Cyclin A와 LATS 유전자들의 난소 내 mRNA 및 단백질 발현에 관한 연구

박창은 ( Chang Eun Park ),김대중 ( Dae Jung Kim ),홍성노 ( Sung No Hong ) 대한임상검사과학회 2008 대한임상검사과학회지(KJCLS) Vol.40 No.1

Despite of the importance of the primordial follicle (PMF) recruitment, factors and mechanisms for process are poorly understood. To evaluate expression and role of the follicular transition from PMF to PMF/primary follicles (PMIF) in the present study, we evaluated expression of lats1, lats2, cyclin A1, and cyclin A2 mRNA and protein, and elucidated and role of lats1-cyclin A in the follicular transition from PMF to PRIF. To analysis of differential expression in PMF and PMIF, each stage follicles were collected by day1 and day5 of immuno-compromised rats (ICR) and analyzed by real-time PCR for the genes. For localization of mRNAs and proteins of the genes, in situ hybridization and immunohistochemistry were performed. We confirmed that the lats1, lats2, cyclin A1, and cyclin A2 mRNA were more expressed in PMF than PMIF. Localization of the four genes expression were observed in nuclei of oocytes from the arrested primordial, and in the surrounding granulosa cells of the growing follicles. The mRNA expressions were gradually decreased with follicular development. From immunohistochemistry studies, Cyclin A1 protein expression were observed in oocyte cytoplasmas of early stage follicles, while observed in granulose cells and oocyte nucleoli during growing follicles. This study suggested that the presence of lats gene family might perform negatively regulation of cell proliferation by modulation of the CDC2/Cyclin A complex activity. lats-cyclin A genes in oocytes of the early stage follicles might play a role in the meiotic cell cycle arrest of the primary oocytes at the primordial follicle stage as well as the follicular growth.

세포주기와 관련된 유전자들의 난소 내 mRNA 및 단백질 발현

박창은 ( Chang Eun Park ),홍성노 ( Sung No Hong ) 대한임상검사과학회 2006 대한임상검사과학회지(KJCLS) Vol.38 No.1

Wee1 is a kinase regulator of the M-phase promoting factor (MPF; a complex of cdc2 and cyclin B1). The present study was undertaken to determine the role(s) of wee1 in the early stages of mouse ovarian follicles. The expression of wee1 and the correlated cell-cycle components, namely cdc2, cyclin B1, and cdc25C, were evaluated by immunohistochemistry. In addition, the expression of Tyr15-phosphorylated cdc2 (cdc2-p) was also examined to determine whether wee1 kinase phosphorylates cdc2 existed. Each component except cdc25C was found cytoplasmic in the oocytes at all stages of follicles, while cdc25C was not detected in primordial follicles. It was found primarily in ovarian somatic cells and to a small extent in granulosa cells of the growing follicles. To further confirm the expression of cell-cycle components in the primordial follicular oocytes, day1 ovaries were enzymatically and mechanically dissociated, then oocytes were isolated from somatic including pre-granulosa cells, and we confirmed that cdc2-p was expressed in oocytes of primordial follicles. From the results of the present study, we concluded wee1, without the counteracting cdc25C, would cause meiotic arrest of oocytes by the inhibitory phosphorylation of cdc2. The expression of all these proteins in the granulosa cells of growing follicles may regulate their mitosis concurrently with the growth of oocytes and follicles.

류마티스 관절염 진단을 위한 검사의 유용성 비교

박창은 ( Chang Eun Park ) 대한임상검사과학회 2015 대한임상검사과학회지(KJCLS) Vol.47 No.4

류마티스 관절염은 주로 관절의 활막 세포의 증식, 병리학적 면역 반응과 관절의 진행성 파괴를 특징으로 하는 만성 염증성 질환이다. 이 질환은 중요한 사회적 건강 문제로 대두된다. 이 논문을 통해 분자수준에서 변화와 류마티스 관절염의 진단 또는 질병의 진행에 대한 발병 기전을 새로운 관점에서 발병기전을 제공한다. 또한 조기 진단과 류마티스 관절염의 예후에 도움이 될 것으로 보인다. 새로운 혈청학적 및 면역학적 바이오마커에 초점을 맞추고 있다. 따라서 넓게 질병 진행의 위험 환자를 식별하는 혈청학적, 생체 면역학적 요인 등을 규명하고 적용시키는 것을 도출할 수 있다. 진단검사 의학을 기반으로 하는 증거는 환자에게 최선의 결과를 제공할 수 있다. 마지막으로. 최근의 연구 데이터는 이 접근을 통해서 치료를 최적화하기 위해 조기 진단 및 치료에 도움이 되는 새로운 접근 방식으로 궁극적인 유용성을 정립하는데 도움이 될 것으로 사료된다. Rheumatoid arthritis (RA) is a chronic inflammatory disease, which is mainly characterized by disease of joints affected with synovial hyperplasia, pathological immune response, and progressive destruction; all of which represent an important social health problem. These provide new insights in its pathogenesis of rheumatoid arthritis diagnosis and disease progression in molecular changes. This review focuses on new serological and immunological markers which seem to be useful in early diagnosis and prognosis of rheumatoid arthritis. Therefore, such tests are widely conducted for serological biomarkers and the developments with such immunological factors to identify patients who are at risk for disease progression. This evidence of the disease based on laboratory medicine could provide the best outcome for patients. Finally, data from recent studies will help to refine the ultimate usefulness of this novel approach for early diagnosis, treatment, and helping clinicians to optimize therapy by using this approach.

PI3K/MAPK 경로 조절을 통한 Isoscopoletin의 U46619 유도의 혈소판 응집 억제 효과

박창은(Chang-Eun Park),이동하(Dong-Ha Lee) 한국식품영양과학회 2021 한국식품영양과학회지 Vol.50 No.3

혈관이 손상되면 출혈을 최소화하고 정상적인 순환을 유지하기 위해 빠른 지혈 반응이 일어나야 하는데 초기반응에서 혈소판 활성화 및 응집이 필수적이다. 그러나 비정상적이거나 과도한 혈소판 응집은 혈전증, 동맥 경화증 및 뇌졸중과 같은 심혈관 질환의 원인으로 작용하기도 한다. 따라서 심혈관 질환의 예방 및 치료를 위해서는 혈소판의 활성을 조절하여 혈소판 응집을 저해시킬 수 있는 물질을 찾는 것이 중요하다. 일반적으로 Scopolia 또는 Artemisia 속의 식물 뿌리가 함유하는 isoscopoletin은 알츠하이머병과 암에 대한 약리학적 효과가 보고되어 있지만 혈소판 응집 및 그 기전에 대한 효과는 불명확한 실정이다. 본 연구는 U46619에 의해 유도된 사람 혈소판 응집에 대한 isoscopoletin의 효과와 TXA2 생성, ATP 및 serotonin을 포함한 과립 분비에 미치는 영향을 연구하였다. 또한, 혈소판 응집에서 신호전달 과정에서 작용되는 인산화 단백질인 PI3K/Akt 및 MAPK 경로의 조절에 대한 isoscopoletin의 효과를 살펴보았다. 그 결과, isoscopoletin은 PI3K/Akt 및 MAPK의 인산화를 억제하여 TXA2 생성을 통한 혈소판 활성화를 억제하였고, ATP 및 serotonin을 포함한 세포 내 과립 분비를 감소시켰으며 최종적으로 혈소판 응집을 농도 의존적으로 억제시켰다. 따라서 본 연구는 isoscopoletin이 PI3K/Akt 및 MAPK의 인산화를 조절하는 항 혈소판 물질이며, 혈소판 유래 심혈관 질환의 예방 및 치료제로 가치가 있다고 판단된다. When blood vessels are damaged, platelet activation and aggregation reactions are essential for the hemostasis process. On the other hand, abnormal or excessive platelet aggregation can cause cardiovascular disease. Therefore, it is important to find a substance that can inhibit platelet aggregation for the prevention and treatment of cardiovascular disease. This study examined the effects of isoscopoletin on U46619-induced human platelet aggregation and the effect on TXA2 production and granule secretion, including ATP and serotonin, which are the major controls. Furthermore, this study examined the effects of isoscopoletin on the regulation of PI3K/Akt and MAPK, which are phosphoproteins that act in the signaling process in platelet aggregation. As a result, isoscopoletin inhibited the phosphorylation of PI3K, Akt, and MAPK, which dose-dependently inhibited platelet aggregation via TXA2 production and decreased intracellular granule secretion, including ATP and serotonin. This paper proposes isoscopoletin as an antiplatelet substance that regulates the phosphorylation of PI3K/Akt and MAPK, which is valuable as a prophylactic and therapeutic agent for cardiovascular diseases derived from platelets.

Mobile transposon-like element, clone MTi7:RNA interference를 이용한 역할 규명

박창은,신미라,전은현,조성원,이숙환,김경진,김남형,이경아,Park, Chang-Eun,Shin, Mi-Ra,Jeon, Eun-Hyun,Cho, Sung-Won,Lee, Sook-Hwan,Kim, Kyung-Jin,Kim, Nam-Hyung,Lee, Kyung-Ah 대한생식의학회 2003 Clinical and Experimental Reproductive Medicine Vol.30 No.4

Objectives: The present study was conducted to evaluate the mobile transposon-like element, clone MTi7 (MTi7) expression in the mouse ovary and to determine its role(s) in the mouse oocytes by RNA interference (RNAi). Methods: MTi7 mRNA expression was localized by in situ hybridization in day5 and adult ovaries. Double stranded RNA (dsRNA) was prepared for c-mos, a gene with known function as control, and the MTi7. Each dsRNA was microinjected into the germinal vesicle (GV) stage oocytes then oocyte maturation and intracellular changes were evaluated. Results: In situ hybridization analysis revealed that MTi7 mRNA localized to the oocyte cytoplasm from primordial to preovulatory follicles. After dsRNA injection, we found 43-54% GV arrest of microinjected GV oocytes with 68%-90% decrease in targeted c-mos or MTi7 mRNA. Conclusions: This is the first report of the oocyte-specific expression of the MTi7 mRNA. From results of RNAi for MTi7, we concluded that the MTi7 is involved in the germinal vesicle breakdown in GV oocytes, and MTi7 may be implicated with c-mos for its function. We report here that RNAi provides an outstanding approach to study the function of a gene with unknown functions.

Laser Captured Microdissection을 이용한 유전자 발현에 대한 연구 (I): RT-PCR을 위한 난자의 RNA 추출 및 증폭을 위한 최소한도의 확립

박창은,고정재,차광렬,이경아,Park, Chang-Eun,Ko, Jung-Jae,Cha, Kwang-Yul,Lee, Kyung-Ah 대한생식의학회 2001 Clinical and Experimental Reproductive Medicine Vol.28 No.3

Objective: Recently, microdissection of tissue sections has been used increasingly for the isolation of morphologically identified homogeneous cell populations, thus overcoming the obstacle of tissue complexity for the analysis cell-specific expression of macromolecules. The aim of the present study was to establish the minimal conditions required for the RNA extraction and amplification from the cells captured by the laser captured microdissection. Methods : Mouse ovaries were fixed and cut into serial sections (7 im thickness). Oocytes were captured by laser captured microdissection (LCM) method by using PixCell $II^{TM}$ system. The frozen sections were fixed in 70% ethanol and stained with hematoxylin and eosin, while the paraffin sections were stained with Multiple stain. Sections were dehydrated in graded alcohols followed by xylene and air-dried for 20 min prior to LCM. All reactions were performed in ribonuclease free solutions to prevent RNA degradation. After LCM, total RNA extraction from the captured oocytes was performed using the guanidinium isothiocyanate (GITC) solution, and subsequently evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) for glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). Results: With the frozen sections, detection of the GAPDH mRNA expression in the number of captured 25 oocytes were not repeatable, but the expression was always detectable from 50 oocytes. With 25 oocytes, at least 27 PCR cycles were required, whereas with 50 oocytes, 21 cycles were enough to detect GA PDH expression. Amount of the primary cDNA required for RT-PCR was reduced down to at least 0.25 $\grave{i}$ l with 50 oocytes, thus the resting 19.75 il cDNA can be used for the testing other interested gene expression. Tissue-to-slide, tissue-to-tissue forces were very high in the paraffin sections, thus the greater number of cell procurement was required than the frozen sections. Conclusion: We have described a method for analyzing gene expression at the RNA level with the homogeneously microdissected cells from the small amount of tissues with complexity. We found that LCM coupled with RT-PCR could detect housekeeping gene expression in 50 oocytes captured. This technique can be easily applied for the study of gene expression with the small amount of tissues.

Relationship of Stress Hormone and Neuroimmunological Response

Chang-Eun Park(박창은) 한국엔터테인먼트산업학회 2014 한국엔터테인먼트산업학회논문지 Vol.8 No.3

심리적 스트레스는 신경과 호르몬의 경로를 통해 면역 및 염증 세포의 기능을 조절할 수 있다. 스트레스는 또한 장관내 신경계의 내인계 인자에 의해 펩티드 호르몬 및 신경 전달 물질의 반응을 촉진시킨다. 세포 매개 면역 반응의 주된 근원은 백혈구이며 이로 인해 B세포, T 세포 그리고 이들로부터 방출되는 사이토카인이 가장 중요한 역할을 한다. 코르티솔과 연관된 사이토카인은 스트레스 매개성 면역 조절에 의해 방출된다. 스트레스에 의해 유도된 인자는 혈장에서 코르티솔을 증가시킨 반면에 인터루킨 (IL)-12/인터페론 (IFN)-γ, NK세포활성은 면역반응을 억제시킨다. 따라서 스트레스는 면역반응에 영향을 미치는 연관성이 있지만 향후 연구에서는 이들의 연관성을 위한 기전 연구가 요구된다. 향후, 스트레스와 관련된 면역반응의 질병관리 및 치료를 위한 바이오마커 적용의 구조체계에 대한 연구가 필요하다. Psychological stress can modulate immune and inflammatory cell function through neural and hormonal pathways. Stress also triggers the response of peptide hormones and neurotransmitters by intrinsic factors of the enteric nervous system. The major sources of cell-mediated immune response are leukocytes, whereby B and T cells and their release of cytokines play the most important role. A cortisol-related cytokine released by stress-mediated immune modulation. Stress-induced factors were increase in cortisol plasma levels, whereas, interleukin (IL)-12/Interferon (IFN)-γ and NK cell actiity were inhibits the immune system. Therefore, the job stresses are related to influenced immune responses but further research is needed to demonstrate for its mechanism relationships. Furthermore, the research needs framework the biomarker adoption for disease management, and treatment with stress related immune response.

프로칼시토닌의 수준에 따른 C-반응성단백과 혈구산정검사의 변화

김진산 ( Jin-san Kim ),박창은 ( Chang-eun Park ) 대한임상검사과학회 2022 대한임상검사과학회지(KJCLS) Vol.54 No.1

프로칼시토닌(procalcitonin, PCT)은 세균성과 바이러스성의 감염을 구별하기 위한 과학적 근거와 진단상의 중요한 단서를 제공한다. 프로칼시토닌이 높은 환자의 임상적 특성과 예후를 조사하고 임상적 진단을 향상시키고자 한다. 프로칼시토닌이 일반적인 집단에서 패혈증의 유병률과 연관성이 있는지를 결정한다. 이에 본 연구는 1년 동안 프로칼시토닌을 검사한 외래 환자(127명)의 C-반응성단백과 혈구산정검사의 결과를 기반으로 후향적 조사연구이다. 분석에 사용된 데이터는 프로칼시토닌, C-반응 단백 그리고 혈구산정검사이다. PCT의 결과가 높은 군의 CRP와 WBC의 양성율은 정상군보다 높게 나타났다(P<0.05). 프로칼시토닌 수준의 특이성과 민감도는 C-반응단백과 백혈구 수준보다 높게 나타났다(P<0.05). 또한. 프로칼시토닌의 결과값을 삼분위한 그룹의 분석에서는 낮은 군과 중간군의 값에 비해 높은 군의 값이 평균보다 높은 것으로 나타났다(P<.001). 상관분석에서는 프로칼시토닌은 C-반응단백, 백혈구(호중구, 림프구 포함)에서 양의 상관성을 보였다(P<.001). 이 연구의 주된 발견은 높은 프로칼시토닌은 C-반응성단백, 백혈구들의 수준에서 높은 연관성을 보이며 진단적 가치를 확인하였다. 따라서 이러한 관련 인자들은 환자의 감염진단과 항생제 치료에 중요한 진단적 및 치료적 영향을 미칠 것으로 사료된다. Procalcitonin (PCT) can provide an experimental rationale and a diagnostic lead to distinguish between bacterial and viral infections. This study sought to investigate the clinical characteristics and prognosis of patients with high PCT levels, to improve clinical diagnosis, and to determine whether PCT levels were associated with the subsequent development of sepsis in the general population. This was a retrospective observational study conducted on outpatients (N=127) over a year. The general data and laboratory parameters studied were PCT, C-reactive protein (CRP), and complete blood count (CBC). The positive rates of CRP and white blood cells (WBCs) in the elevated PCT group were higher than those of the normal group (P<0.05); the specificity and sensitivity of the PCT levels were obviously higher than those of the CRP and WBC levels at diagnosis (P<0.05). The mean PCT levels in the low group were significantly higher than those in the high or moderate group (P<0.001). There was a significant positive correlation with CRP, total WBCs, and neutrophils (P<.001). The main finding of this study was the significant association between an elevated PCT level and CRP and WBC levels, signifying a high diagnostic value. This has important implications for the diagnosis of bacterial infections and therapeutic implications for the use of antibiotic treatment in specific patients.

Collagen 유도의 사람 혈소판에서 Cyclic Nucleotides 생성 및 Ca<SUP>2+</SUP> 동원 조절을 통한 Hydroxygenkwanin의 항혈소판 작용

박창은(Chang-Eun Park),이동하(Dong-Ha Lee) 한국식품영양과학회 2023 한국식품영양과학회지 Vol.52 No.7

심장과 혈관에 영향을 미치는 것으로 알려진 심혈관 질환은 전 세계적인 주요 사망 원인으로 심혈관계 질환 치료를 위해 다양한 항혈소판제가 사용되고 있다. 최근에는 항혈소판제가 갖는 부작용 때문에 부작용이 적은 천연 생물 활성 화합물을 항혈소판 물질로 개발하기 위한 노력이 이루어지고 있다. 팥꽃나무(Daphne genkwa)의 주요 성분인 hydroxygenkwanin은 항균, 항염증 및 항암 효과와 함께 조직 인자를 억제하여 혈전증을 예방한다고 보고된 바 있지만 항혈소판 효과와 그 작용기전에 대해서는 거의 알려진 바가 없다. 본 연구는 hydroxygenkwanin이 collagen 유도의 사람 혈소판에 미치는 효과를 확인하였고, hydroxygenkwanin이 cAMP 및 cGMP 생성 증가 및 의존성 kinase의 활성화를 통해 VASP 및 IP3R이 인산화를 일으키는 것을 알 수 있었다. 이를 통해 세포질 내로의 Ca<SUP>2+</SUP> 동원을 억제하였고 fibrinogen 결합을 감소하였으며, hydroxygenkwanin에 의해 thrombin으로 유도한 fibrin clot의 형성이 억제되었다. 그러므로 본 연구를 통해 hydroxygenkwanin이 비정상적인 혈소판 활성화 및 혈전 형성으로 인하여 초래되는 심혈관 질환의 효과적인 예방 및 치료제로 개발될 가치가 있음을 제안한다. Cardiovascular disease (CVD) is a major cause of death and is treated using various antiplatelet agents. Recently, several efforts have been made to develop antiplatelet agents from natural biologically active compounds due to the side effects of the antiplatelet agent currently used. Hydroxygenkwanin is a major component of Daphne genkwa and has been reported to prevent thrombosis by inhibiting tissue factors and to have antibacterial, anti-inflammatory, and anticancer effects. However, little is known about its antiplatelet effect or mode of action. This study confirms the effect of hydroxygenkwanin on collagen-induced human platelets and reports that hydroxygenkwanin induces the phosphorylations of VASP and IP3R by increasing the productions of cAMP and cGMP and activating dependent kinases and thus inhibiting the translocation of Ca<SUP>2+</SUP> to cytoplasm, fibrinogen binding, and fibrin clot formation. Our findings suggest that hydroxygenkwanin has potential for the treatment of CVD by inhibiting platelet activation and thrombus formation.

진공채혈관의 상태에 따른 평가

박창은 ( Chang Eun Park ) 대한임상검사과학회 2016 대한임상검사과학회지(KJCLS) Vol.48 No.2

분석전 변이로 인하여 검사실은 오류에 직면하게 되고 많은 요인들은 환자의 검사결과에 영향을 미치게 된다. 다양한 진공채혈관은 신속하게 분리하고 방치된 검체의 용혈을 방지한다. 그러나 진공채혈관의 상태에 따라 몇 가지 제한점들이 발생하는데 이러한 문제점을 알아보기 위해 다양한 진공채혈관을 이용해 혈구산정검사로 비교 평가하였다. 유통기한별 검사결과에서는 백혈구(WBC)와 혈소판(PLT)의 값에서 0.24, 0.21로 큰 변이계수를 나타냈고 상관성에서는 백혈구(WBC), 혈소판(PLT), 적혈구(RBC)의 값이 유의 성을 보였다( p<0.01). 한편 각 3사의 비교평가에서는 백혈구(WBC), 혈소판(PLT)의 값에서 0.27, 0.21로 가장 큰 변이계수를 나타냈고. 상관성에서는 각 3사가 모두 백혈구(WBC), 혈소판(PLT), 혈소판분포폭(PDW)의 값에서 높은 유의한 차이를 보였다( p<0.01) 그러나 G사와 B사는 혈소판(PLT), 혈소판분포폭(PDW)의 값에서는 낮은 유의한 차이를 보였다( p<0.05). 결론적으로 진공채혈관의 상태에 따른 분석의 안정성을 위해 적절한 진공채혈관의 분석평가가 이루어져야 한다. Pre-analytical variables account for most laboratory errors and many factors affect the results from a patient. Type of tubes facilitated rapid separation and prevented hemolysis upon prolonged storage. However, there were some limitations associated with vacutainer conditions. To circumvent the problems, the comparability of complete blood cell count values was examined using various vacutainers. The results of the analysis showed a large coefficient variation of 0.24, 0.21 in the value of white blood cells and platelets, and significant correlation was observed between white blood cells, platelets, and the value of red blood cells ( p<0.01). In each of the three tubes, compared to the value of platelets, white blood cells, the greatest coefficient variation was 0.27, 0.21. In correlation of the three companies, significant difference was observed in values of white blood cells, platelets, and platelet distribution width ( p<0.01), however G and B, the value of platelets, and platelet distribution width were significantly lower ( p<0.05). In conclusion, analysis of vacutainers showed that they were suitable for stability of these analytes under vacutainer conditions.