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.

인체 소장상피세포주(HT-29)의 분화단계에 따른 타우린수송체 활성의 변화

박태선(Park Tae Sun),이해미(Lee Hae Mee),김정아(Kim Jung-A),김하원(Kim Ha-Won),박건구(Park Kun-Koo) 韓國營養學會 2000 Journal of Nutrition and Health Vol.33 No.6

고지혈증 랫트를 이용한 수종 전통 한약제의 항고지혈 효과

노환성,고우경,김운자,박건구,조영환,박형섭,Ro, Hwan-Seong,Ko, Woo-Kyoung,Kim, Oon-Ja,Park, Kun-Koo,Cho, Young-Hwan,Park, Hyung-Sup 한국약제학회 1995 Journal of Pharmaceutical Investigation Vol.25 No.4

Three herbal medicine recipes were tried on the animal model of diet induced hypercholesterolemia to screen for the lipid-lowering effect. The recipes adopted were based on the prescription for atherosclerosis-related symptoms by folk-medicine practioners. Hyperlipidemia was induced in rats by giving the high lipid/cholesterol diet for 7 days. Then, the recipes started with the normal diet. Blood was sampled at 1,2,3 and 5 week-points of the treatment, and levels of total cholesterol, triglyceride, high density lipoprotein and lower density lipoprotein were measured. Dae-shiho-tang decreased total cholesterol level significantly. Hwangryun-haedok-tang and Samhwang-sasim-tang slightly decreased total cholesterol level, although it was not statistically significant.

고지혈증 랫트를 이용한 작약의 수종 용매 추출물에 의한 항고지혈 효과

노환성,고우경,양현옥,박건구,조영환,박형섭 ( Hwan Seong Ro,Woo Kyoung Ko,Hyun Ok Yang,Kun Koo Park,Young Hwan Cho,Hyoung Sup Park ) 한국약제학회 1997 Journal of Pharmaceutical Investigation Vol.27 No.2

N/A Hexane, chloroform, methanol and water extracts of Paeoniae Radix were tested on the experimentally induced hypercholesterolemia in rats for lowering effect of serum lipoprotein contents. Hyperlipidemia was induced on male Wistar rats by feeding high cholestetrol diet for 7 days. Serum lipid profile was verified on these rats by measuring total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL), and low density lipoprotein cholesterol (LDL). Then, the diet was changed to normal. At the same time, hexane, chloroform, methanol and water extract of Paeoniae Radix were given orally on daily basis, and the changes in the serum lipid profile were assessed for 4 weeks. Methanol extract of Paeoniae radix decreased TC level at 1. 2, and 4 week point significantly, and water extract decreased TC level at 4 week point significantly comparing with the control group.

Drynariae Rhizoma추출물이 백서 두개관세포 및 골수세포 성상에 미치는 영향

임경석,권영혁,박준봉,김성진,정세영,박건구,Lim, Kyung-Seok,Kwon, Young-Hyuk,Park, Joon-Bong,Kim, Sung-Jin,Choung, Se-Young,Park, Kun-Koo 대한치주과학회 1998 Journal of Periodontal & Implant Science Vol.28 No.2

This study was performed to evaluate the effects of extracts of Drynariae Rhizoma on the characteristics of rat calvaria cells(RCV) and bone marrow cells(RBM) which have the important role on the bone formation in vitro. Drynariae Rhizoma has been known as the useful herbal medicament for treatment of the wound healing including regeneration of bone fracture, and also has been used to treat the periodontal lesions, tooth mobility, gingival bleeding and pus discharge via sulcus in Oriental Medicine. In control group, the cells were cultured alone with Dulbeco's Modified Eagle's Medium contained with 10% fetal bovine serum, 100U/ml penicillin, $100{\mu}g/ml$ streptomycin, $0.5{\mu}g/ml$ amphotericin-B. In experimental group, extracts of Drynariae Rhizoma(0.1, 1, 5, 10, $50{\mu}g/ml$) were added into the above culture condition. And then each group was characterized by examing the cell proliferation at 1, 3, 7, 14, 21, 30th day, the amount of total protein synthesis and alkaline phosphatase activity of RCV at 2,4th day and those of RBM at 3, 6th day. And also, the calcified nodule of RCV was examed at 3, 5th day in three goup, control, experimental, culture with the PDGF group. The results were as follow ; 1. Both RCV and RBM cells in Drynariae Rhizoma-treated experimental group proliferated more rapidly than nontreated control group. The experimental group below $5{\mu}g/ml$ Drynariae Rhizoma-treated showed more prominent cell proliferation from the 7th day to the 21st day than the control group and above $10\;{\mu}g/ml$ treated group in RCV. 2. Amount of total protein synthesis was more increased in Drynariae Rhizomatreated group than in control group. In $5{\mu}g/ml$ Drynariae Rhizoma-treated group showed most prominent protein synthesis of the any other exrperimental group and control group. 3. Alkaline phosphatase activity also more increased in Drynariae Rhizomatreated group than control group. 4. Mineralized nodules in Drynariae Rhizoma-treated group were more than not in control group but also in PDGF-treated group. From the above results, Drynariae Rhizoma appeared to enhanced the proliferation, protein synthesis, alkaline phosphatase activity and cellular ability of mineralized nodule formation than PDGF. So that, we conclude that Drynariae Rhizoma enhances the activities of bone cells which have the important role on the periodontal regeneration and optimal application of Drynariae Rhizoma was thought to be useful as the means in bone regeneration.

우슬 추출물의 경조직 재생 촉진효과

김성진 ( Sung Jin Kim ),박준봉 ( Joon Bong Park ),권영혁 ( Young Hyuk Kwon ),박건구 ( Kun Koo Park ),정세영 ( Se Young Choung ) 한국응용약물학회 2002 Biomolecules & Therapeutics(구 응용약물학회지) Vol.10 No.4

홍화자 추출물과 키토산 병용처리에 의한 경조직 재생촉진 효과

정세영(Se Young Choung),박준봉(Joon Bong Park),박건구(Kun Koo Park),권영혁(Young Hyuk Kwon),김성진(Sung Jin Kim) 한국응용약물학회 2001 Biomolecules & Therapeutics(구 응용약물학회지) Vol.9 No.4

N/A This study was performed to investigate therapeutic effects of Carthami Semen, Paeoniae Radix extracts and chitosan on the growth and differentiation of human periodontal ligament cell. We found that co-treatment of methanol extracts of Carthami Semen and chitosan significantly increased the growth of human periodontal ligament cell. However, the sigle treatment groups of the extracts showed only 20∼30% of the growth increase. Alkaline phosphase activity, one of differentiation markers, was increased approximately 1.5-fold by co-treatment of methanol extract of Carthami Semen and chitosan and calcified nodule formation was also increased at the similar levels as the alkaline phosphatase. But the single treatment groups showed only 20∼30% increases. These results suggest that Carthami Semen and chitosan co-treatment can be used efficiently for periodontium regeneration.

고지혈증 랫트를 이용한 시호 , 작약 , 조구등의 항고지혈 효과

노환성(Hwan Seong Ro),고우영(Woo Kyoung Ko),박건구(Kun Koo Park),조영환(Young Hwan Cho),박형섭(Hyung Sup Park) 한국응용약물학회 1997 Biomolecules & Therapeutics(구 응용약물학회지) Vol.5 No.1

Effects of Bupleuri Radix, Paeoniae Radix or Uncariae Ramulus et Uncus on experimental hyperlipidemia were studied. Hyperlipidemia was induced on male Wistar rats by feeding them on high cholesterol diet for one week, as previously described by the authors. Blood lipid profile was varified on these rats by measuring total cholesterol(TC), triglyceride(TG), high density lipoprotein cholesterol(HDL) and low density lipoprotein cholesterol(LDL). Then, the rats were followed by feeding normal diet. At the same time, methanol extracts of the three individual herb medicines were orally administered to the rats for 4 weeks and the parameters above mentioned were monitered. Methanol extract of Bupleuri Radix reduced the TC value significantly at 2 week point and Paeoniae Radix reduced the TC value at 4 week point in compare to control group, suggesting the antihyperlipidemic effect of the two herbal medicines in vivo. The extract of Uncariae Ramulus et Uncus however did not show antihyperlipidemic effect in our experiment.

Prote in Kinase C Inhibitor (PKCI)에 의한 방사선 민감도 변화와 c - fos Proto-oncogene의 전사 조절

최은경(Eun Kyung Choi),장혜숙(Hyesook Cha ng),이연희(Yun-Hee Rhee),박건구(Kun-Koo Park) 대한방사선종양학회 1999 Radiation Oncology Journal Vol.17 No.4

목 적 : Ataxia-Telangiectasia (AT) 증은 여러 가지 유전적 결함을 갖는 질병으로 방사선 민감도가 비정상적으로 상승되어 있는 것이 특징이다. AT 환자에서 공통적으로 존재하는 ATM 유전자는 현재까지 방사선 신호전달에 관여하는 것으로 알려진 PI-3 kinase와 유사한 구조임이 알려져 ATM이 방사선 신호전달경로에 중요한 작용을 할 것으로 추정하게 되었다. 본 연구에서는 AT 세포와 정상세포에 PKCI를 과발현 시킴으로써 방사선 신호전달에 관여하는 PKC를 억제하여 이것이 방사선 민감도에 미치는 영향을 관찰하고, 방사선에 의해 유도되는 early response gene인 c- fos transcription의 차이를 측정하여 ATM과 PKCI에 의한 신호전달이 c- fos 유전자 전사에 미치는 영향을 분석하고자 하였다. 대상 및 방법 : PKCI expression vector를 작제한 후 정상세포인 LM217과 AT세포인 AT5BIVA에 transfection 시킨 후 plasmid의 genomic DNA에 결합된 것은 polymerase chain reaction (PCR) 방법으로 확인하였고 PKCI의 mRNA 발현 여부는 northern blotting으로 확인하였다. 방사선 민감도는 아포토시스로 측정하였으며 PKCI가 과발현된 각 세포주에 5 Gy의 방사선을 조사한 후 48시간에 세포를 모아 TUNEL 방법으로 아포토시스 세포의 수를 측정하였다. c- fos 유전자의 전사는 reporter 유전자로 c- fos CAT plsmid를 β- gal expression vector와 같이 각 세포주에 transfection 시키고 36시간이 지난 후 CAT assay를 하여 activity를 측정하고 동시에 β- gal assay를 시행하여 transfection 효율을 보정해 주었다. PKCI, Ras의 영향을 보기 위하여는 PKCI, Ras expression vector와 c- fos CAT plasmid를 cotransfection하고 CAT activity로 측정하였다. 결 과 : 이 실험의 결과 LM과 AT 세포에서 PKCI가 방사선 민감도에 미치는 영향과 c- fos 전사에 미치는 영향을 처음으로 보여주었다. PKCI의 과발현이 LM 세포에서는 방사선 민감도를 증가시켰지만 AT 세포에서는 오히려 약간 감소시키는 작용을 나타내었다. c- fos 전사는 AT 세포에서 LM 세포에 비하여 70배 낮게 나타났는데 PKCI가 과발현 됨으로써 LM 에서는 c- fos의 전사가 감소되었지만 AT 세포에서는 영향이 없었다. Ras 단백으로 c- fos를 유도시키고 여기에 PKCI 발현 백터를 contransfection 하면 LM 세포에서는 induction 이 감소되었지만 AT 세포에서는 영향이 없었다. 즉 LM과 AT 세포에서의 PKCI에 의한 반응의 차이는 Ras와 관련된 signal transduction pathway라는 것을 알 수 있었다. 결 론 : PKCI는 정상세포에서는 방사선에 의한 세포 손상을 증가시키지만 AT 세포에서는 별 영향을 보이지 않는 것을 알 수 있었으며, 두 세포간의 이러한 차이는 c- fos proto- oncogene의 전사차이로 설명할 수 있겠다. 이러한 차이가 AT 세포의 방사선 민감도의 한 원인일 것으로 생각된다. Purpose :The human genetic disorder ataxia- telangiectasia (AT) is a multisystem disease characterized by extreme radiosensitivity. The recent identification of the gene mutated in AT, ATM, and the demonstration that it encodes a homologous domain of phosphatidylinositol 3- kinase (PI3- K), the catalytic subunit of an enzyme involved in transmitting signals from the cell surface to the nucleus, provide support for a role of this gene in signal transduction. Although ionizing radiation was known to induce c- fos transcription, nothing is known about how ATM or PKCI mediated signal transduction pathway modulates the c- fos gene transcription and gene expression. Here we have studied the effect of PKCI on radiation sensitivity and c- fos transcription in normal and AT cells. Materials and Methods : Normal (LM217) and AT (AT5BIVA) cells were transfected with PKCI expression plasmid and the overexpression and integration of PKCI was evaluated by northern blotting and polymerase chain reaction, respectively. 5 Gy of radiation was exposed to LM and AT cells transfected with PKCI expression plasmid and cells were harvested 48 hours after radiation and investigated apoptosis with TUNEL method. The c- fos transcription activity was studied by performing CAT assay of reporter gene after transfection of c- fos CAT plasmid into AT and LM cells. Results :Our results demonstrate for the first time a role of PKCI on the radiation sensitivity and c- fos expression in LM and AT cells. PKCI increased radiation induced apoptosis in LM cells but reduced apoptosis in AT cells. The basal c- fos transcription activity is 70 times lower in AT cells than that in LM cells. The c- fos transcription activity was repressed by overexpression of PKCI in LM cells but not in AT cells. After induction of c- fos by Ras protein, overexpression of PKCI repressed c- fos transcription in LM cells but not in AT cells Conclusion :Overexpression of PKCI increased radiation sensitivity and repressed c- fos transcription in LM cells but not in AT cells. The results may be a reason of increased radiation sensitivity of AT cells. PKCI may be involved in an ionizing radiation induced signal transduction pathway responsible for radiation sensitivity and c- fos transcription. The data also provided evidence for novel transcriptional difference between LM and AT cells.