• 대한의생명과학회지
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• 제8권3호
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• pp.179-184
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• 2002

We investigated the morphological changes and TUNEL reaction of apoptotic cells in the liver of D-galactosamine (20 mg/mouse) and lipopolysaccharide (5 $\mu\textrm{g}$/mouse)-treated 30 mice (BALB/c), and in additioa also of apoptotic cells in kidney and spleen. The livers and other some organs of mice at 6, 12, 24, 48 and 72 hrs after treatment were collected and were fixed with 10% neutral formalin and paraffin sections were stained with hematoxylin-eosin or terminal deoxynucleotidly transferase-mediated dUTP nick end labeling (TUNEL) method. Morphological changes in apoptotic hepatocytes were chondensation of nuclei and density of cytoplasms, then the margination and pyknosis of chromatin, the formation of half-moon- or horse-shoe- or ship-like shapes of condensed chromatin mass, lastly formation of apoptotic bodies, disappearance of nuclear envelopes, decrease of stainability, then lysis and disappearance of apoptotic bodies. TUNEL positive reactions of hepatocytes were appeared first moderate in uncondensed hepatocytes, severe in condensed hepatocytes, moderate in chromatin-marginated hepatocytes. These reactions also were appeared moderate in hepatocytes with half-moon- or horse-shoe- or ship-like pyknotic chromatin mass or apoptotic bodies, and mild or negative in hepatocytes with lysed apoptotic bodies or with disappeared nuclear envelopes. Consequently these results suggested that TUNEL positive reactions of hepatocytes appeared at more early stages than appearance of chromatin condensation and disappeared at more early stage than disappearance of histological findings of apoptosis. We also confirmed that the differentiation of apoptotic cells from normal healthy cells of Kupffer cells and vascular endothelial cells in liver, reticular cells and lymphocytes in spleen and epithelial cells of tubules and ducts in kidney was impossible in H-E preparations but was possible in TUNEL preparations.

• 대한의생명과학회지
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• 제4권2호
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• pp.121-128
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• 1998

본 연구에서는 genomic DNA 전기영동과 TUNEL labeling법을 이용하여 정상대장조직, 대장암조직, 대장암 인접림프절조직과 대장암환자 혈액에서 apoptosis 발현을 분석하였다. 정상대장조직 37례중 4례에서, DNA ladder가 확인되었고, 암조직은 47례중 20례에서, 림프절조직은 15례중 5례에서 나타났으며 대장암환자혈액은 7례 모두에서 발현되지 않았다. TUNEL labeling법을 이용한 조직상의 in situ apoptosis발현은 암조직과 림프절조직에서 확인되었다. 따라서 본 연구에서는 대장암이 진행됨에 따라 apoptosis 발현비가 증가되었으므로 apoptotic index가 대장암 발현과 관련되어 있는 듯 하며 예후예측지표자로서 이용될 가능성이 있다고 사료된다.

• 대한본초학회지
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• 제27권3호
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• pp.89-94
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• 2012

Objects : The purpose of this study was to observe the effect of Salviae Miltriorrhiza Radix(SMR) water-extract on intracerebral hemorrhage(ICH) and neuronal apoptosis in the injured areas. Method : ICH was induced by the stereotaxic intrastriatal injection of bacterial collagenase type IV in Sprague-Dawley rats. The rats were givened oral SMR treatment once a day for three days after the ICH treatment. TUNEL positive cells in the affected regions were performed by TUNEL assay, Bax and Bcl-2 positive cells by immunohistochemistry and the Bax expression by western blotting method. Results : The results are as follow; 1. SMR significantly reduced the number of TUNEL positive cells in the peri-hematoma reigions of ICH-induced rats. 2. SMR significantly reduced the number of Bax positive cells in the peri-hematoma regions of ICH-induced rats. 3. SMR did not affect the number of Bcl-2 positive cells in the peri-hematoma regions of ICH-induced rats. 4. SMR significantly reduced the Bax expressions compared with ICH group in hemorrhagic hemisphere of ICH-induced rats. Conclusion : These results suggest that SMR is effective in reducing neuronal apoptosis.

• 대한의생명과학회지
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• 제8권3호
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• pp.161-165
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• 2002

Apoptosis can be difficult to detect in routine histological sections. Since extensive DNA fragmentation is an important characteristic of this process, visualization of DNA breaks could greatly facilitate the identification of apoptotic cells. Several techniques for the qualitative and quantitative detection of this process have been established; recently, an in situ nick end-labelling technique based on the detection of DNA fragmentation, which is a molecular characteristic of apoptotic cell death, was described. Applying this method to paraffin sections of rat tissues, sensitivity was observed to be inconsistently low with regard to the expected number of apoptotic cells. I describe a new modified method for formalin-fixed, paraffin-embedded tissue sections, pretense pretreatment to permeate the tissue sections that involves an TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) is acknowledged as a method of choice in the rapid identification and quantification of the apoptotic cell fraction in paraffin tissue preparations. TUNEL was performed without apoptosis and with apopotosis samples to each of the three concentrations of proteinase K (10, 25, 40 mg/ml) pretreatments. In this study, I show that chemical pretreatments of the tissue sections in proteinase K (25 mg/ml for 15 min at room temperature) considerably enhances the sensitivity of this nick end labelling technique.

• 대한의생명과학회지
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• 제7권4호
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• pp.205-210
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• 2001

Whereas apoptosis is a critical mode of cell deletion in normal organism development, apoptotic cells are also observed in tumor therapy. We therefore investigated the expression of apoptotic cells induced as a function of time and dose in murine A-20 lymphoma treated with cyclophosphamide in vivo, by H&E and TUNEL method. The percent of apoptotic cells were scored from tumor section using TUNEL method. The expression of apoptotic positive cell was determined over a 10-day period following treatment of the mice with 200 mg/kg. Apoptosis increased further with time, reaching a peak value between 12~24 hr (scored 6.7$\pm$1.0%~6.1$\pm$0.7%), and then slowly declined to background levels by 10 days after treatment. The dependence of induction of apoptosis on the dose of cyctophosphamide was determined by treatment with 50, 100, or 200 mg/kg at 12 hr after treatment. Apoptosis was dose dependent in that as the dose was increased the percentage of apoptosis increased. However, the increase in apoptosis at the lower dose used (50 mg/kg) was higher on a per unit dose basis than that at the higher dose used (200 mg/kg). This result show that the alkylating agent cyclophosphamide strongly induces apoptosis in murine lymphoma.

• Journal of Chest Surgery
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• 제37권2호
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• pp.113-118
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• 2004

혈관재건술에 사용되는 냉동 보존된 동종동맥편에 대한 지금까지의 연구는 주로 냉동방법, 냉동전처치, 보존온도에 관심이 집중되어 있었으나, 최근에 해동방법이 균열발생의 중요한 인자가 된다는 연구가 있었다. 이에 저자들은, 같은 조건으로 냉동 보존된 대동맥조직을 서로 다른 두 가지 방법으로 해동시켜, 조직손상 정도를 비교함으로써 이상적인 조직의 해동방법을 알아보고자 하였다. 대상 및 방법: 2,500 g의 토끼에서 대동맥편을 획득한 후, 일반적인 방법에 따라 냉동 보존한 뒤 37$^{\circ}C$의 온수에 급속 해동한 군(RT)과 1$^{\circ}C$/min의 속도로 완속 해동시킨 군(ST)으로 나누고, 각 군에서 apoptosis 발생을 평가하기 위해 각 군의 전체 세포 중 TUNEL (＋) 세포의 비를 비교하였고, 광학현미경하에서 해동된 조직편의 조직학적 특성을 비교 관찰하였다. 결과: 1. TUNEL test상, RT군에서 ST군에 비해 TUNEL (＋) 세포의 비가 유의하게 높았다. 2. 광학현미경하 조직소견상, RT군에서 세포부종과 혈관내피층의 박탈, 소수포 형성, 그리고 이형성세포 등의 소견이 ST군에 비해 더 많이 관찰되었다. 결론: 냉동 보존된 토끼의 대동맥조직에 대한 해동방법의 차이에서, 완속 해동방법이 조직의 형태학적 손상과 apoptosis발생을 감소시켰다. 따라서, 향후 냉동조직을 이용한 이식술에서 술 후 합병증의 발생을 감소시키고 예후를 개선하기 위해서는 기존의 급속 해동하는 방법보다 완속 해동하는 방법의 사용을 고려해야 할 것이다.

• Restorative Dentistry and Endodontics
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• 제33권4호
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• pp.332-340
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• 2008

본 연구의 목적은 흰쥐 상악 대구치를 발거하여 자기장 저속 냉동보관법을 이용하여 냉동 시 치주인대세포의 환성도 및 세포 사멸도를 MTT 검색법과 TUNEL 검사를 이용하여 측정하고자 하였다. 4주령의 암컷 Sprague-Dawley계 흰쥐의 상악 좌우 제1,2대 구치를 발거하여 각 군 당 12개의 쥐 치아를 MTT검색에 이용하였고 6개의 치아를 TUNEL 검사에 이용하였다. 실험군은 5개군으로 대조군은 즉시 발치군이며 4$^{\circ}C$ 냉장고에서 1주일간 보관한 냉장군, 발치 후 동해방지제 처리과정을 거쳐 -196$^{\circ}C$의 액화질소에 넣어 급속 냉동한 액화질소군, 217 mA, 60 Hz, 1 G의 자기장을 이용하여 -0.3$^{\circ}C$/min 의 속도로 -20$^{\circ}C$까지 냉동 후 -196$^{\circ}C$로 급속 냉동한 자기장군, -0.3$^{\circ}C$/min의 속도로 -20$^{\circ}C$까지 냉동 후 -196$^{\circ}C$에 급속 냉동한 저속 냉동군으로 나누었다. 보존액은 F medium을 사용했으며 동해방지제로 10% dimethyl sulfoxide (DMSO)를 사용하였다. 치근면을 단위면적으로 표준화하기 위해 MTT 측정값을 Eosin 염색 후 530 nm에서 측정 한 흡광도 값으로 나누었다. TUNEL 검사 시 각 조직슬라이드에서 400배 크기의 현미경 시야에서 임의로 세 부분을 지정하여 정상 세포수와 양성 세포수를 세어 그 비율을 계산하여 각 실험군 당 평균치를 구하였다. 통계 분석을 위해 one way ANOVA를 시행하였으며 사후검정으로 Scheffe와 Tukey HSD방법을 썼으며 결과는 다음과 같다. MTT검색에 의한 흡광도를 Eosin염색 후 측정한 흡광도로 나눈 값에서는 자기장군은 즉시 발치군보다 낮은 세포활성을 보였고 (p < 0.05) 액화질소군, 저속 냉동군과는 통계적으로 유의성 있는 차이를 보이지 않았다. 그러나 자기장군은 액화질소군, 저속 냉동군과 함께 냉장군보다는 높은 세포 활성도를 보였다 (p < 0.05) TUNEL 검사 결과도 자기장군은 즉시 발치군보다 치주인대의 세포사멸도가 높았으나 (p < 0.05) 저속 냉동군과 액화 질소군과는 통계적으로 유의한 차이를 보이지 않았다. 자기장군은 냉장군보다 세포사멸도가 낮았으며 냉장군은 모든 군 중에서 세포 사멸도가 가장 높았다 (p < 0.05).

• 대한골관절종양학회지
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• 제3권2호
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• pp.69-79
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• 1997

Objective : The objective of this study was to compare expression of various proto-oncogenes and rates of apoptosis in osteosarcoma patients. Modulation of apoptosis may influence resistance to chemotherapy and therefore affect the outcome of cancer treatment. Osteosarcoma is one of the most fatal malignancies in young adolescents and investigation of the role of apoptotic cell death is warranted in relation to chemotherapy and tumor outcome. Design : The terminal deoxynucleotidyl transferase to exposed 3'-hydroxyl termini of DNA (TUNEL method) staining method has been applied for the in situ detection of DNA double strand breaks. Patients : Thirty-three osteosarcomas in various stages of differentiation from twenty-nine patients were investigated immunohistochemically for p53, Bcl-2 and TUNEL method for apoptosis. Results and conclusion; We have found that higher level of wild type p53 were correlated with enhanced expression of apoptosis. Increased apoptosis rates were found in cases of negative Bcl-2 expression. In the present study, we have concluded that a significant proportion of osteosarcoma, a tumor in which resistance to chemotherapy often occurs, express high levels of p53 and low levels of Bcl-2. Our data provide further evidence for cross-talk between Bcl-2 and p53 and suggests that these genes are important determinants of drug-induced apoptosis.

• 한국수의병리학회지
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• 제3권1호
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• pp.27-33
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• 1999

Phosphamidon(PMD) is orgnophosphate insecticide broadly using in agriculture. In order to study PMD toxicity in the testis, histopathological change and apoptosis were assessed following acute and chronic oral administration in rats and chickens. In acute studies, histopathological changes included necrosis and desquamation of spermatogenic cells, multinucleated giant cells in the lumen of seminiferous tubules, and necrotic cells and the giant cells in the epididymal lumen. Atrophy of seminiferous tubule was seen in the chronic exposure with low doses. The toxic effects of PMD in chronic exposure including clinical signs and histopathological changes were more pronounced in chickens than rats. Apoptosis assessment was performed by TUNEL method and Hoechst staining. TUNEL-positive apoptotic cells were found in spermatocytes of seminiferous tubules, testicular apoptosis was more prominent following acute exposure than control and chronic exposure. Above mentioned result noticed that PMD causes apoptotic death and effects directly the spermatocytogenesis.

• 대한수의학회지
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• 제44권3호
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• pp.329-334
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• 2004

Ischemia/reperfusion(I/R) injury of the rat testis causes germ cell death and infiltration of inflammatory cells. To investigate the mechanism of germ cell death in torsion of the rat testis, apoptosis and macrophage activation were studied using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling(TUNEL) method and immunohistochemistry in the testes of Sprague-Dawley rats subjected to 1.5 h of ischemia, followed by 0, 1, 3, 6, 12, 24, 48 and 96 h of reperfusion. Apoptotic, TUNEL-positive cells were found at the base of the seminiferous epithelia after I/R. TUNEL-positive cells were significantly increased 6 h after repair of the torsion, and there was a significant peak in apoptosis 24 h after reperfusion, as compared with normal or sham-operated controls. In contrast, histological evidence of germ cell necrosis in the seminiferous tubules was first visible 24 h after reperfusion. In the testis of sham-operated rats, ED2-positive resident macrophages were found diffusely in the interstitial space, while ED1-positive monocyte-like macrophages were rarely found. After I/R, ED1-positive cells were significantly increased beginning 12 h after reperfusion, while ED2-positive immunoreactivity did not change during the experimental period. Together, the results of this study confirmed that increased numbers of ED1-positive macrophages, but not resident ED2-positive macrophages, infiltrated the interstitial space surrounding damaged tubules and induced germcell death.