Cloning and Expression of Genes regulated by CadmiumTreatment(PT 자료)

박건구 환경독성보건학회 2002 한국독성학회 심포지움 및 학술발표회 Vol.- No.-

변경된 MATLAB Simulink 모델로부터 재사용 가능 테스트 케이스 도출

박건구,한헤진,정기현,최경희 한국정보처리학회 2019 정보처리학회논문지. 소프트웨어 및 데이터 공학 Vol.8 No.6

본 논문에서는 제어기 기능이 표현 된 변경된 MATLAB Simulink/Stateflow(SL/SF) 모델의 재사용 가능한 테스트 케이스 도출 기법을 제안한다. 자동차의 ECU(Electrical Control Unit)와 같이 복잡한 SL/SF 모델의 테스트 케이스를 작성하는데 많은 시간과 노력이 필요하다. 모델이 수정 될 때마다 새로 만들어낼 테스트 케이스를 줄이기 위한 직관적인 방법은 수정 전 모델에서 생성한 테스트 케이스 중 일부를 재사용하는 것이다. 본 논문에서는 모델 행동을 정의하고 테스트 케이스 별 모델 동등성을 판단하여 수정 후 SL/SF에 재사용 가능한 테스트 케이스를 도출하는 방법을 제안한다. 제안된 테스트 케이스 재사용 기법은 상용 자동차 제어기 모델을 이용하여 성능을 평가한다. This paper proposes a reusable test case extraction technique for modified MATLAB Simulink/Stateflow (SL/SF) model. Creating test cases for complicated SL/SF model like ECU(Electrical Control Unit) of automotive, requires a lot of time and effort. An intuitive way to reduce to create new test cases whenever the model changes, is to reuse some test cases which have been generated for the original model. In this paper, we propose a method to define reusable test cases in SL/SF after defining model behavior and judging model equality by test cases. The proposed technique is evaluated using a commercial automotive controller model.

아시아 전사학술대회 ( THE 5TH ASIAN CONFERENCE ON TRANSCRIPTION , ACT-V )

박건구 한국독성학회 1998 한국독성학회보 Vol.12 No.2

Gene Analysis Technology

박건구 한국생물공학회 2005 한국생물공학회 학술대회 Vol.2005 No.4

암세포에서 송엽의 AP-l (c-fos/c-jun)에 미치는 영향

박건구,장혜숙,이정교,최승훈 대한약학회 1999 약학회지 Vol.43 No.1

Phorbol ester, growth factors activities are mediated by unclear transcription factors, the c-Fos and c-Jun, which can regulate transcriptional activation through specific DNA sites and by forming the transcription factor AP-l, which usually mediates cell proliferation and differentiation signals. We explored effects of Pini Folium extract (API-l) on AP-l activity. Western blot analysis confirmed that API-l decreased levels of c-Fos or c-Jun protein induced by the tumor promoter Phorbol 12-myristate 13-acetate (PMA; 200 nM). Transient transfection assays with a c-fos promoter reporter construct showed that API-l decreased transcription activity by ore than 50~60%. However, treatment of API-l activity studied further. The main substances were fractionated into dichloromethane layer. Futhermore, API-l extract repressed the [$^3H$]-thymidine uptake in C6 glioma cells, indicating that this extract could be included in a new type of modulator in the mitogenesis.

DNA 유전자칩을 이용한 인체오염 바이오마커 탐색기법 ( Development of Biomarker for toxic Chemicals using cDNA Microarray )

박건구 환경독성보건학회 2001 한국독성학회 심포지움 및 학술발표회 Vol.- No.-

갑상선암 세포주에서 아데노바이러스-hNIS(Ad-hNIS) 유전자 이입에 의한 방사성 요오드 포획 증진 효과

박건구,진정선,이성진,박정윤,이희란,문대혁,안일민,장혜숙 대한내분비학회 2000 Endocrinology and metabolism Vol.15 No.4·5

갑상선암 세포주에서 아데노바이러스 - hNIS ( Ad - hNIS ) 유전자 이입에 의한 방사성 요오드 포획 증진 효과

박건구,박정윤,이성진,장혜숙,이희란,문대혁,안일민,진정선 대한내분비학회 2000 Endocrinology and metabolism Vol.15 No.4

Background: The sodium-iodide-symporter (NIS) is a plasma membrane glycoprotein with 13 putative transmembrane domains, which is responsible for concentrating iodide into the thyroid by an active transport and provides the mechanism for radioactive-iodine (RAI) therapy for thyroid cancer. However, undifferentiated thyroid cancers and about 2050% of differentiated thyroid cancers do not take up the RAI at therapeutic dose. The NIS has been cloned from rat and human (hNIS) and characterized recently. In an attempt to develop a new therapeutic strategy using hNIS gene for improving the efficacy of RAI therapy in thyroid cancers, we have constructed a recombinant adenovirus encoding the hNIS (Ad-hNIS) and tested its function by an iodide uptake by infecting human thyroid cancer cells. Methods: RT-PCR was performed to measure an intrinsic hNIS expression in thyroid cancer cell lines, such as NPA, FRO and ARO. To generate the hNIS adenovirus, hNIS cDNA was isolated and ligated into Swa I site of cosmid shuttle vector (pAxCAwt). We have produced recombinant adenovirus by co-transfecting the cosmid with DNA-TPC to 293 cell line. Adenovirus that express (β-Galactosidase (LacZ) was also prepared by the similar strategy. Adenovirus infection efficiency was measured in three thyroid cancer cell lines. Finally, 24 hours after infection of ad-hNIS into the cells, I125-uptake was measured. Results: Endogenous hNIS expression was detected only in FRO cells but not in NPA, ARO and Hela cells by RT-PCR. X-Gal staining after infection of Ad-LacZ to thyroid cancer cell (NPA, ARO, FRO) showed that an infection rate in ARO cells was 98.5+0.5%, 97.0+0.2% in FRO cells and 75.5+5.0% in NPA cells. We selected ARO cells for the infection of Ad-hNIS due to the highest infection efficiency and the absence of endogenous hNIS expression. When ARO cells were infected with the ad-hNIS, I125 uptake was increased 504+6.4%. Conclusion: Overexpression of hNIS gene in thyroid cancer cells elicited over 5 fold increase in I-uptake, suggesting that th Ad-hNIS infection to the thyroid cancer cells may improve the efficiency of radioactive iodine therapy (J Kor Soc Endocrinol 15:522-53l, 2000).

SHR(Spontaneously Hypertensive Rat)를 이용한 송엽, 익모초 추출물의 항고혈압 작용

박건구 ( Kun Koo Park ),류재원 ( Je Won Ryu ),최은경 ( Eun Kyung Choi ),노환성 ( Hwan Seong Ro ) 한국응용약물학회 2000 Biomolecules & Therapeutics(구 응용약물학회지) Vol.8 No.1

이온화 방사선에 의한 TIMP1 , TIMP2 유전자 발현 측정

박건구(Kun-Koo Park),진정선(Jung Sun Jin),박기영(Ki Yong Park),이연희(Yun Hee Lee),김상윤(Sang Yoon Kim),노영주(Young Ju Noh),안승도(Seung Do Ahn),김종훈(Jong Hoon Kim),최은경(Eun Kyung Choi),장혜숙(Hyesook Chang) 대한방사선종양학회 2001 Radiation Oncology Journal Vol.19 No.2

목 적 :Tissue inhibitor of matrix metalloproteinase (TIMP)는 matrix metalloproteinase (MMP)에 작용하여 암세포의 침윤과 전이를 억제하고 염증, angiogenesis, fibros is에 중요한 역할을 한다. TIMP 유전자는 여러 cytokine 및 signal molecule에 의하여 조절되는 유전자이므로 방사선에 의한 TIMP의 발현을 측정하고 전사 조절 기전을 연구하고자 하였다. 대상 및 방법 :두경부암 환자의 병변에서 유도하여 확립한 두경부암 세포주를 이용하여 방사선에 의한 TIMP 유전자 발현을 측정하였다. 각 세포주의 방사선 민감도를 측정하고 transwell을 이용한 invasion assay로 전이성을 측정하였다. TIMP1, TIMP2 발현은 conditioned medium을 취해 ELISA assay로 측정하였다. 방사선조사는 2 Gy, 10 Gy군으로 나누어 관찰했고 조사 후 시간 간격은 24, 48시간이었다. MTT assay로 생존세포 수를 측정하여 방사선 세포치사로 인한 발현 변화를 보정하였다. hTIMP1 promoter region을 PCR하여 pGL2- basic luciferase reporter vector에 cloning하여 인간 두경부암 세포주에 이입하여 functional TIMP1 발현이 증가하는지 확인하였고 protein kinase C(PKC) activator인 PMA (phorbol 12-myristate 13- acetate)와 Ras에 의한 TIMP1 발현이 유도되는지 확인하였다. 결 과 :HN- 1, HN-2, HN-3, HN-5, HN-9 세포주의 D0는 각각 1.55 Gy, 1.8 Gy, 1.5 Gy, 1.55 Gy, 2.45 Gy 이었다. 각 세포주의 방사선조사 후 MTT assay에 의한 cell viability는 24, 48시간에서 2 Gy인 경우 모두 94% 이상 그리고 10 Gy에서는 73% 이상의 생존 세포를 확인하였다. TIMP1, TIMP2 단백의 basal 농도는 24시간 48시간에서 점점 증가하여 세포에서 계속 합성되어 분비되고 있음을 확인하였다. 2 Gy 조사 후 24시간에서 TIMP2는 HN- 1, HN-9 세포주에서 감소하였으나, 10 Gy 조사 후에는 두 세포주에서 모두 증가하여 방사선량에 따라 반응이 달랐고, 방사선조사 후 48시간에는 HN- 1세포주에서는 증가하나 HN-9 세포주에서는 감소하여 세포주에 따라 반응이 달랐다. 그러나 방사선에 의한 TIMP1 발현 변화는 미미하였다. TIMP1 reporter gene을 인간 두경부암 세포주에 transfection하고 PMA (100 ng/ml)을 가한 경우 HN- 1세포주에서는 유의하게 증가하고 HN-9 세포주에서는 감소하였다. Ras 발현 벡터와 co- transfection한 경우 TIMP1 promoter가 활성화 되었다. 결 론 :모두 두경부 암에서 유래된 세포주 이지만 방사선에 의한 TIMP의 발현 및 전사조절 기전은 세포주 마다 차이가 있었고 이온화 방사선의 용량에 따라서, 방사선조사 후의 시간 경과에 따라서도 TIMP 발현에 차이가 있었 다. 이 결과는 TIMP의 전사 및 발현이 여러 종류의 signal molecule에 의하여 영향을 받고, 이 signal molecule들이 각 세포주 마다 다르기 때문으로 사료된다. Purpose : Express ion of TIMP, intrins ic inhibitor of MMP, is regulated by s ignal transduction in response to genotoxins and is likely to be an important step in metastas is , angiogenes is and wound healing after ionizing radiation. Therefore, we studied radiation mediated TIMP express ion and its mechanism in head and neck cancer cell lines . Materials and Me thods : Human head and neck ca ncer cell lines established at Asan Medical Center were used and radiosens itivity (D0), radiation cytotoxicity and metastatic potential were measured by clonogenic assay, MTT assay and invas ion assay, respectively. The conditioned medium was prepared at 24 hours and 48 hours after 2 Gy and 10 Gy irradiation and express ion of TIMP protein was measured by Elisa assay with specific antibodies against human TIMP. hTIMP1 promotor region was cloned and TIMP1 luciferase reporter vector was constructed. The reporter vector was transfected to AMC- HN- 1 and-HN-9 cells with or without express ion vector Ras , then the cells were exposed to radiation or PMA, PKC activator. EMSA was performed with oligonucleotide (- 59/- 53 element and SP1) of TIMP1 promotor. Results : D0 of HN- 1, - 2, - 3, - 5 and - 9 cell lines were 1.55 Gy, 1.8 Gy, 1.5 Gt, 1.55 Gy and 2.45 Gy respectively. MTT assay confirmed cell viability, over 94% at 24hrs , 48hrs after 2 Gy irradiation and over 73% after 10 Gy irradiation. Elisa assay confirmed that cells secreted TIMP1, 2 proteins continuous ly. After 2 Gy irradiation, TIMP2 secretion was decreased at 24hrs in HN- 1 and HN- 9 cell lines but after 10 Gy irradiation, it was increased in all cell lines . At 48hrs after irradiation, it was increased in HN- 1 but decreased in HN-9 cells . But the cha nge in TIMP secretion by RT was mild. The transcription of TIMP1 gene in HN- 1 was induced by PMA but in HN-9 cell lines , it was suppressed. Wild type Ras induced the TIMP- 1 transcription by 20 fold and 4 fold in HN- 1 and HN- 9 respectively. The binding activity to - 59/- 53, AP1 motif was increased by RT, but not to SP1 motif in both cell lines . Conclusions :We observed the difference of expresson and activity of TIMPs between radiosens itive and radiores istant cell line and the different s ignal transduction pathway between in these cell lines may contribute the different radiosens itivity. Further research to investigate the radiation response and its s ignal pathway of TIMPs is needed.