the coupling factor(s). Because bone tissue consists of several types of cells, isolation of distinct bone cell populations is prerequisite for studying the mechanism of bone resorption in cellular level. This experiment was performed ⅰ) to isolate the metabolically distinct bone cell populations from fetal rat calvaria by sequential enzyme digestion and biochemical characterization and ⅱ) to identify the factor(s) produced by osteoblast that stimulate resorption employing organ culture of bone. Calvaria from rat fetus at 19 day of gestation, were sequentially digested by enzyme solution consisted of collagenase, trypsin and EDTA for 10 (population I), 10(II), 10(III), 20(IV) and 20 minutes (V). Each bone cell population was primarily cultured for 6-7 days and effects of PTH, calcitonin and PGE_2 on acid and alkaline phosphatase activity and cAMP level were determined. Basal level of acid phosphatase in populations released early were higher than late population. In contrast, basal level of alkaline phosphatase was reversed. PTH(0.4 unit/ml) increased the acid phosphatase activity only in population I with no effect on alkaline phosphatase. Calcitonin(150ng/ml) had no effect on acid and alkaline phosphatase activity in all bone cell populations. cAMP level of population IV and V were increased by PTH significantly while CT had no effect in all bone cell populations at all. PGE_2 increased cAMP in all populations, the acid phosphatase activity in population I and alkaline phosphatase activity in population IV and V. Taken together, these results indicate that population IV and V express typical osteoblastic phenotype while population I revealed some characteristics of osteoclast. Bone cell population IV and V were incubated with fresh MEM or MEM containing 0.4U/ml PTH for 2 hours. After 2 hour-incubation, both the control-conditioned media(control-CM) or PTH-conditioned media(PTH-CM) were collected. Both conditioned media were lyophyllized and redissolved as 2 fold concentrate. Ulnae and radii were removed from 19-day old fetal rats, prelabelled by subcutaneous injection of 200μCi ^45CaCl_2 into their mothers on the 17th day of gestation. After 24 hours, media was changed with fresh BGJb media or BGJb media containing 300μl of control-CM or PTH-CM and cultured for 5 days. Effects of control-CM or PTH-CM were observed by the ratios of %-release of ^45Ca between paired control and experimental group. Control-CM obtained from population IV and V had no or very little effect on bone resorption but PTH-CM obtained from population IV and V increased the ^45Ca release significantly after 3 and 5 days of culture. This result provides the evidence indicating that osteoblastic cells mediate osteoclastic bone resorption stimulated by PTH.
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흰쥐 해마박편에서 콜린성 수용체와 glutamate 유리와의 상호관계에 관한 연구
신동인,김형룡,고성희,김관식,정동균 대한구강생물학회 1990 International Journal of Oral Biology Vol.14 No.2
Present study was performed to clarify the effect of cholinergic agents on the release of glutamic acid employing hippocampal slices. Hippocampal slices(300∼400㎛ thick) were prepared by the method of Kim et al.(1988) and pre-equilibrated in Krebs-bicarbonate medium(KBM, pH 7.4) for 1hr at 37℃. Pre-equilibrated slices were incubated in fresh KBM and then potssium(50mM)-containing KBM for 5 min period. Basal and potassium-induced release of GABA and glutamic acid were determined from recovered medium by HPLC. After 30 min resting period, slices were reincubated in cholinergic agents-containing KBM and cholinergic agent plus potassium-containing medium consecutively for 5 min period each to investigate the effect of cholinergic agent on basal or potassium-induced glutamic acid release from hippocampal slices. The observed results were as follows: 1. The release of glutamic acid induced by the first and second 5 min-exposure of 50mM potassium was 139.7±14.05 nmol and 114.5±10.01 nmol, respectively. When compared with released amounts of glutamic acid during the corresponding spontaneous periods, these were 5.3 and 5.6-fold increase respectively. 2. Acetylcholine(10-1000μM) inhibited potassium-induced glutamic acid release in dose-dependent manner. 3. The inhibition of glutamic acid release caused by acetylcholine(1mM) was antagonized by atropine(50μM) but not by mecamylamine(50μM).
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cAMP 농도에 영향을 미치는 수종약물이 파골세포형성에 미치는 영향
소영,고성희,백정화,김관식,정동균 대한구강생물학회 1992 International Journal of Oral Biology Vol.16 No.2
To study the effect of cAMP on the generation of osteoclasts from their precursor cells, the bone marrow cells were isolated from 7 to 9 week-old male mice. The femur and tibia were dissected aseptically and the marrow cavity was flushed with 1 ㎖ of α-minimum essential medium by slow injection. Collected marrow cells were cultured at 1.5-2.0×10^6 cells/well in 24-well plate for 8 days. In experimental group, PGE_2(5×10^-6, 10^-5M), forskolin(10^-5M) or IBMX(10^-4, 10^-5M) were added singly or in combination from the 1st day culture. After cultures, staining for tartrate-resistant acid phosphatase(TRACP)-marker enzyme of osteoclast-was performed according to the modified method of Burstone. The TRACP-positive multinucleated cells(MNC), which have 3 or more nuclei, were counted. The observed results were as follows. 1. In control group. TRACP-positive mononuclear cells were present, but no TRACP-positive multinucleated cells appeared. 2. PGE_2(5×10^-6M) or forskolin(10^-5M) significantly stimulated the formation of TRACP-positive MNC. Moreover, forskolin potentiated the TRACP-positive MNC formation induced by PGE_2 when added simultaneously. 3. IBMX(10^-4, 10^-5M), when added alone, significantly stimulated the formation of TRACP-positive MNC. However, IBMX(10^-5M) added in combination of PGE_2(10^-5M) partially inhibited the TRACP-positive MNC formation induced by PGE_2.
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Platelet-Derived Growth Factor가 백서두개관 세포군의 증식 및 교원합성에 미치는 영향
김기수,고성희,백정화,민병무,김관식,정동균 대한구강생물학회 1991 International Journal of Oral Biology Vol.15 No.2
To study the effect of platelet-derived growth factor(PDGF) on the replication and collagen synthesis of rat calvarial cells, five bone cell populations(I-V) were prepared from fetal rat calvaria by sequential enzyme digestion. After primary culture for 6-7 days, each bone cell population was collected and then population Ⅰ and Ⅱ, Ⅳ and Ⅴ were pooled together. And the cells were resuspended at 6-8×10^4 cells/㎝^2 and cultured for 2-3 days. The medium was changed to serum-free medium prior to addition of growth factor. The effect of PDGF on the cell proliferation was measured by the incorporation of [^3H]thymidine into DNA. Protein synthesis was determined by measurement of [^3H]proline incorporation into collagenase-digestible protein(CDP) and noncollagenous protein(NCP) according to the method of Peterkofsky and Die-gelmann(1971). The observed results were as follows. 1. PDGF at 10 ng/㎖ significantly increased the [^3H]thymidine incorporation into DNA in all bone cell populations. 2. PDGF at 30 ng/㎖ significantly increased the synthesis of NCP in population Ⅰ, Ⅱ and Ⅳ, Ⅴ. 3. PDGF had no effect on the synthesis of CDP but percent collagen synthesis was decreased significantly in population Ⅳ, Ⅴ. Taken together, the increase of protein synthesis by PDGF in rat calvarial cells was due to the incraese of NCP synthesis.
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