• 대한수의학회지
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• 제41권4호
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• pp.571-578
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• 2001

방사선 피폭선량의 예측을 위한 방사선 민감 지표 모델 개발의 일환으로 apoptotic fragment assay법이 방사선에 피폭된 후 체내 피폭선량을 예측할 수 있는 지표로의 이용 가능성을 평가하기 위하여 코발트-60 감마선과 의료용 싸이크로트론 50MeV($p{\rightarrow}Be^+$) fast neutron 을 0.25Gy에서 1Gy의 선량을 마우스에 각각 전신 조사한 후 소장 음와세포내 apoptotic crypt cell의 수적 변화를 관찰하였다. 저선량 조사군에서 apoptotic crypt cell의 출현 빈도가 1Gy까지 급격하게 증가한 것으로 보아 방사선이 stem cell 지역에 있는 crypt cell의 형태학적 변화를 유발하는 것으로 나타났다. 이상의 결과는 아포토시스가 손상된 세포를 제거하므로 손상된 방사선 민감 표적 장기의 항상성 유지에 중요한 역할을 하는 것으로 판단되었다. Apoptotic fragments의 발생빈도에 대한 선량-반응 곡선에 있어서 음와세포는 중성자조사군이 $y=0.18+(9.728{\pm}0.887)D+(-4.727{\pm}1.033)D^2$ ($r^2=0.984$)으로, 반면에 감마선조사군은 $y=0.18+(5.125{\pm}0.601)D+(-2.652{\pm}0.7000)D^2$ ($r^2=0.970$)의 식을 얻었다. 이와 같이 중성자조사군과 감마선조사군은 공히 linear quadratic model 로 관찰되었다. apoptotic fragments 의 발생빈도와 조사 선량간에 유의한 효과가 있는 것으로 확인되었다. 이상의 결과에서 조사선량의 증가에 비례하여 방사선 민감 세포의apoptotic fragments 가 수적으로 증가하였으며, 고준위 방사선과 저준위 방사선은 선량 반응 관계식과 시간 경과에 따른 영향이 매우 유사하였으며, 마우스 음와세포의 apoptosis 유도에 대한 중성자선의 방사선 생물학적 효과비(RBE)는 2.072이였다. 그리고 모든 방사선조사군에서 방사선피폭 후 4시간과 6시간에 apoptosis 유도가 가장 많았으며, 음와세포의 형태학적 소견은 정상 대조군에서 관찰되지 않는 전형적인 apoptotic fragments 가 나타났다. 따라서 음와 세포에서의 아포토시스 유도는 방사선 피폭으로 발생된 세포 손상의 생물학적 영향 평가검색, 방사선 방호제의 민감도 검사, 방사성 동위원소의 체내 오염에 대한 체내 피폭선량 예측의 지표 및 방사선 민감 표적장기의 손상정도 파악에 이용 가능할 것임. Apoptotic fragment assay 법은 0.25Gy에서 1Gy 까지의 선량에서 간편하고 빠르며 재현성이 있는 지표로서 방사선 민감 표적 장기의 선량 반응 평가와 방사선 피폭후 조기 피폭선량 예측을 위한 방사선 생물학적 선량측정법의 좋은 지표로 사용할 수 있을 것으로 사료됨.

• 대한수의학회지
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• 제41권2호
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• pp.203-210
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• 2001

방사선 피폭선량의 예측을 위한 방사선 민감 지표 모델 개발을 위하여 코발트-60 감마선과 의료용 싸이크로트론 50 MeV($p{\to}RBe^+$) fast neutron을 0.25 Gy에서 1 Gy의 선량을 랫드에 각각 전신 조사한 후 말초혈액내 임파구와 소장에 음와세포의 형태학적 변화를 apoptotic fragment assay법을 이용하여 관찰하였다. 모든 방사선조사군에서 음와세포와 말초 임파구에 아포토시스의 유도가 증가된 것이 관찰되었으며, 이것은 방사선이 방사선 민감세포의 아포토시스 유도를 자극한 것으로 보인다. 상기의 결과는 아포토시스가 손상된 세포를 제거하므로 손상된 방사선 민감 표적 장기의 항상성 유지에 중요한 역할을 하는 것으로 판단되었다. Apoptotic fragments의 발생빈도에 대한 선량-반응곡선에 있어서 음와세포는 중선자 조사군이 $y=0.3+(6.512{\pm}0.279)D(r^2=0.975)$으로, 반면에 감마선 조사군은 $y=0.3+(4.435{\pm}0.473)D+(-1.300{\pm}0.551)D^2(r^2=0.988)$의 식을 얻었다. 그리고 말초 임파구에서는 감마조사군이 $y=3.5+(118.410{\pm}10.325)D+(-33.548{\pm}12.023)D^2(r^2=0.992)$의 식으로 나타났다. 이와 같이 감마선조사군은 공히 linear quadratic model 이였으나 중성자조사군은 linear model 로 관찰되었다. 조사된 세포의 종류와 상관없이 apoptotic fragments의 발생빈도와 조사 선량간의 유의한 효과가 있는 것으로 확인되었다. 이상의 결과에서 조사선량의 증가와 비례하여 방사선 민감 세포의 apoptotic fragments가 수적으로 증가하였으며, 고준위 방사선이 저준위 방사선보다 선량 반응 곡선과 시간 경과에 따른 영향이 보다 강한 것으로 인지되었으며, 음와세포의 apoptosis 유도에 대한 중성자선의 방사선 생물학적 효과비(RBE)는 1.919 였다. 그리고 모든 방사선조사군에서 방사선피폭 후 4시간과 6시간에 apoptosis 유도가 가장 많았으며, 음와세포의 형태학적 소견은 정상 대조군에서 관찰되지 않는 전형적인 apoptotic fragments가 나타났다. 따라서 음와 세포와 말초 임파구에서의 아포토시스 유도는 방사선 조사에 의한 세포 손상의 생물학적 영향 평가를 위한 검색 및 방사선 피폭선량 예측의 지표로 이용 가능할 것으로 사료됨.

• 대한한의학회지
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• 제25권2호
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• pp.138-150
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• 2004

Objectives : Alzheimer's disease (AD) is a geriatric dementia that is widespread in old age. In the near future AD will be the biggest problem in public health service. Although a variety of oriental prescriptions, including Sesim-tang, have been traditionally utilized for the treatment of AD, their pharmacological effects and action mechanisms have not yet been fully elucidated. The present study investigated the effects of Sesim-tang on apoptotic cell death induced by CT105 (carboxy terminal 105 amino acid peptide fragment of APP) overexpression in SK-N-SH neuroblastoma cell lines. Methods: We studied the regenerative and inhibitory effects on Alzheimer's disease in CT105-induced SK-N-SH cell lines by Sesim-tang water extract. We examined for cell morphological pattern, DNA fragmentation, LDH activity assay, zymography assay, and immunohistochemistric analysis. Additionally, we investigated the association between the CT105 and neurite degeneration caused by CT105-induced apoptotic response in neurone cells. Results: Findings from our experiments have shown that Sesim-tang inhibits the synthesis or activities of CT105, which has neurotoxicities and apoptotic activities in the cell line. In addition, pretreatment with Sesim-tang ($>50\mu\textrm{g}/ml$ for 12 hours) partially prevented CT105-induced cytotoxicity in SK-N-SH cell lines. SK-N-SH cell lines overexpressed with CT105 exhibited remarkable apoptotic cell damage. Based on morphological observations by phase-contrast microscope and LDH activity measurements in the culture media, the CT105-induced cell death was significantly inhibited by Sesim-tang water extract. Sesim-tang was found to reduce the expression of APP and caspase-3 induced by CT105 in SK-N-SH cell lines and in rat hippocampus. Conclusions: As the result of this study, in the Sesim-tang group, apoptosis in the nervous system is inhibited, the repair against the degeneration of SK-N-SH cell lines by CT105 expression is promoted. Hence, Sesim-tang may be beneficial for the treatment of AD.

• 동의생리병리학회지
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• 제18권2호
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• pp.507-516
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• 2004

Alzheimer's disease(AD) is a geriatric dementia that is widespread in old age. In the near future AD will be the commom disease in public health service. Although a variety of oriental presciptions in study POD(Polygala tenuifolia extracted from dichlorometan) have been traditionally utilized for the treatment of AD, their pharmacological effects and action mechanisms have not yet fully elucidated. It has been widely believed that AP peptide divided from APP causes apoptotic neurotoxicity in AD brain. However, recent evidence suggests that CT105, carboxy terminal 105 aminoacids peptide fragment of APP, may be an important factor causing neurotoxicity in AD. SK-N-SH cells expressed with CT105 exhibited remarkable apoptotic cell damage. Based on morphological observations by phase contrast microscope and NO formation in the culture media, the CT105-induced cell death was significantly inhibited by POD. In addition, AD is one of brain degeneration disease. So We studied on herbal medicine that have a relation of brain degeneration. From old times, In Oriental Medicine, PO water extract has been used for disease in relation to brain degeneration. We were examined by ROS formation, neurite outgrowth assay and DPPH scravage assay. Additionally, we investigated the association between the CT105 and neurite degeneration caused by CT105-induced apoptotic response in neurone cells. We studied on the regeneratory and inhibitory effects of anti-Alzheimer disease in pCT105-induced neuroblastoma cell lines by POD. Findings from our experiments have shown that POD inhibits the synthesis or activities of CT105, which has neurotoxityies and apoptotic activities in cell line. In addition, treatment of POD(>50 ㎍/㎖ for 12 hours) partially prevented CT(105)-induced cytotoxicity in SK-N-SH cell lines, and were inhibited by the treatment with its. POD(>50 ㎍/㎖ for 12 hours) repaired CT105-induced neurite outgrowth when SK-N-SH cell lines was transfected with CT105. As the result of this study, In POD group, the apoptosis in the nervous system is inhibited, the repair against the degerneration of Neuroblastoma cells by CT105 expression is promoted. Decrease of memory induced by injection of scopolamin into rat was also attenuted by POD, based on passive avoidance test. Taken together, POD exhibited inhibition of CT105-induced apoptotic cell death. POD was found to reduce the activity of AchE and induced about the CA1 in rat hippocampus. Base on these findings, POD may be beneficial for the treatment of AD.

• 약학회지
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• 제48권2호
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• pp.153-158
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• 2004

Paecilomyces tenuipes is one of the famous Chinese medicinal entomopathogenic fungi that parasite in the lavae of silkworm. A cytotoxic compound, 4$\beta$-acetoxyscirpendiol (ASD) was isolated from a methanolic extracts of Paecilomyces tenuipes. The ASD compound belongs to scirpenol subfamily of trichothecene mycotoxin. In a continuation of the elucidation of the mechanism of ASD, we report here the evidences of induction of apoptosis by ASD in human Jurkat T cell line. In MTT reduction assay for monitoring cell viability, ASD showed strong toxicity. The 50 percent inhibitory concentrations of ASD against human T lymphoid Jurkat cell was 59.5 ng/$m\ell$. Phosphatidylserine externalization was increased by ASD at 3 and 6 hrs when compared with that of 6 hrs in the cell line showing in a time-dependent manner. When whole lysates of cells treated with ASD were subjected to western blot assay, 113 kDa poly(ADP-ribose) polymerase (PARP) was significantly cleaved to 89 kDa fragment. Time-dependent DNA fragmentation was also observed when Jurkat T cells were treated with ASD at 100 ng/$m\ell$ for 6 hrs and 18 hrs at the ratios of 8.5% and 15.0%, respectively. From these data, Jurkat T lymphocytes treated with ASD from Paecilomyces tenuipes underwent typical cascades of apoptotic cell death.

• BMB Reports
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• 제37권4호
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• pp.480-486
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• 2004

Brain ischemia brings about hypoxic insults. Hypoxia is one of the major pathological factors inducing neuronal injury and central nervous system infection. We studied the involvement of mitogen-activated protein (MAP) kinase in hypoxia-induced apoptosis using cobalt chloride in C6 glioma cells. In vitro cytotoxicity of cobalt chloride was tested by MTT assay. Its $IC_{50}$ value was $400\;{\mu}M$. The DNA fragment became evident after incubation of the cells with $300\;{\mu}M$ cobalt chloride for 24 h. We also evidenced nuclear cleavage with morphological changes of the cells undergoing apoptosis with electron microscopy. Next, we examined the signal pathway of cobalt chloride-induced apoptosis in C6 cells. The activation of extracellular signal-regulated protein kinase 1/2 (ERK 1/2) started to increase at 1 h and was activated further at 6 h after treatment of 400 M cobalt chloride. In addition, pretreatment of PD98059 inhibited cobalt chloride-induced apoptotic cell morphology in Electron Microscopy. These results suggest that cobalt chloride is able to induce the apoptotic activity in C6 glioma cells, and its apoptotic mechanism may be associated with signal transduction via MAP kinase (ERK 1/2).

• 대한한방내과학회지
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• 제31권2호
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• pp.331-345
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• 2010

Objectives : This study was designed to investigate the effects of Saenggantanggami-bang (SG) on nonalcoholic fatty liver disease. Methods : HepG2 cells were used in an in vitro model. HepG2 cells were divided into three groups. The Normal group was incubated with no fatty acid. The Control group was incubated with 1mM palmitic acid to introduce fat overloading. The PA-SG group was incubated with 1mM palmitic acid and various concentrations of Saenggantanggami-bang (SG). Cell viability and cytotoxicity were analyzed by MTT assay and LDH assay. Intracellular triglyceride (TG) levels, reactive oxygen species (ROS) levels, ATP amount, and GST activity were measured. Cell death pattern and protective effect of SG on cell death were studied by DNA fragmentation and caspase-3 intensity (western blot). Results : Compared with the Control group, cell viability of the PA-SG group significantly increased (P<0.01), cytotoxicity of the PA-SG group decreased (P<0.01), and intracellular TG levels and ROS levels of the PA-SG group decreased (P<0.05). In DNA fragmentation assay, necrotic pattern was observed and DNA fragment decreased in the PA-SG group. In western blot, apoptotic pattern was observed, caspase-3 intensity of the PA-SG group was reduced significantly, but there were no significant differences in intracellular ATP amount and GST activity between the control group and the PA-SG group. Conclusion : The results suggest that Saenggantanggami-bang can be a potential candidate for the clinical treatment of nonalcoholic fatty liver disease.

• 대한한방부인과학회지
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• 제24권3호
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• pp.1-13
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• 2011

Objectives: This study was designed to investigate the analysis of apoptosis by Scirpi Tuber in Hela cell and MCF-7 cell. Methods: For cytotoxic effect of Scirpi Tuber extract, Scirpi Tuber extract were cultured on NIH3T3 cell in vitro. After treatment with various concentration of Scirpi Tuber, cell growth was evaluated in Hela cell and MCF-7 cell. Hoechst 33342 staining was performed to estimate DNA fragment effect of Scirpi Tuber on the apoptosis in Hela cell and MCF-7 cell. Annexin V/PI apoptosis assay was used to estimate the effects of Scirpi Tuber on the early apoptosis in Hela cell MCF-7 cell. All the stained cells were analyzed by a FACS. RT-PCR was used to estimate the apoptosis gene expression effect of Scirpi Tuber extract on Hela cell and MCF-7 cell. Results: Cytotoxic effect of Scirpi Tuber extract was not found on per NIH3T3 cell. The viability of Hela cell was significantly decreased Scirpi Tuber (500, $1000{\mu}g/m\ell$) in Hela cell 1day, 3day and 5days after treatment (p<0.01). The viability of MCF-7 cell was significantly decresed Scirpi Tuber ($1000{\mu}g/m\ell$) in MCF-7 cell (p<0.01), Scirpi Tuber ($500{\mu}g/m\ell$) in MCF-7 cell only 3days after treatment (p<0.01). In RT-PCR analysis, after treatment of $100{\mu}g/m\ell$ of ACR extract, BCL-2 were decreased and BAX, caspase-3 were increased both in Hela cell and MCF-7 cell. DNA fragmentation was observed the Scirpi Tuber on Hela cell and MCF-7 cell. As time goes on DNA fragmentation incresed. In Annexin V/PI apoptosis assay, after treatment of $1mg/m\ell$ of Scirpi Tuber, the early apoptotic cell increased both in Hela cell and MCF-7 cell. As time goes on apoptotic cell increased. Conclusion: Scirpi Tuber appears to have considerable activity on the apoptosis in Hela cell and MCF-7 cell.

• 대한한방종양학회지
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• 제6권1호
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• pp.81-97
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• 2000

Objectives: This experimental study was carried out to evaluate the effects of aqueous and methanol extracts of Hedyotis diffusa which has long been used for cancer treatment in oriental medicines on the induction of apoptotic cell death in human lymphoid leukemia cell line, HL-60. Methods: Cells were treated with various concentrations (200 to $0.4{\mu}g$) and periods (6 to 30 hr) of $H_2O$ and methanol extracts of Hedyotis diffusa. Then, cells were tested for viability by MTT assay. Cells wrere treated with $200{\mu}g/ml$ of methanol extract fork various periods. Genomic DNA was isolated, separated, on 1.5% agarose gels, stained with ethidium bromide and visualized under UV light. Cells were treated with $200{\mu}g/ml$ of each extract for 16 hr. Then, cells were treated with Hoechst dye 33342 and observed by fluorescence microscopy. Cells were treated with various doses of each for 12 hr and $100{\mu}g/ml$ of methanol extract for various periods. Lysate from the cells used to measure the activity of Caspase-1 and-3 proteases by using fluorogenic peptide substrates including acetyl-YVAD-AMC and acetyl-DEVD-AMC, respectively. Cells were treated with $200{\mu}g/ml$ of each extract for various periods. Cell lysates were immunoprecipated with anti-JNKl antibodies. The immune complex was reacted with $32^p-ATP$ and c-Jun as a substrate. The phosphotransferase activity of JNKI was measured by using PhosphoImage analyzer (Fuji Co., Japan). Nuclear extracts were isolated and incubated with oligonucleotide probe of $NF-{\kappa}B$. Transcriptional activation of ${\kappa}B$ was measured by using EMSA and visualized by PhosphoImage analyzer (Fuji Co, Japan). Cell lysates were prepared and analyzed by Western blotting with anti-Bc12 antibodies and anti-Bax antibodies. Cells were pretreated with various doses of methanol extract for 2 hr. Then, the extract was removed by centrifugation. Cells were resuspended with RPMI-1640 media containing 0.3% agarose, 10% FBS, overlayred onto bottom layer agarose and incubated at $CO_2$ incubator for 6 days. The number of colony was counted under light microscopy ($\time100$). Results: The death of HL-60 cells was markedly induced by the addition of methanol extract of Hedyotis diffusa in a dose and time-dependent manners. The apoptotic characteristic ladder pattern of DNA strand break was observed in death of HL-60 cells. In addition, it was shown nucleus chromatin condensation and fragmentation under Hoechst staining. Therefore, Hedyotis diffusa extract-induced death of HL-60 cells is mediated by apoptotic signaling processes. The activity of Caspase 3-like proteases remained in a basal level in HL-60 cells treated with aqueous extract of Hedyotis diffusa. However, it was markedly increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. In addition, the phosphotransferase activity of JNKl was increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. Furthermore, the activation of transcriptional activator, $NF-{\kappa}B$ was markedly induced by methanol extract of Hedyotis diffusa. Anti-apoptotic Bc12 was cleaved into 23Kda fragment by treatment of methanol extract of Hedyotis diffusa. However, expression of proapoptotic Bax protein was increased by treatment of methanol extract of Hedyotis diffusa in a time-dependent manner. Furthermore, methanol extract markedly inhibited the colony forming efficiency of HL-60 cells in semisolid agar culture. Conclusions: Above results suggest that methanol extract of Hedyotis diffusa induces the apoptotic death of human leukemic HL-60 cells via activations of Caspase-3 proteases, JNKI, transcriptional activator $NF-{\kappa}B$, In addition, our results also suggest that methanol extract of Hedyotis diffusa reduces the malignant potential of HL-60 cells via down regulation of colony forming effciency through cleavage of Bc12 as well as induction of Bax.

• 대한한방부인과학회지
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• 제24권3호
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• pp.14-27
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• 2011

Objectives: This study was designed to investigate the effects of Acanthopanacis Cortex Radicis extract(ACRE) on the apoptosis in HeLa cell and MCF-7 cell. Methods: After treatment with various concentration of ACRE, cell growth was evaluated in HeLa cell and MCF-7 cell. Hoechst 33342 staining was performed to estimate DNA fragment effect of ACRE on the apoptosis in HeLa cell and MCF-7 cell. Annexin V/PI apoptosis assay was used to estimate the effects of ACRE on the early apoptosis in HeLa cell and MCF-7 cell. RT-PCR was used to estimate the apoptosis gene expression effect of ACRE on Hela cell MCF-7 cell. Results: Under $0.1mg/m\ell$ of ACRE, cytotoxic effect was not found per NIH3T3 cell. The viability of HeLa cell and MCF-7 cells was significantly decreased ACRE ($100{\mu}g/m\ell$) in HeLa cell and MCF-7 cell, ACRE ($50{\mu}g/m\ell$) in HeLa cell 3 days after treatment, in MCF-7 cell 1&3 days after treatment (p<0.01). DNA fragmentation was observed 3 days after treatment of cl of ACRE on HeLa cell and MCF-7 cell. In Annexin V/PI apoptosis assay, after treatment of $100{\mu}g/m\ell$ of ACRE, the early apoptotic cell increased both in HeLa cell and MCF-7 cell. In RT-PCR analysis, after treatment of $100{\mu}g/m\ell$ of ACRE, bcl-2 were decreased and bax, caspase-3 were increased both in HeLa cell and MCF-7 cell. Conclusions: ACRE appears to have considerable activity on the apoptosis in HeLa cell and MCF-7 cell.