비만증에서 체중감소가 뇌척수액 및 혈중 Neuropeptide-Y, α-Melanocyte Stimulating Hormone 과 Leptin 농도에 미치는 영향

남수연,김경욱,이준희,윤수지,김경래,차봉수,임승길,이현철,허갑범 대한내분비학회 2001 Endocrinology and metabolism Vol.16 No.2

Background: Although leptin and its principal mediators, neuropeptide Y (NPY) and α-melanocyte stimulating hormone (MSH) are postulated to play a pivotal role in the energy balance in experimental animals, the physiologic roles of leptin and its molecular targets are not fully identified in cases of human obesity. Methods: The subjects consisted of 16 obese women (mean BMI 35.6 ㎏/㎡) before and after weight loss that was induced by a 2 week-very low caloric diet (800 kal/day) and 14 normal weight women (who had a mean BMI of 20.4 ㎏/㎡). We evaluated the plasma and cerebrospinal fluid (CSF) leptin, NPY and -MSH levels and their relationship in normal weight and obese women. Additionally, changes of these peptides during a negative energy balance (800 kcal/day) were assessed in causes of human obesity. Results: Obese subjects exhibited a 6.3-fold higher plasma leptin level (21.9±1.2 vs 3.5±0.4 ng/mL, p<0.05) and a 2.8-fold higher CSF leptin level (0.29±0.02 vs 0.10±0.01 ng/mL, p<0.05) compared to control subjects. The CSF/plasma leptin ratio in normal weight subjects was 2.3-fold higher than that in obese subjects. After a weight loss in obese subjects, the plasma leptin level decreased by 40% and the CSF level decreased by 51%. The CSF/plasma leptin ratio was slightly lower than the baseline level. There was a positive linear correlation between CSF and plasma leptin level at the baseline in obese subjects (r= 0.74, p<0.05) and a positive logarithmic correlation in normal weight subjects and in obese subjects after a weight loss (r= 0.66, p<0.05). The BMI negatively correlated with the CSF/plasma leptin ratio (r=-0.86, p<0.05) in any subjects. Neither the baseline plasma levels nor the baseline CSF levels of NPY were different between the normal weight subjects and obese subjects. After a weight loss the CSF NPY level decreased significantly compared to the baseline values in obese subjects. The α-MSH levels in plasma and CSF did not differ significantly from controls in obese subjects at the baseline or after a weight loss. The baseline CSF leptin level neither correlated with the baseline CSF NPY level nor the baseline CSF α-MSH level. Conclusion: These results demonstrated that the efficiency of leptin delivery to the CNS is reduced in human obesity and that the CNS leptin uptake involves the combination of saturable and unsaturable mechanisms. A marked reduction in the CSF leptin levels compared to the plasma level after a weight loss in obese subjects can be a potent stimulus for the body to regain weight. In contrast to the results that were observed in experimental animals, the CSF NPY and α-MSH did not differ from the controls in human obesity and there was no significant correlation between the CSF leptin and CSF of these neuropeptides. This could have resulted from leptin resistance in cases of human obesity although the mechanisms for this resistance remain to be determined.

가와사키병 환아의 혈장내 G-CSF와 GM-CSF 농도 및 과립구에서의 이들 수용체의 발현 변화

유영경,이기범,김현희,김소영,김유정,이원배,Yoo, Young-Kyoung,Lee, Gibum,Kim, Hyun-Hee,Kim, Soo-Young,Kim, You-Jeong,Lee, Wonbae 대한소아청소년과학회 2003 Clinical and Experimental Pediatrics (CEP) Vol.46 No.4

목 적 : G-CSF와 GM-CSF는 과립구생성에 중요한 사이토카인으로서, 각각의 수용체(이하 G-CSFr과 GM-CSFr)에 결합하여 기능을 하게 되며, 이들 수용체들은 미성숙 골수 세포로부터 성숙된 말초 과립구까지 발현된다. 일반적으로 혈중 G-CSF와 GM-CSF의 농도 및 이들 수용체의 발현은 과립구가 증가하는 감염질환에서 변화한다고 알려져 있으나, 가와사키병에서 과립구 수 증가와 관련된 변화에 대해서는 아직 연구된 바가 없다. 본 연구에서는 가와사키병 환아와 정상아의 혈중 G-CSF와 GM-CSF의 농도를 측정하고 말초혈액의 과립구 표면에 존재하는 이들의 수용체를 정량분석 하였으며, 또한 수용체를 충분히 포화시킬 수 있는 과량의 CSF에 수용체를 노출시켰을 때 결합하지 않고 남은 수용체 발현양에는 어떤 변화가 있는 지를 분석하여 가와사키병 환자의 과립구 수 증가와 G-CSF와 GM-CSF 및 이들 수용체 변화의 관련성을 규명하고자 하였다. 방 법 : 정상 대조군으로서 비감염성질환으로 입원한 같은 연령대의 아동 중 말초혈액 백혈구 수 및 중성구 수가 정상인 아동 13명과 면역글로불린을 쓰기 전 급성기 초기의 가와사키병 환아 14명, 총 27명의 혈중 G-CSF와 GM-CSF의 농도를 측정하였고 세포표면 G-CSFr와 GM-CSFr의 발현양은 각각 항 G-CSF 수용체 단클론항체, 항 GM-CSF 수용체 단클론항체와 혼합 후 유세포 분석기를 이용하여 정량적으로 분석하였으며, 활동성 G-CSFr, GM-CSFr의 양적변화를 유세포 분석기를 이용하여 측정하였다. 결 과 : 가와사키병 환아의 총 백혈구 수는 정상대조군과 차이가 없었으나 중성구 수, 단핵구 수는 증가하였고 총 백혈구수는 중성구 수 증가에 따라 증가하였다. 혈중 G-CSF의 농도는 정상대조군과 유의한 차이가 없었으나 감소되어 있었고(P=0.133) 혈중 GM-CSF의 농도는 정상대조군에 비해 유의하게 감소되어 있었다(P=0.027). 가와사키병 환아의 중성구의 G-CSFr, GM-CSFr의 발현양은 정상대조군과 통계적으로 유의한 차이가 없었다(P=0.721, P=0.912). 수용체를 포화시키기에 충분한 과량의 CSF와 배양시킨 후 포화농도에도 결합하지 않은 수용체의 양을 측정하면 남은 수용체수는 감소하게 되는데, 가와사키병 환아에서 과량의 G-CSF에 배양한 후 감소된 중성구 G-CSFr의 발현양은 정상대조군과 유의한 차이는 없었고(P=0.554), 혈중 G-CSF 농도와는 무관하였으며(P>0.05) 혈중 GM-CSF 농도와 반비례하였다(정상아; r=-0.589, P=0.044, 가와사키병 환아; r=-0.946, P=0.004). 가와사키병 환아와 정상대조군에서 수용체를 포화시키기에 충분한 과량의 GM-CSF에 배양한 후 중성구의 GM-CSFr의 발현양을 분석한 결과는 발현이 증가된 소견을 보여(P=0.255) 항 GM-CSF항체가 항 G-CSF 항체와는 달리 GM-CSF와 그 수용체의 결합에 무관한 것으로 나타났다. 증가된 중성구의 GM-CSFr의 양은 정상아에서는 총 백혈구 수와 비례하였으나(r=0.788, P=0.035), 가와사키병 환아에서는 상관관계가 없었다(P=0.644). 결 론 : 가와사키병 환아의 백혈구 수 증가의 원인은 중성구 수의 증가로 보이고 G-CSF, GM-CSF의 농도는 감소되어 있었으며 G-CSFr, GM-CSFr 발현양 및 활동성 G-CSFr, GM-CSFr 발현양은 정상아와 유의한 차이가 없어서 이는 중성구 수 증가가 G-CSF, GM-CSF의 증가나 그 수용체 수 변동에 의한 것이 아닌 것으로 나타났고 GM-CSF 농도의 감소가 활동성이 있는 G-CSFr의 증가를 일으켜 가와사키병 급성기 총 백혈구 수 증가의 원인인 중성구 수 증가를 유도했을 가능성을 나타낸다. Purpose : This study aimed to demonstrate the possible pathogenesis of granulopoiesis in patients of Kawasaki disease(KD) using quantitative analysis of G-CSF, GM-CSF and their CSFr. Methods : The plasma levels of G-CSF, GM-CSF, G-CSFr and GM-CSFr were studied in 14 patients in the acute phase of KD; 13 children with normal peripheral white blood cell counts were used as the normal control group. The plasma concentration of G-CSF, GM-CSF were analyzed by ELISA. The G-CSFr and GM-CSFr on the peripheral granulocytes were analyzed by a quantitative flow cytometric assay and QuantiBRITE, and the quantitative changes of receptors which did not combine with G-CSF and GM-CSF were measured. Results : The total number of leukocytes in KD was similar to normal control group, but the leukocytes increased according to the number of neutrophils. The plasma concentration of G-CSF were decreased similar to normal control group(P=0.133), but that of GM-CSF decreased more than the normal control group(P=0.227). The quantity of G-CSFr, GM-CSFr were revealed to be no less than the normal control(P=0.721, P=0.912). After incubation with excessive G-CSF, the expressed G-CSFr on the neutrophils were decreased in both groups(P=0.554). The quantities of expressions of GM-CSFr on the neutrophil after incubation with the excessive GM-CSF were always increased in both groups(P=0.255). The amount of GM-CSFr of neutrophils are in proportion to total white blood cells (r=0.788, P=0.035), but it wasn't in the case of KD(P=0.644). Conclusion : The leukocytosis in KD that mediated by increasing neutrophil was not correlated with the plasma concentrations of G-CSF and GM-CSF, and the amount of expression of G-CSFr and GM-CSFr on granulocyte. It is possible that the reduction of concentration of GM-CSF results by increasing the active GM-CSFr.

백혈구 증가증 환아의 혈장내 G-CSF와 GM-CSF의 농도 및 과립구에서의 이들 수용체의 발현

최원석,유경환,김유정,김소영,김현희,이원배,Choi, Won Seok,Ryu, Kyung Hwan,Kim, You Jeong,Kim, So Young,Kim, Hyun Hee,Lee, Wonbae 대한소아청소년과학회 2003 Clinical and Experimental Pediatrics (CEP) Vol.46 No.3

목 적 : G-CSF와 GM-CSF는 과립구생성에 중요한 사이토카인으로서, 각각의 수용체 G-CSFr과 GM-CSFr에 결합하여 기능을 하게 되며, 이들 수용체들은 미성숙 골수 세포로부터 성숙 된 말초 과립구까지 발현된다. 일반적으로 혈중 G-CSF와 GM-CSF의 농도 및 이들 수용체의 발현은 과립구가 증가하는 감염질환에서 변화한다고 알려져 있으나, 백혈구 증가증에서 과립구수 증가와 관련된 변화에 대해서는 아직 연구된 바가 없다. 본 연구에서는 백혈구 증가증 환아와 정상아의 혈중 G-CSF와 GM-CSF의 농도를 측정하고 말초혈액의 과립구 표면에 존재하는 이들의 수용체를 정량분석하였으며, 활동성이 있는 수용체 발현 양에는 어떤 변화가 있는 지를 분석하여 백혈구 증가증 환아의 과립구 수 증가와 G-CSF와 GM-CSF 및 이들 수용체 변화의 관련성을 규명하고자 하였다. 방 법 : 정상 대조군으로서 비감염성 질환으로 입원한 같은 연령대의 아동 중 말초혈액 백혈구 수 및 중성구 수가 정상인 아동 13명과 급성기 초기의 백혈구 증가증 환아 14명, 총 27명의 혈중 G-CSF와 GM-CSF의 농도를 측정하였고 세포표면 G-CSFr와 GM-CSFr의 발현양은 각각 항 G-CSF 수용체 단 클론항체, 항 GM-CSF 수용체 단클론항체와 혼합 후 유세포 분석기를 이용하여 정량적으로 분석하였으며, 활동성 G-CSFr, GM-CSFr의 양적변화를 유세포 분석기를 이용하여 측정하였다. 결 과 : 백혈구 증가증 환아의 총 백혈구 수는 $24,100{\pm}6960/{\mu}L$로 정상 대조군 $8,680{\pm}378/{\mu}L$에 비해 유의하게 증가되었으며 중성구 수는 백혈구 증가증 환아에서 유의하게 증가되어 있었으나($20,800{\pm}2120/{\mu}L$ vs $3,360{\pm}2,120/{\mu}L$) 단구수는 차이가 없었다. 혈중 G-CSF의 농도 $164.0{\pm}187.5pg/mL$는 정상 대조군 $71.9{\pm}94.1pg/mL$과 감소된 경향을 보였으나 통계적으로 유의한 차이가 없었고(P=0.217) 혈중 GM-CSF의 농도도 유의한 차이가 없었다(P=0.968). 백혈구 증가증 환아의 중성구의 G-CSFr 발현양 $963.6{\pm}575.7$은 정상대조군 $1711.1{\pm}452.6$에 비해 유의한 감소를 보였으며(P=0.012), 백혈구 증가증 환아의 중성구의 GM-CSFr의 발현양 $471.7{\pm}217.0$은 정상 대조군의 $854.8{\pm}383.0$과 통계적으로 유의한 차이가 없었다(P=0.220). 항 G-CSFr항체는 수용체가 G-CSF와 결합하고 나면 수용체에 결합하지 못하는 epitope에 대한 항체로 보여지며, 수용체를 포화시키기에 충분한 과량의 CSF와 배양시킨 후 포화농도에도 결합하지 않은 수용체의 양을 측정하면 남은 수용체 즉, 활동성 수용체 수는 감소하게 되는데, 백혈구 증가증 환아에서 과량의 G-CSF에 배양한 후 감소된 중성구 G-CSFr의 발현양은 $407.8{\pm}405.1$로 정상 대조군의 $1,012.2{\pm}488.5$에 비해 유의한 감소를 보였고(P=0.050), 혈중 G-CSF 농도 및 혈중 GM-CSF 농도와 무관하였다(P=0.735, P=0.087). 백혈구 증가증 환아와 정상대조군에서 수용체를 포화시키기에 충분한 과량의 GM-CSF에 배양한 후 중성구의 GM-CSFr의 발현양을 분석한 결과는 발현이 증가된 소견을 보였다. 증가된 중성구의 GM-CSFr의 발현 양은 정상아 및 백혈구 증가증 환아에서 유의한 차이가 없었다(P=0.828). 결 론 : 백혈구 증가증에서는 중성구 수 증가에 의하여 총 백혈구 수가 증가되고 혈중 G-CSF의 농도는 중성구 증가의 원인으로 생각되며 G-CSFr과 결합하여 백혈구 증가증을 일으키는 것으로 보인다. GM-CSF 농도 및 GM-CSFr은 백혈구 증 Purpose : Granulocyte-colony stimulating factor(G-CSF) and granulocyte macrophage-colony stimulating factor(GM-CSF) are principal cytokines in granulopoiesis and their physiologic effects are mediated through binding to specific cell surface receptors. Although it is known that the level of serum G-CSF and GM-CSF, and presentation of the receptors are increased in infectious diseases, there have been no studies to find the correlation between the granulopoiesis and leukocytosis. This study was designed to measure G-CSF and GM-CSF in leukocytosis and in control and to demonstrate the possible pathogenesis of granulopoiesis in leukocytosis using quantitative analysis of G-CSF, GM-CSF and their CSFr. Methods : The plasma levels of G-CSF, GM-CSF of 13 children without leukocytosis and 14 children with leukocytosis were measured. Counts of cell surface G-CSFr and GM-CSFr were measured by combining anti G-CSFr and anti GM-CSFr monoclonal antibodies to their respective receptors by using quantitative flow cytometric assay. Results : There was no significant difference betweeen the plasma concentration of G-CSF and GM-CSF in acute leukocytosis and in the control group. However, levels of G-CSFr in acute leukocytosis decreased significantly compared to the control(P=0.012) and the levels of GM-CSFr in both groups revealed no significant difference. Conclusion : Increase in the number of leukocyte in leukocytosis was mediated by increasing the number of neutrophil, and increased plasma concentration of G-CSF may be the cause of neutrophilia. But GM-CSF did not have any influence on leukocytosis.

FIV-Tet-On Vector System을 이용한 hG-CSF 유전자의 효율적인 발현 조절

권모선,구본철,김태완 한국동물생명공학회(구 한국동물번식학회) 2007 Reproductive & developmental biology Vol.31 No.3

본 연구에서는 hG-CSF의 발현을 유도적으로 조절하기 위한 FIV-Tet-On lentivirus vector system을 구축하고자 하였다. hG-CSF는 호중성구 계열 세포의 증식과 분화, 생존에 영향을 미치는 물질로서, 이 유전자의 발현을 증가시키기 위하여 FIV-Tet-On vector 상의 hG-CSF나 rtTA2SM2 서열의 3' 위치에 WPRE 서열을 도입하였다. 구축된 각각의 vector는 293FT 세포에 일시적으로 transfection하여 virus를 생산하였으며, 이 virus를 일차 배양 세포인 CEF와 PFF에 감염시켰다. 각 세포에 전이된 hG-CSF의 발현 양상을 관찰하기 위하여 doxycycline을 첨가하거나 첨가하지 않은 배지에서 배양한 후 quantitative real-time PCR, Western blot과 ELISA를 이용하여 hG-CSF 유전자의 발현 정도를 비교 측정한 결과, CEF에서는 WPRE가 hG-CSF의 3' 위치에 도입된 경우에 발현량과 유도율이 가장 높은 것으로 나타났고, PFF에서는 rtTA 서열의 3'위치에 도입된 경우에 발현량과 유도율이 가장 큰 것으로 확인되었다. 이 FIV-Tet-On vector system은 형질 전환 동물의 생산이나 유전자 치료에서 문제시되는 외래 유전자의 지속적인 과다 발현에 의한 개체의 생리적인 부작용을 최소화하기 위한 해결 방법으로 제시될 수 있을 것이다. In this study, using FIV-based lentivirus vector system, we tried to express hG-CSF in tetracycline-controllable manner. hG-CSF influences the proliferation, differentiation, and survival of cells in the neutrophil lineage. To enhance stability and translation of hG-CSF transcript, WPRE sequence was also introduced into FIV-Tet-On vector at downstream region of either the hG-CSF gene or the sequence encoding rtTA. Primary culture cells (CEF, chicken embryonic fibroblast; PFF, porcine fetal fibroblast) infected with the recombinant FIV were cultured in the medium supplemented with or without doxycycline for 48 hours, and induction efficiency was measured by comparing the hG-CSF gene expression level using quantitative real-time PCR, Western blot and ELISA. Higher hG-CSF expression and tighter expression control were observed from the vector in which the WPRE sequence was placed at downstream of the hG-CSF (in CEF) or rtTA (in PFF) gene. This FIV-Tet-On vector system may be helpful in solving serious physiological disturbance problems which has continuously hampered successful production of transgenic animals and gene therapy.

Structural Integrity Evaluation for Initial Characteristic Measurement Equipment and CANDU Spent Fuel

Jae-Jun Lee,Seong-Ki Lee,Jun Kim,Yong-Chan Kim 한국방사성폐기물학회 2023 한국방사성폐기물학회 학술논문요약집 Vol.21 No.2

A lot of CANDU Spent Fuels (CSFs) have been stored in spent nuclear fuel pools and dry storage facilities. In accordance with the enhanced nuclear regulations, the initial characteristics of CSF should be inspected to ensure the integrity of CSF and the reliable operation of storage system before loading it into a cask for long-term dry storage. For the inspections, an initial characteristics measurement equipment was designed, which is used for Pool-Side Examination (PSE) in the spent fuel pool of the pressurized heavy water reactor nuclear power plant. Measurements using the equipment consist of non-contact inspections and contact inspections. The non-contact inspections do not affect CSF integrity, whereas the integrity of CSF can be reduced during the contact inspections under abnormal operating conditions because the probe of equipment may apply specific loads to the CSF. Therefore, the structural integrity evaluations of equipment and CSF are performed using Finite Element (FE) analyses for four combinations based on two abnormal conditions and two probe positions. The used abnormal conditions are the pressing load condition and the scratching load condition, and two probe positions are the center and bottom of the fuel rod in the longitudinal direction, respectively. In this evaluation, the bottoms of the fuel rod or CSF are defined as the regions facing the bottom surface of equipment. The analysis of the pressing load condition is performed by pressing the probe of the equipment in radial direction of the CSF fuel rod. That of the scratching load condition is carried out by applying a specific radial load to the CSF fuel rod using the probe and then applying the load to the surface of the fuel rod while moving axially along the surface. All combinations are analyzed considering geometric, boundary and material non-linearity under the dynamic load, which is dependent on the equipment operating velocity. The stresses of CSF and equipment components were obtained from these analyses. The maximum stress of each component was generated at the combination on the scratching load condition for the bottom position among the four combinations. The obtained maximum stresses are lower than the yield stress for each component material. Also, the CSF is not overturned due to the support plate of the equipment in all analyses. Therefore, the structural integrity and safety of the equipment and the CSF are maintained under abnormal operating conditions during the inspection using the initial characteristic measurement equipment.

Field evaluation of the safety and immunogenicity of a classical swine fever virus E2 subunit vaccine in breeding and nursery animals on Jeju Island, South Korea

장규환,Eun-Joo Kim,Seong-Cheol Cho,Sung-Up Moon,Byeong Soo Kim,Jinhee Kim,Kyoung Ju Jeong,송경옥,Seong Hwan Mun,Won-Myoung Kang,Jonghoo Lee,Changnam Park,Hyoung-Seok Yang,이창희 대한백신학회 2022 Clinical and Experimental Vaccine Research Vol.11 No.3

Purpose: Classical swine fever (CSF) reemerged on CSF-free Jeju Island where vaccination is not practiced by the unintentional injection of a live attenuated vaccine (modified live attenuated vaccines–low-virulence Miyagi [MLV-LOM]) in 2014. Since the Jeju provincial authority is considering adopting a voluntary immunization policy using a CSF-E2 subunit vaccine to combat LOM-derived CSF endemic, this study aimed to evaluate in Jeju herds. Materials and Methods: Two vaccination trials using the Bayovac CSF-E2 vaccine licensed for use in South Korea assessed the safety and humoral immunity of the CSF-E2 vaccine in breeding (trial 1) and nursery animals (trial 2) under farm application conditions. Results: Neither local nor systemic (including reproductive) adverse effects were objectively observed in pregnant sows and young piglets following a respective vaccination regime at pregnancy or weaning, respectively. Trial 1 showed that sows immunized with the CSF-E2 vaccine possessed high and consistent E2-specific and neutralizing antibody levels. The CSF-E2 vaccine-immunized pregnant sows subsequently conferred appropriate and steady passive immunity to their offspring. In trial 2, a double immunization scheme of the CSF-E2 vaccine in piglets at 40 and 60 days of age could elicit a consistent and long-lasting adequate antibody response. Additionally, the two trials detected no Erns-specific antibody responses, indicating that CSF-E2 vaccine can differentiate infected from vaccinated animals (DIVA). Conclusion: Our trial data collectively provide invaluable information on applying the CSF-E2 subunit vaccine to circumvent the possible drawbacks associated with the MLV-LOM concerning the safety, efficacy, and DIVA, in the LOM-endemic field farms and contribute to advanced CSF eradication on Jeju Island.

Actazolamide와 Fluphenazine의 뇌척수액 생성에 대한 효과

송준혁,박윤관,정흥섭,서중근,이훈갑,이기찬,주정화 대한신경외과학회 1993 Journal of Korean neurosurgical society Vol.22 No.10

The purpose of this study was to investigate the effect of acetazolamide and fluphenazine on the formation of CSF. Studies were performed in 12 cats those were divided into 2 groups ; A-F group included animals received initial acetazolamide infusion and additional infusion of fluphenazine to the initial infusion and the F-A group for vice versa. The rate of CSF formation was measured at 3㎝ above zero outflow pressure by force transducer which connected to personal computer. After obtaining steady value of CSF formation rate, the drugs were infused intravenously according to the protocol. Base line CSF formation rate, 18.87±6.52μl/min, is reduced to 6.67±2.45μl/min after acetazolamide infusion and further reduced to 3.48±4.06μl/min after additional fluphenaxine. In fluphenazine group, the base line CSF formation rate, 16.34±4.58μl/min is reduced to 9.63±4.58μl/min after initial infusion of fluphenazine and further to 6.45±3.64μl/min, after additional infusion of acetazolamide. Mean reduction of CSF formation after initial intravenous infusion of acetazolamide and fluphenazine were 59% and 37% respectively. Although statistically insignificant, the CSF formation reduction in A-F group revealed more even and profound value comparing with that of F-A group. These date suggest that in addition to the effect of acetazolamide to reduce the formation of CSF, some other mechanism may exist in CSF formation that major tranquilizer exert the effect on CSF formation.

Roles of Macrophage Colony Stimulating Factor in White and Brown Adipocytes

Sulagna Mukherjee,Kanikkai Raja Aseer,윤종원 한국생물공학회 2020 Biotechnology and Bioprocess Engineering Vol.25 No.1

Macrophage colony stimulating factor (M-CSF), commonly known as CSF1, is a cytokine produced by osteoblast cells, and is well known for its important role in differentiation of osteoclasts and white adipocyte hyperplasia. However, the role of M-CSF in lipid metabolic activity is lacking insight. In the current study, we explore unidentified actions of M-CSF in adipocytes. Levels of M-CSF were determined for white and brown adipose tissues and cells, and the expression levels of the important protein markers in lipid metabolism were evaluated through immunoblot analysis, subsequent to blocking M-CSF in fat cells by silencing its receptor Csf1r. Interestingly, we observed presence of M-CSF in brown fat cells. In addition, the lack of M-CSF in HIB1B cells attenuated the expressions of protein markers responsible for lipid metabolism in the brown adipocytes. Further, deficiency of M-CSF resulted in decreased adipogenesis along with reduced expression of lipogenic markers for both white and brown adipocytes. Moreover, lipid metabolism was balanced by M-CSF via the PPARγ-mediated LPL activity in white adipocytes. Taken together, the results from this study indicate that MCSF is an important regulatory protein in both white and brown adipocytes.

Effects of EGF, IGF-I, VEGF and CSF2 on development of porcine conceptus trophectoderm during early pregnancy

Wooyoung Jeong 한국발생생물학회 2014 한국발생생물학회 학술발표대회 Vol.2014 No.9

The majority of early conceptus mortality in pregnancy occurs during the peri-implantation stage, suggesting that this period is important for conceptus viability and the establishment of pregnancy. Successful establishment of pregnancy in all mammalian species depends on the orchestrated molecular events that transpire at the conceptus-uterine interface during the peri-implantation phase. This maternal-conceptus interaction is especially crucial in pigs because in them non-invasive epitheliochorial placentation occurs, in which the pre-implantation phase is prolonged. During the pre-implantation period, conceptus survival and the establishment of pregnancy are known to depend on the developing conceptus receiving an adequate supply of histotroph, which contains a wide range of nutrients and growth factors. Evidence links epidermal growth factor (EGF), insulin- like growth factor-I (IGF-I), vascular endothelial growth factor (VEGF), and colony-stimulating factor 2 (CSF2) to embryogenesis or implantation in various mammalian species; however, in the case of pig, little is known about such functions of these growth factors, especially their regulatory mechanisms at the maternal-conceptus interface. Therefore, the objectives of this study were to determine: 1) the temporal and cell-specific expression of EGF, IGF-I, VEGF, and CSF2 signaling systems in the porcine endometrium during the estrous cycle and early pregnancy; 2) the potential intracellular signaling pathways responsible for the activities of these four factors in primary porcine trophectoderm (pTr) cells; and 3) the changes in cellular activities induced by these promising factors. First, the functional effect and cellular signaling cascades in pTr cells induced by EGF, which exhibits potential growth-promoting activities on the conceptus and endometrium, were investigated. EGFR mRNA and protein were abundant in endometrial luminal epithelia (LE) and glandular epithelia (GE), stratum compactum stroma, and conceptus trophectoderm on Days 13-14 of pregnancy, but not in any other cells of the uterus. EGF treatment of pTr cells increased the abundance of phosphorylated (p)-AKT1, p-ERK1/2 MAPK and p-P90RSK in the nucleus and/or cytoplasm when compared with the levels in control cells. Furthermore, EGF-stimulated phosphorylation of AKT1 and ERK1/2 MAPK were inhibited in pTr cells transfected with an EGFR siRNA, and compared with control siRNA- transfected pTr cells, the EGFR siRNA-transfected pTr cells exhibited an increase in the expression of gene encoding interferon (IFN)-δ and transforming growth factor (TGF) β-1; by contrast, no effect was detected on the expression of the gene encoding IFN-γ. Moreover, EGF stimulated the proliferation and migration of pTr cells, but these stimulatory effects were blocked by pharmacological inhibitors such as SB203580 (a p38 inhibitor), U0126 (a MAPK inhibitor), rapamycin (an MTOR inhibitor), and LY294002 (a PI3K inhibitor). Second, IGF-I was examined. IGF-1 is another promising growth factor that is known to play key roles in reproductive processes; however, little is known about IGF-I-induced functional effects and regulatory mechanisms during peri-implantation in pigs. In this study, endometrial type I IGF receptor (IGF-IR) mRNA was determined to increase substantially during early pregnancy relative to the level during the estrous cycle, and the mRNAs of both IGF-I and IGF-IR were abundant in endometrial LE and GE, stroma and conceptus trophectoderm on Day 12 of pregnancy. Moreover, IGF-I treatment potently increased the amounts of p-AKT1 and, ERK1/2 MAPK in the nucleus and cytoplasm and of RPS6 in the cytosol when compared with the amounts in untreated pTr cells, and IGF-I-induced activation of AKT1 and ERK1/2 was blocked by LY294002. Furthermore, IGF-I strongly stimulated both the proliferation and the migration of pTr cells, but these effects were inhibited by SB203580, U0126, rapamycin and LY294002. Third, this study focused on VEGF, which was identified as a potential mediator of the fetal-maternal dialog that regulates the development of the peri-implantation porcine conceptus. In addition to its known angiogenic effects, VEGF has been suggested to play roles in the development of the early embryo, but VEGF-induced effects on the peri-implantation conceptus remain unknown. Results of this study revealed that endometrial VEGF, VEGF receptor (VEGFR)-1, and VEGFR-2 mRNA levels in endometrial LE and GE, endothelial blood vessels, and scattered cells in the stroma were more abundant during the peri-implantation period of pregnancy than during the estrous cycle. Moreover, VEGF treatment of pTr cells increased the abundance of p-AKT1, p-ERK1/2, p-p70RSK, p-RPS6 and p-4EBP1, and the addition of LY294002 suppressed VEGF-induced phosphorylation of ERK1/2 and AKT1. Furthermore, VEGF potently stimulated both the proliferation and the migration of pTr cells, but these effects were inhibited in the presence of SB203580, U0126, rapamycin and LY294002. The fourth promising cytokine studied was CSF2, which is also known as granulocyte-macrophage colony-stimulating factor (GM-CSF). CSF2 plays a role in facilitating mammalian early embryonic development. In this study, endometrial CSF2 mRNA expression was determined to be increased during the peri-implantation period relative to the mRNA level during the estrous cycle. In pTr cells, CSF2 significantly induced the activation of AKT1, ERK1/2, MTOR, p70RSK, and RPS6, but not of STAT3, and the addition of LY294002 abolished CSF2-induced increases in p-ERK1/2, p-MTOR, and p-AKT1 levels. Furthermore, CSF2 strongly stimulated pTr cell proliferation, an effect that was blocked by U0126, rapamycin and LY294002. Collectively, these results provide new insights into the potential mediators that regulate the development of the peri-implantation conceptus at the fetal-maternal interface. These results indicate that endometrial- and/or conceptus derived EGF, IGF-I, VEGF, and CSF2 critically affect the growth and development of porcine trophectoderm cells, and that these stimulatory effects are coordinately regulated by multiple cellular signaling cascades including the PI3K-AKT and ERK1/2 MAPK pathways during early pregnancy in pigs.

뇌척수액에서 항 Glutamic acid decarboxylase 항체검사의 참고치 설정

박민호,신선영,윤태석,신희정,노경운,Park, Min-Ho,Shin, Sun-Young,Youn, Tae-Seok,Shin, Hi-Jung,Noh, Gyeong-Woon 대한핵의학기술학회 2017 핵의학 기술 Vol.21 No.2

본 연구를 통해 항 GAD 항체검사에서 뇌척수액에 대한 정상치 범위를 추정하였다. 본원에서 211명을 대상으로 혈청과 뇌척수액에 대한 항 GAD 항체검사를 함께 의뢰받은 환자 데이터를 분석해 본 결과 혈청은 1.0 U/mL 이상의 값을 가진 환자가 약 5%를 차지하였고. 뇌척수액의 경우에는 1.0 U/mL이상의 값을 가진 환자는 약 98% 이상을 차지하였다. 혈청의 참고치 (1.0 U/mL 이상 양성)를 뇌척수액에 적용했을 경우에는 대부분의 환자들이 강직증후군 및 간질을 진단 받게 된다. 이에 혈청과 뇌척수액의 참고치를 병용해서 사용하는 것은 부적합하다고 판단하였고 뇌척수액에 대한 참고치를 본원에서 자체적으로 설정하고자 하였다. 실험 대상으로는 정상인의 뇌척수액을 채취하는 데는 어려움이 있어 이와 가장 유사한 생리식염수를 음성대조군으로 선정하였다. 총 70개의 생리식염수를 검사한 결과 Mean값이 1.97 U/mL, SD 값이 0.72 U/mL가 나왔다. 이 값들에 Hoffmann법으로 Mean값에서 ${\pm}2SD$를 정상범위로 하여 0.54 U/mL ~ 3.40 U/mL로 Expected reference range를 설정할 수 있었다. 본 실험에서 아쉬운 점은 참고치 설정에 있어 정상인의 뇌척수액으로 검사가 이루어지지 못한 부분이다. 앞으로 정상인의 뇌척수액으로 참고치 설정을 할 수 있다면 보다 더 정확하게 임상적으로 적용할 수 있을 것이라 생각 된다. Purpose Anti-Glutamic acid decarboxylase antibody test (GAD Ab) has been used as a predictor of type 1 diabetes. GAD Ab has also been shown to be highly potent in cerebrospinal fluid (CSF) of patients with suspected diabetic peripheral neuropathy. Recently, it has been known that clinical significance of GAD Ab using CSF is useful for the neurological disorders. However, the reference value of anti-GAD Ab has been provided only for serum. In this experiment, we estimated the reference value of anti-GAD antibody for CSF in neurological patients. Materials and Methods A total of 211 neurological patients were enrolled. Serum and CSF were analyzed by radioimmunoassay (RIA) using commercial RIA anti-GAD Ab kit (RSR, London, United Kingdom). Normal saline was used as the normal CSF control because CSF is most similar to 0.9% normal saline. Results The mean value of normal CSF control was 1.97 U/mL, and two standard deviations (2SD) was 1.44 U/mL. Based on this data, the expected reference range of CSF could be estimated from 0.54 U/mL to 3.40 U/mL Conclusion The reference range of normal CSF control using normal saline obtained with Hoffmann's method. However, there will be a need to validate the CSF reference values using human normal CSF.