냉동 제대혈 세포의 체외 증폭

김삼용,김철희,배광봉,김현수,박상준,김종숙,윤환중,조덕연 충남대학교 암연구소 1998 癌共同硏究所 硏究誌 Vol.2 No.1

Background : Cord blood(CB), which has no HLA restriction, is an alternative to bone marrow for hematopoietic stem cell transplantation. The use of cord blood, however, is limited by the number of progenitor/stem cells necessary to reconstitute the older child or adult. Therefore, ex vivo expansion of CB could have tremendous impact on diverse clinical settings. We studied the ex vivo expansion of isolated population of CD34_(+) cells from cryopreserved CB cells. Methods : CD34 cells were isolated from cryopreserved CB mononuclear cells. Purified cells were cultured with various combinations of hematopoietic growth factors including erythropoietin(EPO), stem cell factor(SCF), granulocyte-colony-stimulating factor(G-CSF), gra-nulocyte, macrophage-colony-stimulating factor(GM-CSF), interleukin-1β(IL-1β), 1L-3, and IL-6. After 7, 10 or 14 days of culture, the fold increases of colony-forming unit- granu-locyte, macrophage(CFU-GM), burst-forming unit-erythroid(BFU-E), colony-forming unit-mix (CFU-Mix), and high proliferative potential colony-forming cell(HPP-CFC) were evaluated. Results : Ten-day culture with the combination of EPO, SCF, G-CSF, IL-1β, and IL-3 resulted in a median of 60-fold increase of CFU-GM, which was greater than those with the combinations of less than 5 growth factors. The addition of IL-6 or GM-CSF to this combination did not enhance CFU-GM expansion. Ten-day culture was significantly superior to 7-day culture for CFU-GM expansion. Prolongation of culture to 14 days, however, revealed decreased expansion of CFU-GM compared to 10 days. BFU-E and CFU-Mix were expanded to 2~5 folds in 7-day culture with the combination of EPO, SCF, and G-CSF. Further expansion was not achieved in 10-day culture and colonies disappeared in 14-day culture. HPP-CFC was expanded to a median of 7.5 folds in 7-day culture with the combination of EPO, SCF, G-CSF, IL-1β, IL-3, and IL-6. Neither 10-day or 14 day-culture enhanced expansion of HPP-CFU. Conclusion : Cryopreserved cord blood cells maintain ex vivo expansion potential. In our system, 10-day culture with the combination consisting of EPO, SCF, G-CSF, IL-1β, and IL-3 seems to be adequate for hematopoietic progenitor/stem cell expansion from cryopreserved cord blood cells.

단기배양을 통한 말초혈액 CD34 양성세포의 체외증폭

박상준,김철희,배광봉,김현수,김종숙,윤환중,조덕연,김삼용 충남대학교 의과대학 지역사회의학연구소 1997 충남의대잡지 Vol.24 No.1

Background: It is suggested that clinical practice in the areas of bone marrow transplantation and gene therapy might rely on the ex vivo expansion of hematopoietic stem/progenitor cells. However, the condition for ex vivo expansion of hematopoietic progenitor cells is not well established. The authors pursued a series of experiments to define the proper conditions for the expansion of hematopoietic cells in the short-term liquid suspension culture of mobilized peripheral blood CD34+ cells. Methods: 1.0ml cultures were initiated with 9×10^3 PB CD34+ cells, which were isolated from PB mononuclear cells (MNCs) by high-gradient cell sorting, in 12 well plates with the various combinations of hematopoietic growth factors(HGF). The following recombinant human HGFs were used: stem cell factor(SCF) 100ng/ml, granulocyte colony-stimulating factor(G-CSF) 100ng/ml, GM-CSF(granulocyte, macrophage colony-stimulating factor) 100ng/ml, interleukin-1 beta(IL-1B) 1ng/ml, interleukin-3(IL-3) 20ng/ml, interleukin-6 (IL-6) 100ng/ml. At the end of culture, colony-forming cells were evaluated by semisolid clonogenic assay. Results: 1) Using the high-gradient magnetic sorting system, CD34^+ cells were isolated with a yield of 40 3% 2) In 7 day culture of PB CD34^+ cells(9×10^3 cells), nucleated cells expanded mean 10×10^3(range, 9 to 20×10^3) with the addition of SCF alone, 35×10^3(range, 10 to 60×10^3) with SCF plus G-CSF plus GM-CSF, and 130×10^3(range, 40 to 300×10^3) with the combination of SCF, G-CSF, IL-1, IL-3, IL-6, GM-CSF. In 14 day culture, nucleated cells expanded 10×10^3 to 1,860×10^3 with combination of human hematopoietic growth factors. 3) In 10 day culture without medium change of PB CD34^+ cells, CFU-GM numbers expanded 16. 5 fold(range, 7 to 59 fold) with the addition of SCF plus G-CSF plus Il-1 plus IL-3, 31.3 fold(range, 20.5 to 101.1 fold) with the combination of SCF, G-CSF, IL-1, IL-3, GM-CSF. In 14 day culture with or without medium change of PB CD34^+ cells was inferior to 10 day culture for CFU-GM expansion. 4) There was no significant difference for CFU-GM expansion between five growth factors(SCF,G-CSF,IL-1,IL-3,GM-CSF) and six growth factors(five growth factors plus IL-6). Conclusion: The authors could confirm that short-term suspension culture of peripheral blood CD34+ cells could expand hematopoietic progenitor cells. Ten-day culture with medium change of CD34+ cells with the addition of five growth factors, i.e. SCF, G-CSF, IL-1B, IL-3, and GM-CSF, might be the most efficient in this system.

실리콘 겔에 활성화된 복강 대식세포의 interleukin-6 및 tumor necrosis factor-α에 의한 섬유모세포 중식 자극

김환묵,한상배,이백권,이종원,한기택,천지훈 大韓成形外科學會 1998 Archives of Plastic Surgery Vol.25 No.5

Silicone gel breast implants may induce local(fibrous capsular contracture) or systemic(rheumatoid arthritis, systemic sclerosis, etc) complications. The exact mechanism of fibrous capsular contracture has not been fully understood. In the present study, we tried to find out the effect of silicone gel on the fibroblast proliferation which has been known as a major contributing factor in fibrous capsular contracture formation. In vitro, activated macrophages are known to secrete monokines which affect fibroblast proliferation and collagen synthesis. And tumour necrosis factor-α(TNF-α) and interleukin-6(IL-6), which were released by macrophages, were reported as potent stimulator of fibroblast proliferation. The goal of this study is to investigate the role of macrophages and tumour necrosis factor-αor interleukin-6 in the interaction of fibroblasts and silicone gel. We designed four groups, two experimental and two control, using Institute for Cancer Research(ICR) mouse peritioneal macrophage and silicone gel. For the preparation of the conditioned medium of macrophages, peritoneal macrophages were prepared and cultured for 24 hours on the silicone gel-coated and naked (not coated) surface [silicone gel-macrophage conditioned medium(SCM; experimental group) and normal polystyrene-macrophage conditioned medium(NCM; control group) respectively]. To correct the effect of 10% fetal bovine serum which was included in Rapid Prototyping and Manufacturing Institute (RPMI) 1640 medium and draw the effect only by macrophages, the RPMI 1640 medium with 10% fetal bovine serum was cultured by the same method on the silicone gel-coated and naked surface (silicone gel-macrophage free conditioned medium; SFM and normal polystyrene-macrophage free conditioned medium; NFM respectively). Each conditioned medium was added onto NIH 3T3 fibroblasts culture at a final 25% concentration of total culture medium and followed by the cultivation for 24 hours. For antibody neutralizing experiments, each conditioned medium was preincubated with polyclonal rabbit anti-mouse TNF-α antibody or polyclonal rat anti-mouse IL-6 antibody for 1 hour and then, conditioned medium with antibody was added to the culture medium of NIH 3T3 fibroblasts by the same method. After 24 hours cultivation, total number of viable fibroblast(cell growth), DNA synthesis and collagen synthesis of fibroblasts with each medium were measured by sulforhodamine B(SRB) assay, 3H-thymidine and 3H-proline incorporation respectively. The results were as follows: 1. In the experiment about the effect of the conditioned medium on the fibroblast activity, the experimental group(SCM), compared with the control group(NCM), showed a significant increase of the cell growth (p<0.01), a significant decrease of DNA synthesis(p<0.001), but no significant difference in the collagen synthesis. 2. In the experiment about the effect of polyclonal rabbit anti-mouse TNF-α antibody on the fibroblast activity, after the addition of antibody the experimental group, compared with the control group, showed a significant decrease of the cell growth(p<0.001), a significant increase of DNA synthesis(p<0.01), but no significant difference in the collagen syn thesis. 3. In the experiment about the effect of polyclonal rat anti-mouse IL-6 antibody on the fibroblast activity, after the addition of antibody the experimental group, compared with the control group, showed a significant decrease of the cell growth(p<0.001), a significant increase of DNA synthesis(p<0.0001), but no significant difference in the collagen synthesis. In conclusion, culture supernatants (conditioned medium) of peritoneal macrophages, activated by silicone gel, stimulate the NIH 3T3 fibroblast proliferation. TNF-α and IL-6, products of macrophage, are involved in the stimulation of NIH 3T3 fibroblast proliferation in an in vitro condition.

충돌제트의 속도변화에 따른 실험적 연구

김동균,김정환,배석태,김시범,이영호 동아대학교 생산기술연구소 2001 生産技術硏究所硏究論文集 Vol.6 No.1

The flow characteristics of impinging jet flow are affected greatly by nozzle plate to distances. An sharp edge nozzle was used to achieve uniform mean velocity at the nozzle inlet, and its diameter is 10mm(d). Therefore, the flow characteristics on the impinging jet plate can be changed largely by the control of main flow. In the parent study, we investigate the effects of main flow velocity, its variable is nozzle inlet velocity(1.5m/sec, 3m/sec, 5m/sec)

산에 의한 응고화에 영향을 미치는 인자들과 형성된 응고물의 물리학적 특징

김명환,김병용,배혁진 한국농화학회 1992 Applied Biological Chemistry (Appl Biol Chem) Vol.35 No.2

Viscosity changes of acidified milk at the various pH ranges(5.2∼4.2) was measured as a function of temperature. The rate and extent of acid-induced coagulation of milk protein were monitored by turbidity changes as a function of temperature, preheating and salt. Relative viscosities of acidified milk were also measured. The coagulation of casein occurred in a specific pH range and was accompanied by a sharp increase in viscosity at pH of 5.0∼5.2, depending on the heating temperatures. Onset pH of coagulation and maximum coagulation rate were enhanced by increasing temperatures and preheating process and reduced by addition of salt. Relative viscosity of acidified milk was reversed at the same conditions, reflecting the size of casein coagulum formed was related to the coagulation rate.

골다공증 환자 혈청이 정상인 조골세포의 성장과 분화에 미치는 영향

김진,이재훈,김경욱,이광원,한상배,김환묵 대한악안면성형재건외과학회 2002 Maxillofacial Plastic Reconstructive Surgery Vol.24 No.4

Osteoporosis is a condition in which an imbalance appears between bone resorption and formation, with bone resorption exceeding formation. Recent studies have shown that bone formation abnormalities in various forms of osteopenia result mainly from defective recruitment of osteoblastic cells. These abnormalities in osteoblast function and bone formation are associated with alterations in the expression or production of several growth factors, such as TGF-β which modulate the proliferation and activity of bone-forming cells. Bone transplantation is an absoulte requirement in several patholigical conditions. The growth factors, such as TGF-β, PDGF, were the effective in promoting growth of the bone in vitro and in animal models. We have investigated the effects of PDGF and TGF-β on the proliferation and differentiation of the normal human osteoblast in vitro culture. The normal human osteoblast from iliac bone were primarily cultured. The serums obtained from the osteoporotic patients and the normal was used to quantify PDGF and TGF-β from the osteoporotic patients serum and the normal serum. To clarify the effects of the various different the culture conditions such as 1×10 exp(4) cells/㎖, 2.5×10 exp(4) cells/㎖, 5×10 exp(4) cells/㎖, 10×10 exp(4) cells/㎖ (0.2∼2×10 exp(4) cells/well) of the osteoblast at 24 hours, 48 hours, 72 hours under 10% FBS, 10% normal human serum, 10% osteoporotic human serum, 3% normal human PRP, 3% osteoporotic human PRP. The cell proliferation and differentiation was determined by [^3H]-thymidine and SRB assay, and the magnitude of differentiation to osteoblast was confirmed by von Kossa staining and alkaline phosphatase stain and measuring the alkaline phosphatase activity during 48 hours and 72 hours. Statistical differences were evaluated using the scheffe's test. The ANOVA procedure of the SAS system. The obtained results were as follows ; 1. The age distribution of normal human was 36.9±4.4 old, osteoporotic human was 72.5±0.2 old with statistical significant difference(p<0.05). 2. The quantification of TGF-β of the normal human serum was 39658.38±11630.43 pg/㎖, the osteoporotic human serum was 30459.40±1704.92 pg/㎖ with statistical significant difference. The quantification of PDGF of the normal human serum was 3064.13±709.51 pg/㎖, the osteoporotic human serum was 2514.13±140.21 pg/㎖ with no statistical significant difference(p<0.05). 3. The DNA synthesis and protein assay of human osteoblast at 24 hours, 48 hours, 72 hours was similar increased to 10% normal human seurm, 10% osteoporotic human serum. There was no statistical significant difference between the normal human and the osteoporotic patients in 3% normal human PRP and 3% osteoporotic human PRP. 4. The optimal cell concentration was 5×10 exp(4) cells/㎖ among 1×10 exp(4) cells/㎖, 2.5×10 exp(4) cells/㎖, 5×10 exp(4) cells/㎖, and 10×10 exp(4) cells/㎖. The DNA synthesis was decreased after 72 hours in the normal human serum and PRP, the osteoportic serum and PRP. 5. The alkaline phosphatase activity was as the same result 10% FBS, 10% osteoportic serum and 10% normal human serum at 48 hours with no statistical significant, but the alkaline phosphatase activity was increased in 10% osteoportic human serum and 10% normal human serum except 10% FBS at 72 hours. From above result, the amount of TGF-β of the normal human growth factor was higher than the osteoporotic patients, but the growth factors of the osteoportic patients were enough the proliferation and differentiation of normal human osteoblasts such like the same effects of normal human growth factors.

혀에 작용하는 capsaicin에 관한 감각 정보의 특성

이배환,김기석,김기영,진춘조,나흥식,홍승길 한국심리학회 1999 한국심리학회지 생물 및 생리 Vol.3 No.1

본 연구는 혀에 작용하는 capsaicin이 유발하는 감각 정보의 특성을 알아보기 위해 수행되었다. α-chloralose로 마취한 고양이에게 기계적 자극, 미각자극, capsaicin을 혀에 도포하고, 동통 유발 물질과 capsaicin을 혀의 동맥에 주입하면서 고삭신경과 설신경에서 신경의 활동을 기록하였다. 고삭신경에서 혀에 도포한 capsaicin에 대한 반응은 미각 자극 및 혀의 동맥에 주입한 동통 유발 물질에 대한 반응과 상관이 있었다. 혀의 혈관에 주입한 capsaicin에 대한 고삭신경의 반응은 혀에 도포한 capsaicin 및 혀의 혈관에 주입한 동통 유발 물질에 대한 반응과 상관이 있었다. 설신경에서 혀에 도포한 capsaicin에 대한 반응은 동맥에 주입한 동통 유발 물질에 대한 반응과 상관이 있었다. 혀의 혈관에 주입한 capsaicin에 대한 설신경의 반응은 혀에 도포한 capsaicin 및 혀의 혈관에 주입한 동통 유발 물질에 대한 반응과 상관이 있었다. 이러한 결과는 capsaicin이 혀에 작용할 때 동통뿐만 아니라 미각에 관한 정보도 유발한다는 것을 시사한다. 이때 미각에 관한 정보는 고삭신경을 통해 중추로 전달되며, 동통에 관한 정보는 설신경을 통해 전달되지만, 동통에 관한 정보가 고삭신경을 통해 전달될 가능성도 배제하지 못한다. The present study was performed to investigate the characteristics of sensory information produced by capsaicin in the tongue. Activities of the chorda tympani(CN) and lingual nerves(LN) were recorded while taste stimuli and capsaicin were being applied topically, and capsaicin and algesics were being injected intra-arterially to the tongue of cats anesthetized with α-chloralose. Responses of the CN fibers to topically applied capsaicin were correlated with those to taste stimuli and intre-arterially injected algesics. Responses of the CN fibers to intra-arterially applied capsaicin were correlated with those to topically applied capsaicin and intra-arterially injected algesics. Responses of the LN fibers to topically applied capsaicin were correlated with those to intra-arterially injected algesics. Responses of the LN fibers to intra-arterially aplied capsaicin were correlated with those to topically applied capsaicin and intra-arterially injected algesics. These results suggest that capsaicin provoke taste as well as pain sensation in the tongue. Taste information produced by capsaicin is conveyed to the brain via the CN and pain information via the LN. However, the possibility that pain information may be conveyed via the CN can not be excluded.

전측 시개전핵의 하행성 통각 조절계에 대한 하올리브 수준의 복측 연수의 관여

이배환,홍승길,안창일,손진훈,김기석 한국심리학회 한국심리학회지 생물 및 생리 Vol.6 No.1

본 연구는 전측 시개전핵의 자극으로 인한 하행성 통각 억제 체계에 하올리브 수준의 복측 연수가 관여하는가를 알아보기 위해 수행되었다. 펜토바비탈로 마취된 쥐의 꼬리튀기 반사를 사용하여 전측 시개전핵을 전기자극하고 복측 연수를 전기손상시키거나 리도카인을 주입한 결과 전측 시개전핵의 자극으로 유발된 무통 효과가 억제되었다. 반대로 복측 연수를 전기자극하거나 글루타메이트를 주입하여 세포체를 활성화시키면 마찬가지로 무통이 발생하였다. 이 결과는 전측 시개전핵의 하행성 통각 억제 체계에 하올리브 수준의 복측 연수가 관여한다는 것을 시사한다. The present study was conducted to determine whether the ventral medulla (VM) at the level of the inferior olive (IO) is involved in descending pain inhibition system including the anterior pretectal nucleus (APTN). Pain sensitivity was assessed using tail-flick test to radiant heat in the rat anaesthetized with pentobarbital. Electrolytic lesions of the VM or microinjections of lidocaine into the VM inhibited the analgesic effects of stimulating the APTN. Electrical stimulation of the VM or microinjections of glutamate into the VM produced analgesic effects similar to those of stimulating the APTN. These results suggest that the VM including the IO is involved in a descending antinociceptive pathway originating in the APTN.

전측 시개전핵의 하행성 통각 조절계에 대한 중뇌수도주변회백질의 관여

이배환,김현,서영석,홍승길,안창일,김기석 한국심리학회 한국심리학회지 생물 및 생리 Vol.6 No.1

이 연구는 전측 시개전핵의 자극으로 인한 하행성 통각 억제 체계에 중뇌수도주변회백질이 관여하는가를 알아보기 위해 수행되었다. 펜토바비탈로 마취된 쥐의 꼬리튀기 반사를 사용하여 전측 시개전핵을 전기자극하고 중뇌수도주변회백질에 리도카인을 주입한 결과 전측 시개전핵의 자극으로 유발된 무통 효과가 억제되었다. 하올리브 수준의 복측 연수에 WGA-HRP를 주입하였을 때 중뇌수도주변회백질의 복외측 부위에 표지된(labeled) 뉴론이 집중적으로 관찰되었으며, 전측 시개전핵에서도 표지된 뉴론이 발견되었다. 이러한 결과는 전측 시개전핵의 하행성 통각 억제 체계에 최소한 부분적으로 중뇌수도주변회백질이 관여한다는 것을 시사한다. This study was conducted to determine whether the periaqueductal gray (PAG) is involved in descending pain inhibition system mediated by the anterior pretectal nucleus (APTN). Pain sensitivity was assessed using tail-flick test to radiant heat in the rat anaesthetized with pentobarbital. Microinjections of lidocaine into the PAG inhibited the analgesic effects of stimulating the APTN. After WGA-HRP was injected into the ventral medulla at the level of the inferior olivary nuclei, the labeled neurons were detected in the ventrolateral PAG and in the APTN. The results suggest that the PAG is at least partially involved in a descending antinociceptive pathway originating in the APTN.

탄소섬유의 CVD-SiC 증착특성에 미치는 CH₃SiCl₃대 H₂의 비율의 영향

박병배,심환보,이보성,이영석,김종환 忠南大學校 産業技術硏究所 1994 산업기술연구논문집 Vol.9 No.2

Chemical vapor deposition of SiC on carbon fibers was investigated to improve oxidation resistance of carbon fibers, which was performed at the horizontal hot-wall reactor using the reactant mixtures of CH₃SiCl₃and H₂ with carrier gas Ar. The chemical composition, oxidation resistance, and tensile strength of SiC-coated carbon fiber were investigated as functions of CH₃SiCl₃/H₂ ratio, reaction temperature, coating thickness, and so on. The composition of deposited layer revealed C rich β-SiC at low hydrogen concentration and Si rich β-SiC at high hydrogen concentration. Because Si rich β-SiC layer is easy to be converted into SiO₂ favorable to oxidation resistance, high input value of H₂/CH₃SiCl₃ mole ratio resulted in good oxidation resistance in the SiC coating layer of carbon fibers.