• Title/Summary/Keyword: TUNEL method

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Reperfusion Hyperemia Demonstrated on Perfusion MRI: It′s Relationship with Programmed Cell Death

  • 이승구;김동익;김상흠;김시연;인연권
    • Proceedings of the KSMRM Conference
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    • 2001.11a
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    • pp.170-170
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    • 2001
  • Purpose: To evaluate the relationship between reperfusion hyperemia in reversible cerebral ischem and the degree of programmed cell death. Method: We produced the animal models of reversible cerebral ischemia in 10 cats by mean of middle cerebral artery (MCA) occlusion with transorbital approach. MCA was occluded b microvascular clamp for an hour. MR imaging was performed at 0, 1, 2 days after ischemi and reperfusion. Perfusion (PWI) [Contrast enhanced GRE EPI, TR/TE= 1500/40, 40 Phases, 128 matrix, 12 cm FOV] and diffusion (DWI) (SE EPI, b=0, 500, 1000) weighted images were obtained using Philips Intera 1.57 system. rCBV and ADC maps were calculated wi IDL based postprocessing program. Tissue slices were obtained after the last MR imagin TUNEL, Calbin and Acid-Fuscin staining were done for corresponding slices as MR imagin We investigated the differences of degree of apoptosis in the area of reperfusion hyperemia.

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A Novel Chenodeoxycholic Derivative HS-1200 Induces Apoptosis in Human HT-29 Colon Cancer Cells (인체 대장암 세포주(HT-29)에서 담즙산 합성유도체(HS-1200)의 세포 사망 기전)

  • Oh Sin Geun;Yang Kwang Mo;Hur Won Joo;Yoo Young Hyun;Suh Hong Suk;Lee Hyung Sik
    • Radiation Oncology Journal
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    • v.20 no.4
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    • pp.367-374
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    • 2002
  • Purpose : To investigate the growth inhibitory effects, and the underlying mechanism of human colon cancer cell (HT-29) death, induced by a new synthetic bile acid derivative (HS-1200). Materials and Methods : Human colon cancer cells (HT-29), in exponential growth phase, were treated with various concentrations of a new synthetic bile acid derivative (HS-1200). The growth inhibitory effects on HT-29 cells were examined using a frypan blue exclusion assay. The extent of apoptosis was determined using agarose gel electrophoresis, TUNEL assays and Hoechst staining. The apoptotic cell death was also confirmed by Western blotting of PARP, caspase-3 and DNA fragmentation factor (DFF) analysis. To investigate the involvement of mitochondria, we employed immunofluorescent staining of cytochrome c and mitochondrial membrane potential analyses. Results : The dose required for the half maximal inhibition $(IC_{50})$ of the HT-29 cell growth was $100\~150\;{\mu}M$ of HS-1200. Several changes, associated with the apoptosis of the HT-29 cells, were reveal by the agarose gel eletrophoresis, TUNEL assays and Hoechst staining, following their treatment with $100\;{\mu}M$ of HS-1200. HS-1200 treatment also induced caspase-3, PARP and DFF degradations, and the western blotting showed the processed caspase-3 p20, PARP p85 and DFF p30 and p11 cleaved products. Mitochondrial events were also demonstrated. The cytochrome c staining indicated that cytochrome c had been released from the mitochondria in the HS-1200 treated cells. The mitochondrial membrane potential $(\Delta\Psi_m)$ was also prominently decreased in the HS-1200 treated cells. Conclusion : These findings suggest that the HS-1200 - induced apoptosis of human colon cancer cells (HT-29) is mediated via caspase and mitochondrial pathways.

The Effect of Melatonin on Mouse Jejunal Crypt Cell Survival and Apoptosis (멜라토닌이 생쥐 소낭 세포 재생과 아포토시스에 미치는 영향에 대한 연구)

  • Kang, Jin-Oh;Ha, Eun-Young;Baik, Hyung-Hwan;Cho, Yong-Ho;Hong, Seong-Eon
    • Radiation Oncology Journal
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    • v.18 no.1
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    • pp.60-67
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    • 2000
  • Purpose :To evaluate protective mechanism of melatonin against radiation damage and its relationship with apoptosis in mouse jejunum. Materials and Methods: 168 mice were divided into 28 groups according to radiation dose and matatonin treatment. To analysis crypt survival, microcolony survival assay was done according to Withers and Elkind's method. To analysis apoptosis, TUNEL assay was done according to Labet-Moleur's method. Results : Radiation protection effect of melatonin was demonstrated by crypt survival assay and its effect was stronger in high radiation dose area. Apoptosis index with 8 Gy irradiation was 18.4$\%$ in control group and 16.5$\%$ in melatonin treated group. After 18 Gy, apoptosis index was 17.2$\%$ in control group and 15.4$\%$ in melatonin treated group. Apoptosis index did not show statistically significant difference between melatonin treated group and control group. Conclusion : Melatonin shows clear protective effect in mouse jejunum against radiation damage but its protective effect seems not to be related with apoptosis protection effect.

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A Comparison of Surgical Methods of Inducing Femoral Head Osteonecrosis in Rats (랫드에서 대퇴골머리 골괴사 유발 외과적 방법의 비교)

  • Kim, Jun-Soo;Park, Jin-Uk;Choi, Seok-Hwa;Kim, Gon-Hyung
    • Journal of Veterinary Clinics
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    • v.27 no.3
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    • pp.240-245
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    • 2010
  • Osteonecrosis of the femoral head is an idiopathic and progressive disease. It was reported that several animal models have been used for the research of osteonecrosis. However, no standardized animal model for the study of osteonecrosis has been developed to date. This study was conducted to compare the degree of osteonecrosis of three surgically induced osteonecrosis models in rats. Twenty Sprague-Dawley rats (24 weeks old, male) were divided into three experimental groups and a control group, five heads each. Three groups were surgically induced into osteonecrosis; the ligamentum teres were cut and the periosteum of the femoral neck was stripped (Group S), the steel wire was ligated to the neck of the femoral head (Group W), and the femoral neck was tied up with a wire in the same way as in the W group, and burned by attaching the electrode tip to the wire and then the wire was removed (Group B). After two weeks, rats were sacrificed and the femoral head and neck were collected. Histological findings were evaluated with H/E stains, Safranin-O and TUNEL for osteonecrotic lesions in the bones and cartilages of the femoral head. Osteonecrosis was induced successfully in all groups (Group S, W and B) in two weeks, a short period of time. Significant necrotic changes of the cartilage were detected only in Group B. In the modified cautery model in particular, the method of removing the wire after cautery was completed in the experimental model of osteonecrosis more efficiently than any other method.

Morphological Changes of Mouse Ovary by X-Ray Irradiation (방사선 조사선량에 따른 생쥐 난소의 형태학적 변화)

  • Yoon, Chul-Ho;Choi, Jong-Woon;Yoon, Surk-Hwan
    • Journal of Radiation Protection and Research
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    • v.32 no.4
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    • pp.140-156
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    • 2007
  • This research was performed to investigate the morphological changes of folliculus ovary according to the radiation dose. The whole body radiation of 200 cGy, 400 cGy, and 600 cGy was given to the each groups of 5 months-aged female mouse. Various staining methods used in this research are: Hematosylin-Eosin method, and immunohistochemistrical methods using BrdU, TUNEL, p53, p21, PCNA and inhibin. The minute structural changes of folliculus ovary were observed through an electron microscope with high magnification. The morphological changes of growing folliculus ovary became distinct as the dose of X-rays increased. Especially, the nuclei of granular cells showed manifest condensation and the changes of the transparent zone were distinct. As a result of histochemical reaction according to Masson's trichrome method and reticular fiber method, the changed granular cells, the deformed basilar membrane of folliculus ovary and the abnormal arrangement of the reticular fiber were observed. In the reaction of BrdU, the granular cells of normal folliculus ovary with positive reaction rapidly decreased according to the increase of the dose of X-rays. In TUNEL study, granular cells showing positive reaction in retarded folliculus ovary were expanded to growing folliculus ovary and primordial folliculus ovary according to the increase of the dose of X-rays. In case of 600 cGy of X-rays, oocyte underwent apoptosis. In p53 immunohistochemistry, p53 manifested to be stronger as the dose of X-rays increased. p53 reactivity was manifested distinctively in all cells comprising folliculus ovary following irradiation of 600 cGy. p21 was manifested in granular cells of folliculus ovary and showed very positive reaction around follicular antrum according to the increase of the dose of X-rays. In PCNA, positive reaction was manifested in growing folliculus ovary, mature folliculus ovary and primordial folliculus ovary, but the extent of the reaction decreased as the dose of the X-rays decreased. The finding that the reaction of granular cells around folliculus ovary was stronger than that near follicular membrane indicates that what was damaged first by X-ray was the cells near folliculus ovary and follicular antrum. The reactivity of $inhibin-{\alpha}$ showed difference according to the growing stage of folliculus ovary: $inhibin-{\alpha}$ showed the most strong reaction in mature folliculus ovary with follicular antrum. There was strong reaction in granular cells around follicular membrane but $inhibin-{\alpha}$ did not occur at all in theca cells comprising follicular membrane. $Inhibin-{\alpha}$ in ovary tissue exposed to 400 cGy of X-rays was manifested more strongly than in ovary tissue exposed to 600 cGy of X-rays, which was related to the phenomenon that granular cells of mature folliculus ovary underwent necrosis or apoptosis increasingly due to X-rays. In an electron microscope with high magnification, nuclei and protoplasm of granular cells in growing folliculus ovary abruptly underwent minute structural changes according to the increase of dose of X-rays. Cell residue, by-product of cell decease, neutrophil and macrophage around follicular antrum were observed. The minute structural changes in granular cells showed typical characteristics of apoptosis: the increase of electronic density due to nuclear condensation, fragmentation of nuclei and atrophy of protoplasm. Necrosis of cells was identified but it was not so remarkable. Macrophage with apoptotic bodies was scattered. Proportional to the radiation dose, we found that the generation of heterogeneous substance of normal ovary texture's follicular fluid, the emergence of dyeing characteristic in the basilar membrane of folicle, the generation of apoptosis, and the transformation of macrophages, etc. From this results, we can infer the possible radiation hazard on the ovary of cervix cancer patient with radiation therapy.

Detection of Apoptosis by M30 Monoclonal Antibody in Non-small Cell Lung Carcinomas (비소세포 폐암에서 단클론항체 M30를 이용한 세포자멸사 측정)

  • Kim, Gwang-Il;Lee, Gun;Lim, Chang-Young;Lee, Hyeon-Jae
    • Journal of Chest Surgery
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    • v.40 no.2 s.271
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    • pp.114-121
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    • 2007
  • Background: Apoptosis plays a crucial role in carcinogenesis, as well as in development and tissue homeostasis. Terminal deoxyribonucleotidyl transferase mediated neck end labelling (TUNEL) and in situ nick end labelling (ISEL) have been used to investigate the apoptosis in tissues. Since the introduction of the M30 monoclonal antibody to overcome drawbacks of TUNEL and ISEL, the apoptosis in various tumors, with the exception of pulmonary carcinomas, has been studied. In this study, attempts were made to examine the correlation of apoptosis in non-small cell carcinomas, using both M30 and the expression of p53 protein, with the clinicopathological factors. Material and Method: Forty five patients with surgically resected non-small cell carcinomas were included. Immunohistochemical staining with M30 and p53 monoclonal antibody were peformed, and their expressions compared with the clinicopathological features. The overall survival time and recurrence-free survival time were calculated, and the factors influencing the survival time analyzed using a univariate analysis. The effects of the expression stati of M30 and p53 on the risks of cancer related to both death and recurrence were evaluated using a multivariate analysis. Result: The p53 positive group had many more M30 positive cells than the p53 negative group (p53 positive group; $61.7{\pm}26.8$ cells vs. p53 negative group; $45.6{\pm}29.6$ cells, p=0.005) and significantly more p53 positive patients showing at least 10 positive cells (apoptotic index, $Al{\ge}1$) on M30 staining (p53 positive group; 52.4% (11/21) vs. p53 negative group 16,7% (4/24), p=0.025). In the univariate analysis, the survival times in relation to smoking (pack-year), performance status (PS) and Al showed significant differences. The multivariate analysis demonstrated the relative risk (R.R) of cancer death increased almost 7.5-fold (R.R 7.482; 95% Cl $1.886{\sim}29.678$; p=0.004) and the risk of recurrence almost 3,8-fold (R.R 3.795; 95% Cl: $1.184{\sim}12.158$; p=0.025) in the high Al (${\ge}1$) compared to the low Al (<1) group. There was no prognostic effect of p53 expression on the survival time or risk of cancer death and recurrence. Conclusion: In non-small cell lung carcinomas, M30 immunohistochemistry was an excellent method for analyzing apoptosis; the high apoptotic index could be an adverse prognostic predictive factor.

Effect of Radiation Dosage Changes on the Cell Viability and the Apoptosis Induction on Normal and Tumorigenic Cells (방사선의 선량변화가 수종의 정상세포와 종양세포주의 세포활성도와 apoptosis 유발에 미치는 영향)

  • Park In-Woo;Lee Sam-Sun;Heo Min-Suk;Choi Soon-Chul
    • Journal of Korean Academy of Oral and Maxillofacial Radiology
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    • v.29 no.2
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    • pp.435-449
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    • 1999
  • Purpose : The study was aimed to detect the differences in the cell viability and the apoptosis induction after irradiation on normal and tumorigenic cells. Materials and Methods : The study. that was generated for two human normal cells(RHEK, HGF-l) and two human tumor cells(KB. HT-1080). was tested using MTT assay at 1 day and 3 day after irradiation and TUNEL assay under confocal laser scanning microscope at 1 day after irradiation. Single irradiation of 0.5. 1, 2. 4. and 8Gy were applied to the cells. The two fractions of 1. 2. 4. and 8Gy were separated with a 4-hour time interval. The irradiation was done with 5.38Gy/min dose rate using Cs-137 irradiator at room temperature. Results and Conclusions : 1. In 3-day group. the cell viability of HGF-1 cell was significantly decreased at 2. 4 and 8Gy irradiation, the cell viability of KB cell was significantly decreased at 8Gy irradiation and the cell viability of HT-I080 cell was significantly decreased at 4 and 8Gy irradiation. 2. There was significant difference between RHEK and KB cell line in the cell viability of 3-day group at 8Gy irradiation. There was significant difference between RHEK and HGF-1 cell line in the cell viability of 3-day group at 4 and 8Gy irradiation. 3. There was a significantly decreased cell viability in 3-day group than those in 1-day group at 2. 4 and 8Gy on HGF-1 cell. at 4 and 8Gy on HT-I080 cell. at 8Gy on KB cell. 4. We could detect DNA fragmented cells only on KB cell. Number of apoptotic cells of KB cell was significantly increased at 4 and 8Gy irradiation. However, there was no correlation between cell viability and apoptosis. 5. On all 4 cell lines, there were no differences between single and split irradiation method in cell viability and apoptosis.

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A Comparative Study for Effects of Chongmyungtang and Chocolate Mixed Chongmyungtang on Learning and Memory Impairment (총명탕과 초콜릿 첨가 총명탕의 학습 및 기억장애에 대한 효능 비교연구)

  • Kim, Seong-Joon;Park, Won-Sang;Choi, Hyeon;Kim, Bum-Hoi;Shin, Jung-Won;Sohn, Young-Joo;Sohn, Nak-Won;Jung, Hyuk-Sang
    • Herbal Formula Science
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    • v.16 no.1
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    • pp.131-145
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    • 2008
  • With tablets and powder, decoction has been a widely-used method of medicine formula. However, for these formulas have unique bitter tastes and flavors of herbal component materials as it is, the compliance of herbal medicine is severly decreased especially for female and younger patients. Consequently, expected treatment effects can't be acquired completely. If loathsome tastes and flavors of decoction were effectively reduced while pharmacological activity were kept intact, the compliance could be promoted Chong-Myung-Tang has been widely prescribed for student patients with memory This study shows that Chong-Myung-Tang+chocolate have no difference from Chong-Myung-Tang in terms of pharmacological activity. Sensory difference with net chocolate was also surved. In order to observe the difference of Chong-Myung-Tang+chocolate and Chong-Myung-Tang, memory impairment was induced by intraventricular injection of $A{\beta}_{25-35}$ peptides on mice and Chong-Myung-Tang and Chong-Myung-Tang+chocolate were administered orally for 14 days. In water maze task, improvement of learning ability during acquisition period and significant increase of memory score during retention period resulted from the treatment of Chong-Myung-Tang and Chong-Myung-Tang+chocolate with respect to the $A{\beta}-injected$ control animals. Furthermore, the $A{\beta}_{25-35}$ toxicity on the hippocampus was assessed with immunohistochemistry (Bax, TUNEL), and differences in antioxidant activity was observed through TBARS and DPPH test. We employed sensory tests using chocolate flavor, herb flavor, and bitter taste & hardness as standards to show sensory differences with net chocolate. In this study, it is demonstrated that Chong-Myung-Tang+chocolate do not disturb the pharmacological activity of Chong-Myung-Tang, and have no sensory difference with net chocolate. Chong-Myung-Tang+chocolate can be used to enhance the compliance remarkably and thought of as an effective, functional formula to maximize expected treatment.

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Protective Effects of Curcumin on CCl4-Induced Hepatic Fibrosis with High Fat Diet in C57BL/6 Mice (C57BL/6 마우스에서 고지방 식이와 CCl4로 유발한 간섬유증에 미치는 커큐민의 보호효과)

  • Jekal, Seung-Joo;Min, Byung Woon;Park, Ho
    • Korean Journal of Clinical Laboratory Science
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    • v.47 no.4
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    • pp.251-258
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    • 2015
  • Curcumin, a major polyphenolic compound of turmeric, is well known to prevent non-alcoholic steatohepatitis (NASH) related to obesity. The aim of the study was to investigate the effect of curcumin on hepatic fibrosis induced by carbon tetrachloride ($CCl_4$) in obese mice. $CCl_4$ was administrated in mice fed a normal diet (ND) or a high fat diet (HFD) for 7 weeks together with or without curcumin. It was conducted to examine for metabolic profiles, adipocyte size, and liver fibrosis by serum biochemistry, histology and immunohistochemistry. Also, Apoptosis of hepatic cells was determined by the TUNEL method. Treatment with curcumin significantly lowered the body weight, fasting glucose, serum AST and ALT, and decreased the adipocyte size, the number of macrophage and mast cells in adipose tissue, and collagen deposition in liver tissue in the HFD+$CCl_4$ group compared with the findings of the HFD+$CCl_4$ group. In contrast, treatment with curcumin on the ND+$CCl_4$ group did not show a significant difference except the body weight and mast cell number when compared with the ND+$CCl_4$ group. Furthermore, curcumin significantly reduced the number of parenchymal apoptotic cells, whereas it increased the number of non-parenchymal apoptotic cells, especially resembling an activated hepatic stellate cell in the liver. Taken together, this data suggests that curcumin might be an effective antifibrotic drug for the prevention of liver disease progression in obese mice. Thus, the development of curcumin as a therapy for obesity and liver fibrosis is supported.

The Effects of Antioxidant and Anti-Alzheimer on Hydrogen peroxide and $\beta$-amyloid peptid-induced PC 12 cells by Semen Ziziphi Spinosae water extract ($H_{2}O_2$와 A$\beta$로 유도된 pc12 cell에서 생산조인(生酸棗仁) 수추출물의 항산화 및 항치매 효과)

  • Lee, Sang-Won;Kim, Dae-Hyun;Yun, Jong-Hyun;Kim, Jin-Woo;Jung, Ejun-Young;Lee, Seoung-Geun;Lee, Key-Sang;Kim, Tae-Heon;Lyu, Yeoung-Su;Kang, Hyung-Won
    • Journal of Oriental Neuropsychiatry
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    • v.19 no.3
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    • pp.179-193
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    • 2008
  • Objective: The antioxidant and anti-Alzheimer effects of Semen Ziziphi Spinosae (SZS) water extract against the amyloid beta peptide (1-42) or H202-induced oxidative damage and cell death were investigated in rat pheochromocytoma line PC 12. Methods: The cells were incubated with SZS water extract and oxidative damage-inducing materials, amyloid beta peptide (1-42) or H2O2 for 24 h. The cellular viability was assessed by WST-1 assay, cytotoxic damage by LDH activity assay, oxidative damages of cells by fluorescence spectrophotometric method, and apoptosis by TUNEL staining assay. Results and Conclusions: 1. Preincubation of the cells with SZS water extract prior to amyloid beta peptide (1-42) (2 uM) or H2O2 (30 uM) exposure elevated the cell survival close to the control and decreased the level of LDH activity and the fluorescence from the cell homogenates and TUNEL staining of the cells, compared to only amyloid beta peptide (1-42) (2 uM) or H2O2 (30 uM) treated conditions. 2. Our study suggests that Semen Ziziphi Spinosae (SZS) water extract has protective effects against amyloid beta peptide (1-42) or H2O2-induced cell toxicity through the antioxidation mechanism, which might be beneficial for the treatment of Alzheimer's disease.

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