저칼륨혈증 흰쥐 신장에서 Akt, p-Akt, ERK 및 p-ERK 단백발현의 변화

배춘상(Choon Sang Bae),조혜정(Hye Jung Cho),안규윤(Kyu Yoon Ahn) 대한체질인류학회 2017 해부·생물인류학 (Anat Biol Anthropol) Vol.30 No.3

저칼륨혈증은 신장의 형태학적 변화와 대사성 알칼리증을 유발시키는 것으로 알려져 있다. 이전 결과들은 저칼륨혈증의 병태생리학적 변화에 K+평형 조절 이온채널, pump 유전자, NF-E2-related factor 2 (Nrf2) 전사유전자를 포함한 다양한 유전자가 관여할 것이라는 가능성을 제시하였다. 이에 본 연구는 칼륨제한 식이 기간에 따른 흰쥐 신장 내 Akt와 p-Akt 및 ERK와 p-ERK의 발현 및 분포의 변화를 Western 분석과 면역조직화학 방법으로 관찰함으로써, 저칼륨혈증이 신장에서 AKT/ERK 인산화에 영향을 미치는지를 확인하였다. Western 분석소견에서 Akt 및 p-Akt 단백질 발현은 칼륨제한 식이가 진행될수록 증가하는 양상을 보였으며, ERK 및 p-ERK 단백질발현은 칼륨제한 식이 2주군에서 정상 식이군에 비해 약간 증가하는 양상을 보였다. 면역조직화학 소견에서 Akt 단백의 면역반응성은 먼쪽곱슬세관, 겉질곧은부분 및 속질곧은부분에서 중등도의 발현을 보였다. 칼륨제한 식이군의 Akt의 면역반응성은 칼륨제한 식이가 진행될수록 바깥속질집합관에서 현저히 증가하였다. 칼륨제한 식이군의 p-Akt의 면역반응성은 칼륨제한 식이 2주군의 먼쪽곱슬세관, 치밀반점과 속질곧은부분에서 증가하였고 토리쪽곱슬세관에서는 중등도의 발현을 보였다. 칼륨제한 식이군의 ERK의 면역반응성은 칼륨제한 식이 2주군과 3주군의 바깥속질집합관에서 현저히 증가하였으며 먼쪽곱슬세관, 겉질집합관에서는 증등도의 증가를 보였다. 칼륨제한 식이군의 p-ERK의 발현부위는 정상식이군과 비교해 차이가 없었으나 면역반응성은 칼륨제한 식이 2주군의 바깥속 질집합관의 핵에서 현저히 증가하였다. 이상의 결과 저칼륨혈증 시 p-Akt의 발현은 칼륨제한 식이가 길어질수록 점진적으로 증가하였지만, p-ERK의 발현은 칼륨제한 식이 2주군에서 현저히 증가되었다. 따라서 저칼륨 상태에서의 Akt 및 ERK 인산화의 촉진은 이온채널 및 이온수송체 유전자 조절뿐만 아니라 세포 내 신호전달에 있어 중요한 역할에 관여할 것임을 시사해 주었다. Hypokalemia causes metabolic alkalosis and morphological changes of the kidney. K+ balance is regulated not only by ion channels or pump gene, but also by various genes including NF-E2-related factor 2 (Nrf2). Previous study suggested the possibility that Akt and ERK kinase may be involved in Nrf2 transcriptional gene activation. In present study, we investigate the alterations of Akt, p-Akt, ERK, p-ERK protein in both normal kidney and K+-deficient diet kidney using Western blot analysis, and immunohistochemisrty. Our western blot data showed that the expression of Akt and p-Akt was increased gradually in K+-depleted diet (from 1W-3W) compared to normal group. The expression of ERK and p-ERK was markedly increased in K+-depleted diet 2W in comparison with normal group. Based on our immunostaining results, Akt protein immunoreactivity was prominently increased in outer medullary collecting duct, especially in K+-depleted diet 2 weeks. The localization of p-Akt proteins in K+-depleted groups was not different from normal group, but the immunoreactivity was significantly increased in distal convoluted tubule, macula densa and outer medullary thick ascending limb in K+-depleted diet 1 and 2 weeks groups. ERK protein immunoreactivity was prominently increased in outer medullary collecting duct, especially in K+-depleted diet 2 and 3 weeks. The localization of p-ERK proteins in K+-depleted groups was not different from normal group, but the immunoreactivity was prominently increased in the nucleus of outer medullary collecting duct especially in K+-depleted diet 2 weeks. Taken together, we suggest that the expression of p-Akt was gradually increased in K+-depleted groups of kidney, but the expression of p-ERK was markedly increased in K+-depleted diet 2 week group. Hence, the promotion of AKT and ERK phosphorylation in hypokalemic condition may be involved in the regulation of ion channels, ion transporters and subsequent intracellular signal transduction.

당뇨유발 흰쥐 질 조직에서 Insulin 투여 효과

이상녕(Sang Nyeong Lee),이송은(Song Eun Lee),배미옥(Mi Ok Bae),남광일(Kwang IL Nam),박광성(Kwang-Sung Park),배춘상(Choon Sang Bae),김백윤(Baik Yoon Kim),박성식(Sung Sik Park),안규윤(Kyu Youn Ahn) 대한해부학회 2005 Anatomy & Cell Biology Vol.38 No.3

당뇨병을 가진 여성의 주된 성적문제는 질 윤활작용의 감소와 성적 각성장애이고 이는 질 조직의 섬유화와 관련이 있다고 한다. 본 연구는 streptozotocin (65 mg/kg)투여 당뇨유발 군과 당뇨유발 후 insulin 투여 군 흰쥐에서 질 조직의 변화를 조직화학, Western분석 및 면역조직화학 기법을 통해 관찰하고자 하였다.평균 혈당농도는 대조군에서 79±16 mg/dL, 당뇨유발 군에서 453±188.4 mg/dL 그리고 insulin 투여군에서 56.7±20.6 mg/dL 였다. H-E 소견에서 대조군의 질벽은 6~11층의 중층편평상피, 민무늬근육, 결합조직 및 다수의 혈관들로 구성되어 있었다. 당뇨유발 군에서는 중층편평상피 층이 2~6층으로 감소하였으며 또한 민무늬근육과 혈관의 수와 크기도 감소하였다. Insulin 투여 군은 대조군의 소견과 유사하였다. 대조군의 Masson’s trichrome 염색소견에서는 보라색으로 염색되는 민무늬근육이 상당량 관찰되었고 청색으로 염색되는 아교섬유는 규칙적으로 분포되었다. 당뇨유발 군에서는 아교섬유가 증가되어 성긴결합조직이 치밀결합조직으로 변하였으며, 아교섬유의 배열도 불규칙하였다. Insulin 투여 군은 대조군의 소견과 유사하였다. Western분석 결과 당뇨유발 군에서의 TGF-β1의 발현은 현저히 증가하였으나 Ec-NOS의 발현은 감소하였다. TGF-β1에 대한 면역양 성 반응은 섬유모세포와 아교섬유에서 관찰되었고 당뇨유발 군에서 Western분석 소견과 같이 현저히 증가하였으며 대조군과 insulin 투여 군에서는 미약하였다. Ec-NOS에 대한 면역양성 반응은 혈관의 내피세포에서 관찰되었으며, 면역반응성은 Western분석 소견과 같이 대조군과 insulin 투여 군에서는 현저하였으나 당뇨유발 군에서는 상당히 감소하였다. 대조군과 insulin 투여 군의 estrogen receptor α에 대한 면역양성 반응은 중층편평상피의 바닥층과 중간층, 민무늬근육세포 및 신경에서 관찰되었으나 당뇨유발 군에서는 중층편평상피의 중간층, 민무늬근육세포 및 신경에서만 관찰되고 반응성도 감소하였다. 이상의 결과로 미루어 당뇨유발 흰쥐 질 조직의 섬유화는 TGF-β1, NOS 및 estrogen 등과 관련이 있을 가능성을 제시하였으며, 이로 인한 당뇨 여성의 성기능 장애는 혈당을 조절함으로써 예방할 수 있음을 암시하였다. The most commonly reported sexual problems in diabetic women are sexual arousal disorder and a lack of vaginal lubrication. The aims of this study were to investigate the vaginal structural changes and expressions of TGF-β1, Ec-NOS and estrogen receptor α by histochemistry, immunohistochemistry and Western blot analysis in diabetic and insulin-treated diabetic rats. The mean blood glucose levels were significantly increased in the diabetic rats (453±188.4 mg/dL) compared to the control group (79±16 mg/dL) and insulin-treated diabetic rats (56.7±20.6 mg/dL). The vaginal wall in control rat showed 6~11 layered stratified squamous epithelial lining and submucosal smooth muscle, connective tissue and vasculatures. In diabetic rat, the vaginal epithelium was reduced to 2~6 layers and the submucosal vasculatures were decreased in size and number. Collagen fibers were increased and irregularly distorted arrangement. Insulin-treated diabetic rat showed similar morphologic features as control rat. In diabetic rat, TGF-β1 expression was upregulated by 1.65 times and Ec-NOS expression was 40% downregulated compared to control and insulin-treated diabetic rats in Western blot analysis. In control and insulin-treated diabetic rats, TGF-β1 immunoreactivity was detected in fibroblasts and the collagen fibers, Ec-NOS immunoreactivity in the endothelial cells of blood vessels, and estrogen receptor α immunoreactivity in the basal and intermediate cell layers of stratified squamous epithelium, smooth muscle fibers, and nerve fibers. In diabetic rat, expression of TGF-β1, Ec-NOS, and estrogen receptor α was exhibited comparable cellular patterns of labeling, but signal intensity was increased in TGF-β1 and decreased in Ec-NOS and estrogen receptor α. These results suggest that vaginal tissue fibrosis in diabetes mellitus may be caused by altered expression of TGF-β1, NOS and estrogen. It also implies that sexual arousal disorder and lack of vaginal lubrication in the diabetic women could be protected or delayed by controlling blood glucose levels.

원숭이 외측슬상체배측핵에서 칼슘결합단백 Parvalbumin과 Calbindin-D 28K의 분포

고승희,배춘상,박성식,Ko, Seung-Hee,Bae, Choon-Sang,Park, Sung-Sik 한국현미경학회 1994 Applied microscopy Vol.24 No.4

The calcium-binding proteins (CaBP), parvalbumin (PV) and calbindin-D 28K (calbindin) are particularly abundant and specific in their distribution, and present in different subsets of neurons in many brain regions. Although their physiological roles in the neurons have not been elucidated, they are valuable markers of neuronal subpopulations for anatomical and developmental studies. This study is designed to characterize dorsal lateral geniculate nucleus (dLGN) neurons and axon terminals in terms of differential expression of immunoreactivity (IR) for two well-known CaBPs, PV and calbindin. The experiments were carried out on 6 adult monkeys. Monkeys were perfused under deep Nembutal anesthesia with 2% paraformaldehyde and 0.2% glutaraldehyde in 0.1M phosphate buffer. After removal, the brains were postfixed for 6-8 hr in 2% paraformaldehyde at $4^{\circ}C$ and infiltrated with 30% sucrose at $4^{\circ}C$. Thereafter, they were frozen in dry ice. Serial sections of the thalamus, at $20{\mu}m$, were made in the frontal plane with a sliding microtome. The sections were stained for PV and calbindin with indirect immunocytochemical methods. For electron microscopy, after infiltration with 30% sucrose the blocks of thalamus were serially sectioned at $50{\mu}m$ with a Vibratome in the coronal plane and stained immediately by indirect ABC methods without Triton X-100 in incubation medium. Stained sections were postfixed in 0.2% osmium tetroxide, dehydrated and flat-embedded in Spurr resin. The block was then trimmed to contain only a selected lamina or interlaminar space. The dLGN proper showed strong PV IR in fibers in all laminae and interlaminar zones. Particularly dense staining was noted in layers 1 and 2 that contain many stained fibers from optic tract. Neuronal cell body stained with PV was concentrated only in the laminae. In these laminae staining was moderate in cell bodies of all large and medium-sized neurons, and was strong in cell bodies of some small neurons together with their processes. Calbindin IR was marked in the neuronal cell body and neuropil in the S layers and interlaminar zones whereas moderate in the neuropil throughout the nucleus. Regional difference in distribution of PV and calbindin IR cell is distinct; the former is only in the laminae and the latter in both the S layer and interlaminar space. The CaBP-IR elements were confined to about $10{\mu}m$ in depth of Vibratome section. The IR product for CaBP was mainly associated with synaptic vesicle, pre- and post-synaptic membrane, and outer mitochondrial membrane and along microtubule. PV-IR was noted in various neuronal elements such as neuronal soma, dendrite, RLP, F, PSD and some myelinated or unmyelinated axons, and was not seen in the RSD and glial cells. Only a few neuronal components in dLGN was IR for calbindin and its reaction product was less dense than that of PV, and scattered throughout cytoplasm of soma of some relay neurons, and was also persent in some dendrite, myelinated axons and RLP. The RSD, F, PSD and glial elements were always non-IR for calbindin. Calbindin labelled RLP were presynaptic to unlabeled dendrite or dendritic spine and PSD. Calbindin-labeled dendrite of various sizes were always postsynaptic to unlabeled RSD, RLP or F. From this study it is suggested that dLGN cells of different functional systems and their differential projection to the visual cortex can be distinguished by differential expression of PV and calbindin.

생쥐에서 Pilocarpine에 의한 경련이 해마의 신경세포사멸, 이끼섬유발아 및 신경발생에 미치는 영향

이상현(Sang-Hyeon Lee),국지현(Ji-Hyun Kook),황신애(Shinae Hwang),김종근(Jong-Keun Kim),배춘상(Choon-Sang Bae) 대한체질인류학회 2011 해부·생물인류학 (Anat Biol Anthropol) Vol.24 No.4

해마 치아이랑은 지속적인 경련 후 이끼섬유발아(mossy fiber sprouting)가 일어나는 부위일 뿐만 아니라 성체 뇌의 신경발생(neurogenesis)이 일어나는 부위로 많은 연구의 대상이 되고 있다. 본 논문은 pilocarpine에 의한 발작을 이용한 해마 및 치아이랑에서 신경세포사멸, 이끼섬유발아 및 신경발생의 변동을 조사하여 이들 3자간의 상관관계를 규명하고자 하였다. 수컷 ICR 생쥐와 C57BL/6 생쥐에 pilocarpine을 처리하여 발작을 유발하였으며 처음 4시간 동안 발작양상을 관찰하여 Racine의 5등급 scale에 중 적어도 3등급 이상인 동물을 실험에 사용하였다. 신경세포사멸은 Fluoro-Jade C(FJC) 염색으로, 이끼섬유발아는 NeoTimm 염색으로, 신경발생은 bromodeoxyuridine(BrdU) 주사 후 이에 대한 면역조직염색으로 평가하였다. 본 실험에서 사용한 모든 동물에서 pilocarpine(300 mg/kg, ip) 투여에 의해 3등급 이상의 발작을 일으켰으며 발작 반응은 ICR 생쥐에서 C57BL/6 생쥐에서 보다 더 강하게 나타났다. 즉 ICR 생쥐의 평균 발작 등급이 4.37로 C57BL/6 생쥐의 3.22보다 유의하게 강하였다. 그러나 3등급 이상의 발작이 시작하는데 걸린 시간은 15~20분 사이로 차이가 없었다. 약물주입 4시간 후 ICR 생쥐에서는 FJC염색 상 해마 문(hilus)에서 사멸세포가 관찰되었으나, C57BL/6 생쥐에서는 관찰되지 않았다. 24시간 이후부터는 두 strain 모두에서 CA1 및 CA3 피라밋세포의 사멸도 관찰되었다. 이 세포사멸은 약물투여 후 3일에 가장 강하였으며 l주일 후에는 거의 관찰할 수 없었다. 신경발생을 나타내는 해마의 BrdU 양성세포수는 대조동물의 경우 C57BL/6 생쥐가 ICR 생쥐보다 유의하게 많았다. BrdU 양성세포수는 약물투여 2일 후에 대조군에 비해 유의하게 증가하였으며, 8일 후에는 더 큰 증가를 보인 후 15일 후에는 대조군 수준으로 돌아왔다. 두 strain 모두에서 약물투여 후 해마 신경발생의 증가를 보였으나 대조동물과의 비로 비교하면 ICR 생쥐가 C57BL/6 생쥐에 비해 더 큰 증가를 보였다. 이끼섬유발아는 약물투여 2주 후에 약하게 나타나서 4주 후에는 더 강해졌으며 두 strain 간에 차이는 관찰 할 수 없었다. 이상의 성적은 pilocarpine에 의한 발작의 정도와 신경세포사멸 및 신경발생 간에는 서로 상관관계가 있지만 이들과 이끼섬유발아 간에는 직접적인 상관관계가 없음을 시사한다. Present study was performed to delineate the inter-relationship among neuronal death, mossy fiber sprouting (MFS) and neurogenesis in hippocampal formation of pilocarpine-treated mice. Status epilepticus was induced by intraperitoneal administration of 300 mg/kg pilocarpine in male ICR and C57BL/6 mouse. The severity of seizure was evaluated using 5 grades of Racine scales for first 4 hr after pilocarpine injection. Fluro-Jade C (FJC) staing, NeoTimm"s staining and immunohistochemistry for BrdU were employed to evaluate neuronal cell death, MFS and neurogenesis, respectively. All animals in the present study induced seizures over grade 3 of Racine scale by pilocarpine injection. ICR mice show higher seizure severity (mean Racine scale; 4.37) than C57BL/6 mice do (mean Racine scale; 3.22), while the latency times for the first seizure over Racine scale grade 3 are from 15 min to 20 min and showed no difference between the 2 strains. In ICR mouse, numerous FJC-positive cells in hilus of hippocampus were detected at 4 h after pilocarpine injection, while they were not detected at that time in C57BL/6 mouse. The number of FJC-positive neuronal cells, which were densely found in the pyramidal layer of CA1, CA3 and hilus polymorphic regions of hippocampus, reached peak at 3 days after injection and then few cells were found at 7 days after injection in both strains. In control animals, BrdU positive cells in dentate subgranular layer which represent the hippocampal neurogenesis were more numerous in C57BL/6 than in ICR. The number of BrdU positive cells significantly increased at 2 days after pilocarpine injection and reached the peak at 8 days after injection and returned to control level at 15 day after injection in both strains. The percent increase of the BrdU positive cell was more prominent in ICR mouse. MFS was found at 2 weeks after the injection and the intensity of MFS was getting strong at 4 weeks after injection. There was no differences in MFS grading between 2 strains. These results suggest that there are some inter-relationships among the seizure severity, hippocampal neuronal cell death and hippocampal neurogenesis, but they don"t have any significant relationships with the mossy fiber sprouting from dentate granule cells.

고양이 송과체의 전자현미경적 연구

최재권,배춘상,오창석,이정헌,Choi, Jae-Kwon,Bae, Choon-Sang,Oh, Chang-Seok,Lee, Jung-Hun 한국현미경학회 1992 Applied microscopy Vol.22 No.1

Parenchyma of the cat pineal body consisted of pinealocytes and glial cells. The pinealocyte, predominant cell type, was characterized by having large mitochondria with pale matrix, abundant polyribosomes, moderately-developed Golgi apparatus, centrioles and occasional cilia. The pinealocyte had one thick and long cytoplasmic process at the one pole of the cell, and slender and shorter processes at the other pole, and in addition occasional short processes from the cell body. These processes contained longitudinally arranged microtubules, and a few mitochondria. Thick processes teminated as bulgings either in the intercellular process-rich area, or in the perivascular border which was formed by glial cell processes. These endings of pinealocyte processes had many small vesicles, mitochondria, and occasional dense bodies. Glial cells with abundant filaments of intermediate type and clear cytoplasmic matrix were fibrous astrocyte. Perikarya of the astrocytes had small and dense mitochondria, moderately developed Golgi apparatus, dense bodies and variable amount of intermediate filaments. Glial cell processes run through the intercellular spaces among the pinealocyte processes. Glial cell of protoplasmic type had no or a few filaments, but it had well-organized rough endoplasmic reticulum, dense mitochondria, well developed Golgi apparatus and many dense granules. Intercellular canaliculi formed by adjacent pinealocytes and glial cell processes were often noted. Within the parenchyma, sympathetic and parasympathetic axons and their endings were noted. These endings were present mostly in the intercellular spaces without having membrane specialization, however, in rare instances, ending with small clear and dense cored vesicles, and large dense cored vesicles formed specialized synapse with a pinealocyte process. Within the perivascular spaces nerve fibers and endings, Schwann cells and pericyte were noted. In rare case pinealocyte process penetrated into the perivascular space through the interuptions of glial border. These results suggest that pinealocyte of the cat has less significance in secretory function and is rather neural type of cell.

인태아(人胎兒) 수핵(髓核) 발육(發育)에 관(關)한 전자현미경적(電子顯微鏡的) 연구(硏究)

윤재룡,배춘상,김은경,Yoon, Jae-Rhyong,Bae, Choon-Sang,Kim, Eun-Kyung 한국현미경학회 1991 Applied microscopy Vol.21 No.2

The development of notochordal cells of nucleus pulposus was studied with electron microscope in human fetuses ranging from 30 mm to 260 mm crown-rump length. At 30 mm fetus, primitive notochordal cells were large with central nucleus, few organelles, and their cytoplasm usually contained dense glycogen and fine filaments. Notochordal cells at all ages contained bundles of fine filaments of indeterminate nature. One unusual feature of fetal notochordal cells was the consistent presense of rough endoplasmic reticulum surrounding poorly developed mitochondria. At 50 mm fetus, notochordal cells formed dense masses with interdigitating cell membranes connected by a variety of cell to cell junctions. With increasing age, the cell connections became slender threaded cytoplamic extending from cell and enclosed large extracellular space. Chondrocyte-like cells appeared to be separated by large volumes of extracellular matrix. Viable notochordal and condrocyte-like cells existed in specimen from all age. The extracellular spaces were filled with fibrillar and granular material by 90 mm fetus. Necrotic cells were distinguished by loss of their membrane integrity, vacuolization of their organelles, and the presence of dense osmiophilic masses. In adult tissue, notochordal cells became rounded or irregular in shape and developed a pericellular matrix consisting of collagen fibrile, and dense particle. The structure of notochordal cells and their persistance in the nucleus pulposus after fetal life suggested that they may have a significant role in the formation and maintenance of the nucleus pulposus. The presence of Golgi complex and well-developed endoplasmic reticulum in chondrocyte-like cells suggested that they are capable of producing and maintaining the extracellular matrix.

저칼륨혈증 흰쥐에서 GTF IIA 유전자의 발현

이창배(Chang Bae Lee),이용찬(Yong Chan Lee),조혜정(Hye Jung Cho),이송은(Song Eun Lee),배춘상(Choon Sang Bae),김백윤(Baik Yoon Kim),박성식(Sung Sik Park),안규윤(Kyu Youn Ahn) 대한해부학회 2008 Anatomy & Cell Biology Vol.41 No.1

저칼륨혈증시 칼륨평형 조절에 이온채널이나 pump 유전자 이외에도 다양한 유전자가 관여할 것으로 생각된다. 본 연구는 유전자를 탐색하는데 유용하게 쓰이는 DNA chip microassay 방법을 이용하여 General transcription factor IIA(GTF IIA) 유전자를 탐색하고 이 유전자의 발현 및 분포를 Northern 분석과 in situ hybridization 조직화학 방법으로 관찰하였다. Northern 분석소견에서 GTF IIA 유전자는 고환에서 제일높게 발현되었고 심장, 허파, 콩팥, 부신, 지라 및 간에서는 중등도로 발현되었으며, 뇌, 먼쪽주름창자, 침샘, 위 및 샘창자에서는 미약하였다. 칼륨식이 변화에 따른 콩팥, 지라 및 부신에서의 GTF IIA 유전자의 발현은 점차 감소하다가 3주째에는 회복되는 경향을 보였다. 그러나 허파에서는 칼륨제한 식이 1주와 3주에서는 증가하였고 2주에서는 오히려 감소하였다. in situ hybridization 조직화학소견에서 정상 식이 군의 콩팥에서 GTF IIA 유전자의 발현은 겉질의 S3 segment, 먼쪽곱슬세관 및 겉질집합관에서만 관찰되었고, 속질에서는 관찰할 수 없었다. 칼륨제한 식이군에서는 S3 segment, 먼쪽곱슬세관, 겉질집합관, 바깥속질집합관 및 속속질집합관에서도 발현되었다. 발현정도는 겉질에서 칼륨제한 식이 2주까지는 상당히 감소하였으나 3주에서는 회복되는 경향이었고, 바깥속질 및 속속질집합관에서는 1주부터 증가하였다. 부신에서는 토리층에서만 발현되었고 칼륨제한식이군에서는 감소하다가 3주째에 회복되었다. 지라에서는 가장자리 구역과 종자중심의 림프구에서 발현되었고 발현정도는 부신과 유사하였다. 허파에서는 기관지상피세포와기관지 주위 림프구에서 발현되었다. 발현정도는 칼륨제한식이 3일군과 2주군에서는 감소하였으나 1주와 3주에서는 증가하는 소견을 보였다. 정상 식이군의 먼쪽주름창자와 위에서는 기저부의 창자샘에서만 관찰되었고 고환에서는 정세관의 기저부 정조세포와 정모세포에서 관찰되었다. 이상의 소견은 GTF IIA 유전자 발현이 조직에 따라 다르며, 저칼륨혈증 콩팥의 바깥속질 및 속속질집합관에서 증가하여 이들 부위에서 이 유전자가 이온수송체 유전자 조절에 관여하고 있음을 시사해 주었다. Potassium balance in chronic hypokalemia is regulated by ion channels, ion transporters, and various related genes. We isolated general transcription factor IIA (GTF IIA) gene using a DNA chip microassay, a useful method in cloning genes. Northern analysis and in situ hybridization (ISH) were carried out to analyze the expression and localization of GTF IIA mRNA in rat in relation to the amount of potassium in the diet. Isoform-specific 32P-labeled cDNA (Northern analysis) or digoxigenin-labeled cRNA (ISH) probes were used. Northern analysis demonstrated that GTF IIA mRNA was expressed abundantly in testis; modestly in heart, kidney, lung, adrenal gland, liver, and spleen; and weakly in brain, distal colon, duodenum, salivary gland, and stomach. In potassium-restricted animals, GTF IIA expression was decreased in the kidney, adrenal gland, and spleen, but expression was restored to normal levels in L3w. The expression level in the lung was decreased in L3d and L2w, and increased in L1w and L3w. ISH showed that mRNA for the GTF IIA gene was detected in the distal convoluted tubule, S3 segment of the proximal tubule, and cortical collecting duct in the normal group. In potassium-restricted groups, the hybridization signal was detected in the distal convoluted tubule, S3 segment of the proximal tubule, and entire collecting tubule. The signal intensity of the outer and inner medullary collecting ducts was higher in the potassium-restricted group than in the normal group but was decreased in the distal convoluted tubule and S3 segment of the proximal tubule. In the normal group, mRNA of the GTF IIA gene was detected in the zona glomerulosa cells of the adrenal gland, lymphocytes of the marginal zone, germinal center of the spleen, and bronchial epithelium and lymphocytes of the lung. mRNA for the GTF IIA gene was also detected in the cells of the basal portion of the intestinal glands of the distal colon and stomach, and in spermatogonia and spermatocytes of the seminiferous tubule. These results suggest that expression of GTF IIA differs between various tissues and that increased expression of the GTF IIA gene in the outer and inner medullary collecting ducts of the hypokalemic kidney might regulate the ion transporter genes in these segments.

저칼륨혈증 흰쥐 신장에서 Aquaporin mRNA와 단백 발현의 변화

김대성(Dae Sung Kim),이창배(Chang Bae Lee),배춘상(Choon Sang Bae),김백윤(Baik Yoon Kim),박성식(Sung Sik Park),안규윤(Kyu Youn Ahn) 대한해부학회 2009 Anatomy & Cell Biology Vol.42 No.1

There has been a general agreement that potassium depletion causes metabolic alkalosis and substantial morphological changes in kidney structure, and is associated with renal functional abnormalities, including a decrease in urinary concentrating ability. The present study was to examine the alterations of expression and distribution of AQP-1, 2, 3 and 4 mRNAs and proteins in the kidneys of normal and K-depleted rats using RT-PCR, Western blot analysis, and immunohistochemistry. Predicted size of AQP-1, 2, 3, and 4 mRNAs was 119, 822, 539, and 642 bp, respectively. AQP-1 mRNA expression was gradually decreased in K-depleted rats, particularly LK 2W. AQP-2, 3 mRNAs were markedly decreased in K-depleted rats. AQP-4 mRNA expression was markedly increased in K-depleted rats, particularly LK 2W. Western blot analysis demonstrated that AQP-1 protein expression was only decreased in LK 3D and others were comparable with normal rat. AQP-2, 3 proteins expression was markedly decreased in K-depleted rats, compared with normal rat. But, AQP-4 protein expression was markedly increased in K-depleted rats, particularly LK 3W. In immunohistochemistry, AQP-1 was detected in the apical membranes of proximal tubules and thin limb of Henle loop. In potassium-depleted kidney, the pattern of cellular labeling and signal intensity of AQP-1 protein is identical to that of normal rat. AQP-2 was detected in apical region and cytoplasm of the principal cells of entire collecting duct. In potassium-depleted kidney, the pattern of cellular labeling of AQP-2 protein is identical to that of normal rat, but signal intensity is markedly decreased. AQP-3 was detected in the bosolateral plasma membrane of principal cells of entire collecting duct. In potassium-depleted kidney, the pattern of cellular labeling of AQP-3 protein is identical to that of normal rat, but signal intensity is markedly decreased. AQP-4 was detected in the bosolateral plasma membrane of principal cells of entire collecting duct. In potassium-depleted kidney, the pattern of cellular labeling of AQP-4 protein is identical to that of normal rat, but signal intensity is markedly increased in outer and inner medullary collecting ducts. In summary, these results demonstrate that chronic hypokalemia shows the different expression pattern of AQP-1, 2, 3, and 4 mRNAs and proteins. These results suggest that a decrease in urinary concentrating ability is a major factor in the decreased AQP-2, 3 expression, and that is partly compensated by increased expression of AQP-4. 본 연구는 칼륨제한 식이 기간에 따른 흰쥐 신장 내 AQP-1, 2, 3 및 4의 발현 및 분포변화를 RT-PCR, Western 분석과 면역조직화학 방법으로 관찰하였다. RT-PCR 분석 소견에서 AQP-1 mRNA는 예견된 크기인 199 bp 근처에서 관찰되었고 칼륨제한 식이 군에서 정상 식이 군에 비해 감소하였다. Western 분석 소견에서 AQP-1 단백은 칼륨제한 식이 3일 군에서만 정상 식이 군에 비해 감소하였고 그 이후에는 유사하였다. AQP-2, 3 mRNA는 RT-PCR 분석 소견에서 예견된 크기인 822 bp, 539 bp 근처에서 관찰되었으며 정상 식이 군에 비해 칼륨제한 식이 군에서 뚜렷한 감소 경향을 나타냈었다. Western 분석 소견에서도 AQP-2, 3의 단백발현은 칼륨제한 식이 군에서 정상식이 군에 비해 크게 감소하는 것으로 나타났다. AQP-4 mRNA는 RT-PCR 분석 소견에서 예견된 크기인 624 bp 근처에서 관찰되었으며 다른 AQP과는 달리 정상 식이 군에 비해 칼륨제한 식이가 진행될수록 증가하는 양상을 보였다. Western 분석 소견에서도 AQP-4 단백은 발현양이 극히 낮았지만 칼륨제한 식이가 진행될수록 증가하는 경향이였다. 면역조직화학 소견상 AQP-1의 면역반응성은 토리쪽세관과 Henle 고리 내림부분의 첨부세포막에서 관찰되었다. 칼륨제한 식이 군의 AQP-1의 발현부위는 변화가 없었으나 발현 정도는 약간 감소하였다. AQP-2, 3의 면역반응성은 전 집합관에서 관찰되었다. 칼륨제한 식이 군의 AQP-2, 3의 발현부위는 변화가 없었으나 발현 정도는 현저히 감소하였다. AQP-4의 면역반응성은 전 집합관에서 발현되었다. 칼륨제한 식이 군의 AQP-4의 발현부위는 변화가 없었으나 발현 정도는 바깥속질집합관과 속속질 집합관에서 현저히 증가하였다. 이상의 결과로 저칼륨혈증시 신장 AQP-1, 2, 3, 4 mRNA와 단백발현 양상은 상호간에 차이를 나타내며, 요농축능의 감소는 AQP-2, 3에 의해 주로 일어나나 AQP-4가 발현의 증가로 보상적 기능을 할 것으로 생각되었다.

메기 송과체의 광수용세포 및 신경세포 돌기의 3차원적 구조

남광일,이송은,오창석,배춘상,박성식,Nam, Kwang-Il,Lee, Song-Eun,Oh, Chang-Seok,Bae, Choon-Sang,Park, Sung-Sik 한국현미경학회 2000 Applied microscopy Vol.30 No.3

메기 송과체를 대상으로 3차원적 전자현미경법을 이용하여, 송과체를 구성하는 신경세포 및 광수용세포 돌기들의 위치적 상호관계를 3차원적으로 구명하고자 하였다. 광수용세포의 돌기들은 서로 복잡하게 얽혀 있었으며, 다른 세포질내로 돌기의 일부가 파고드는 양상을 보이는 것도 있었다. 신경세포 돌기는 이들 읽혀 있는 광수용세포 돌기 사이를 주행하면서 여러군데서 리본연접을 형성하고 있었는데, 광수용세포내의 이들 리본연접의 수는 위치에 따라 다양하였다. 한편 송과강내에서는 탐식되기 전의 광수용세포 외절에 대식세포가 접근해 있는 것도 관찰되었다. 이상의 관찰결과, 메기 송과체의 광수용세포의 기저돌기는 다양한 형태이며, 특히 인접부위로 주행하는 신경세포의 돌기와 연접을 형성하기 위해 또 다른 가지를 내고 있음을 알 수 있었다. The topographic correlation between the processes of photoreceptor cells and nerve cells in the pineal organ of catfish, Parasiluns asotus, was studied using 3D electron microscopy. Upon examination, one neuronal cell process was found to pass through the intertwined processes of the photoreceptor cells. Interestingly , we observed two photoreceptor processes interlock, after which two buds from one process penetrated the other. Synaptic ribbons were observed in the cytoplasm of the photoreceptor cella, especially near the neuronal process. Macrophages were occasionally found to be contact with the outer segments of the photoreceptor cells in the pineal lumen.

한국인 중족골 골간부 영양공에 관한 연구

천종익(CHEON Jong Ik),배춘상(BAE Choon Sang) 대한체질인류학회 1996 대한체질인류학회지 Vol.9 No.1

간추림. 한국인 성안 중족골 197개를 대상으로 성영 구별 없이 골간부 영양공의 수, 위치 빛 진입망향 둥을 육안적으로 관찰하여 다음과 같은 결과를 얻었다. 충족골 공간부에는 대부분 하나의 영양공이 중간부에 위치하고 있어 공치수는 평균 43~51 이었으며, 제1중즉골의 약 73% 에서는 두 개 이상의 영양공이 출현하였다. 제 1, 2 중족골에서는 영양공이 외측면에 더 많이, 제 3, 4 중족골에서는 내, 외측면에 비슷하게, 제5중족골에서는 주호 내측면에 출현하는 경우가 않았다. 영양공의 진입방향은 제1중족골에서는 두부를, 나머지 중족골에서는 기저부를 향하고 있었으며, 진입방향이 항상 일정함에 대한 설명으로 성장골단설이 타당하였다.