吖啶橙荧光染色法在胃癌诊断中的临床意义

目的：探讨吖啶橙荧光染色检测法对胃癌的诊断价值。方法：对100例胃癌患者进行内窥镜取材，将标本同时做吖啶橙荧光染色检测及组织病理学检查。结果：吖啶橙荧光染色检测胃癌的检出率为46％。结论：对胃癌的诊断和鉴别诊断具有一定的临床意义，具有快速、简便、成本低等优点。     

白杨黄素对人胃癌细胞SGC7901的放疗增敏作用及其相关机制研究

目的:探讨白杨黄素对人胃癌SGC7901细胞增殖的放射增敏作用及其相关机制.方法:应用克隆形成实验分别检测不同浓度白杨黄素及60Co γ射线联合应用对人胃癌细胞系SGC7901细胞生存率的影响;应用吖啶橙(AO)/溴化乙碇(EB)荧光染色和TUNEL法分别检测白杨黄素与60Co γ射线联合应用后对人胃癌细胞系SGC7901细胞凋亡的影响;应用Westen-blot检测与细胞凋亡调控相关的蛋白Caspase-3、Survivin和Bcl-2的表达情况.结果:白杨黄素能够明显增强60Co γ射线对人胃癌SGC7901细胞克隆集落形成的抑制作用;增强60Co γ射线导致的人胃癌SGC7901细胞的凋亡;白杨黄素能够上调60Co γ射线处理过后人胃癌SGC7901细胞内活性Caspase的表达,下调细胞内凋亡抑制蛋白Survivin和Bcl-2的表达.结论:对于胃癌细胞系SGC7901,白杨黄素可以作为一种有效的放射增敏剂.     

鲨鱼软骨制剂对人胃癌细胞增殖抑制作用的研究

目的研究SCP对人胃癌细胞生长、抑制和损伤效应,探讨其抗癌作用机制.方法用不同浓度的SCP加入体外培养的人胃癌细胞(MGC80-3)中,观察加药后细胞生长和有丝分裂指数(MI)的变化;用MTT法检测其细胞增殖抑制作用;用吖啶橙荧光染色方法观察细胞DNA和RNA含量的变化,用透射电镜观察细胞的损伤效应.结果SCP明显抑制MGC80-3细胞生长,IC50值为1mg/ml;MI于加药后12h较对照组明显降低;60%以上细胞DNA和RNA荧光染色较对照组明显减弱,核DNA由黄色荧光变为绿色荧光,胞质红色荧光几乎消失,电镜下可见核染色质凝集边移,细胞器变性.结论SCP可有效抑制人胃癌细胞的增殖.     

罗格列酮诱导人胃癌MGC803细胞凋亡及其对bax和Bcl-2表达的影响

目的:探讨罗格列酮对人胃癌MGC803细胞凋亡的影响及其分子机制。方法:吖啶橙(AO)荧光染色和流式细胞仪分析检测细胞凋亡。同时行bax和bcl-2细胞免疫化学染色和定量分析。结果:罗格列酮能诱导MGC803细胞凋亡,且随着浓度增加,其凋亡率逐渐增加。罗格列酮(25μmol/L、50μmol/L)作用于人胃癌MGC803细胞48h后bax表达的光密度值由0·10±0·02分别升至0·42±0·03、0·75±0·04,P=0·000;bcl-2表达的光密度值由0·51±0·02分别降至0·25±0·04、0·12±0·01,P=0·001。结论:罗格列酮能诱导人胃癌MGC803凋亡,其机制可能与bax基因表达上调、bcl-2基因表达下调有关。     

吖啶橙荧光染色检测法在恶性肿瘤诊断方面的研究

近年来恶性肿瘤的发病率及死亡率不断升高，而死亡率又是与早诊断、早治疗息息相关的，怎样才能早诊断从而获得早治疗，这是肿瘤界长期以来一直在探索的问题、。目前确诊恶性肿瘤主要是依据病理细胞学检查和组织学检查，它们是通过细胞形态和组织结构的改变来作出诊断的。而部分早期癌症病人细胞往往只表现有难以确诊的核酸的增加，还没有形态和结构的改变，这就是造成细胞学和组织学漏诊的主要原因。吖啶橙荧光染色检测法则可弥补细胞学、组织学诊断的这一不足，因为它是通过荧光染色后，观察核酸的变化来判断细胞的良恶性程度。下面就AO荧光染色检测法的原理、诊断标准、现行评价及方法改进等综述如下。     

选择性COX-2抑制剂Celecoxib诱导胶质瘤细胞凋亡作用机制初探

背景与目的：选择性COX-2抑制剂具有诱导多种肿瘤细胞凋亡的作用，但在胶质瘤方面研究还不多见，本研究门的是探讨选择忡COX-2抑制剂塞来昔布（Celecoxib）诱导胶质瘤C6细胞凋亡，及其在诱导胶质瘤细胞凋亡的作用机制方法：应州PGE2检测、吖啶橙染色、流式细胞仪（FCM）检测不同浓度的Celecoxib对胶质瘤C6细胞作用，通过放免法检测不同浓度Celecoxib作用于胶质瘤C6细胞后PGE2的含量，通过吖啶橙染色从形态上观察治疗组细胞的形态学变化，流式细胞仪检测各组细胞发生凋亡的情况。结果：随Celecoxib浓度增加而PGE2合成减少（P〉0．05）．吖啶橙染色荧光染色对照组细胞核DNA为均匀黄色或黄绿色的荧光，治疗组（Celecoxib 50μmol／L）凋亡细胞的细胞核或细胞质内可处致密浓染的黄绿色荧光或黄绿色碎片．Celecoxib浓度为12．5μmol／L和50μmol／L时．FCM法检测凋亡分别为5．83％和15．32％．Celecoxib对C6胶质瘤细胞增殖抑制作用具有浓度依赖性．细胞凋亡随浓度增加而增多。结论：Celecoxib具有诱导胶质瘤C6细胞凋亡的作用并呈剂量依赖性，随PGE2合成下降细胞凋亡增多，PGE2途径是Celecoxib诱导胶质瘤细胞凋亡机制之一．对胶质瘤的治疗有重要意义。     

BaX在维生素E琥珀酸酯诱导人胃癌细胞凋亡中的作用

背景与目的:探讨Bax蛋白在维生素E琥珀酸酯(vitamin E succinate,VES)诱导人胃癌SGC-7901细胞凋亡中的作用.材料与方法:用吖啶橙/溴化乙啶(AO/EB)染色观察细胞凋亡的形态学改变;Westem Blot法检测不同剂量VES对人胃癌SGC-7901细胞中Bax蛋白表达量的影响和VES对Bax蛋白与细胞色素c(cytochrome c,Cyt c)蛋白在细胞内分布的影响;用Mito Tracker RedCMXRos荧光染色观察线粒体膜电位(△Ψm)的变化.结果:VES可引起SGC-7901细胞发生凋亡,提高Bax蛋白表达水平,并使Bax蛋白从胞浆转移到线粒体;VES作用后发生线粒体膜电位下降,Cyt c从线粒体释放到胞浆.结论:VES可能通过Bax蛋白来启动线粒体凋亡途径,诱导SGC-7901细胞发生凋亡.     

特异性环氧合酶-2抑制剂SC236诱导胃癌细胞株凋亡的实验研究

目的:探讨环氧合酶-2(COX-2)特异性抑制剂SC236诱导胃癌细胞株SGC7901凋亡的机制. 方法: SC236对人胃癌来源的SGC7901细胞进行凋亡诱导.以吖啶橙染色后荧光显微镜下观察凋亡细胞形态并计算凋亡指数.Western blot法检测凋亡调节蛋白Bcl-2、Bak、Bax与CED-9的表达情况.流式细胞仪检测细胞凋亡率.结果: SGC7901细胞经SC236作用后,细胞中凋亡促进因子Bak的表达水平明显增高、凋亡抑制因子Bcl-2的表达水平显著下降、而凋亡促进因子Bax和凋亡抑制因子CED-9表达水平无明显变化.细胞凋亡指数或凋亡率则随SC236浓度的增加和作用的时间延长而升高.结论: COX-2特异抑制剂SC236可通过上调凋亡促进因子Bak表达和下调凋亡抑制因子Bcl-2表达而诱导胃癌细胞株SGC7901的凋亡.     

白杨黄素对人胃癌细胞SGC7901的放疗增敏作用及其相关机制研究

目的：探讨白杨黄素对人胃癌SGC7901细胞增殖的放射增敏作用及其相关机制。方法：应用克隆形成实验分别检测不同浓度白杨黄素及^60Coγ射线联合应用对人胃癌细胞系SGC7901细胞生存率的影响;应用吖啶橙（AO）／溴化乙碇（EB）荧光染色和TUNEL法分别检测白杨黄素与^60Coγ射线联合应用后对人胃癌细胞系SGC7901细胞凋亡的影响;应用Westen—blot检测与细胞凋亡调控相关的蛋白Caspase-3、Survivin和Bcl-2的表达情况。结果：白杨黄素能够明显增强^60Coγ射线对人胃癌SGC7901细胞克隆集落形成的抑制作用;增强^60Coγ射线导致的人胃癌SGC7901细胞的凋亡;白杨黄素能够上调^60Coγ射线处理过后人胃癌SGC7901细胞内活性Caspase的表达,下调细胞内凋亡抑制蛋白Survivin和Bcl-2的表达。结论：对于胃癌细胞系SGC7901,白杨黄素可以作为一种有效的放射增敏剂。     

大黄素对人胃癌细胞MKN45增殖抑制作用及其分子机制的探讨

目的:研究大黄素(Emodin)对人胃癌细胞MKN45的作用及探讨其诱导MKN45凋亡的分子机制。方法:用MTT法观察大黄素对MKN45的生长抑制作用;荧光染色法及流式细胞仪分析诱导凋亡作用;蛋白质印迹法检测p53和p21蛋白水平的变化;细胞免疫化学法检测Fas的表达。结果:与对照组相比,大黄素能明显抑制MKN45细胞的生长且呈浓度、时间依赖性,其IC50(48h)为(47.5±0.3)μmol/L;大黄素处理胃癌细胞后,吖啶橙(AO)染色观察示,细胞核内可见浓染致密的颗粒荧光、新月型改变、核固缩或片段化及凋亡小体的形成。流式细胞仪分析显示,大黄素能浓度依赖性诱导MKN45细胞凋亡和G2/M期阻滞。蛋白质印迹法检测显示,随着药物浓度的不断增加,p53和p21WAF1蛋白水平表现出明显增强的趋势;细胞免疫化学方法显示,大黄素处理后的细胞Fas/APO-1表达较对照组明显增多。结论:大黄素对人胃癌细胞MKN45有明显的生长抑制作用,且该作用与p53依赖性诱导凋亡以及Fas表达水平的上调有关。     

A comparison of acridine orange and Feulgen cytochemistry of human tumor cell nuclei.

Specimens of cells derived from tumors of the human female genital tract plus normal cells as standards have been divided into aliquots and stained according to acridine orange or pararosanilin:Feulgen procedures. Acridine orange-stained cells were slit-scanned for 535 nm nuclear fluorescence; Feulgen-stained cells were comb-scanned for 580 nm nuclear absorbance. For each specimen examined, the tumor cell:normal cell ratio of mean nuclear fluorescence following acridine orange staining was greater than the tumor cell:normal cell ratio of mean nuclear absorbance following Feulgen staining. The tumor cell:normal cell ratio of mean nuclear fluorescence ranged from 2.3 for a nonkeratinizing squamous cell carcinoma to 3.9 for a keratinizing squamous cell carcinoma. The tumor cell:normal cell ratio of mean nuclear absorbance ranged from 1.4 for a mixed mesodermal sarcoma to 2.3 for a small cell squamous cell carcinoma. These results indicate that the elevated nuclear fluorescence intensity from acridine orange-stained tumor cells cannot be explained solely on the basis of elevated Feulgen:DNA content. An alternative hypothesis, consistent with these results, is that DNA is the principal binding substrate for intranuclear acridine orange and that the DNA of certain tumor cells is more accessible to acridine orange than is the DNA of normal cells.     

Cell kinetics and histological types of gastric cancer as studied by DNA-RNA cytofluorometry after acridine orange fluorescence staining

In order to clarify the relation between histological classification and biological behavior of individual tumor cells, we quantitatively analyzed the cell kinetics of 56 advanced gastric cancers using DNA-RNA cytofluorometry after acridine orange fluorescence staining and compared the results with histological findings. The gastric cancers examined could be classified into two main groups based on the ploidy patterns constituting the cell population kinetics. In group I, tumor cells were found to be chiefly composed of diploid cells with many S-phase cells even at their advanced growth stages, and their morphological characteristics were shown to correspond to undifferentiated carcinoma (according to Nakamura & Sugano). In group II, tumor cells were composed of various classes of polyploid cells as well as of diploid cells with an increased number of S-phase cells, and the extent of polyploidization appeared to progress with the growth of the tumor. The morphological characteristics of this group tended to correspond to differentiated adenocarcinoma. It is, therefore, concluded that both the histological structures and cell morphology have close relationships with cell kinetics in the human gastric cancers.     

Binding of acridine orange to DNA in situ of cells from patients with acute leukemia

Fluorescence flow cytometry was used to generate DNA histograms of acridine orange stained leukemic cell populations in G0-G1 phase of the cell cycle. Complexes of the intercalating agent, acridine orange, with double-stranded DNA in situ, emit green fluorescence upon excitation with blue laser light. The histograms were evaluated by first determining the standard deviation of the fluorescence intensity relative to the mean channel of fluorescence, i.e., the coefficient of variation, and then dividing the coefficient of variation of a patient's sample by that of a control sample (rCV). The mean rCV of cell populations of acute lymphoblastic leukemia (31 patients) differed significantly from that of nonlymphoblastic leukemia (21 patients). When cells were treated with a solution of citric acid and magnesium sulfate prior to their staining with acridine orange, the mean rCV of cell populations of acute lymphoblastic leukemia increased while that of acute nonlymphoblastic leukemia decreased compared to their respective pretreatment values. The mean difference of rCVs between untreated and treated cells (rCVD) within each disease category was statistically significant. A logistic regression model, based on rCVD, confirmed the conventional classification of acute lymphoblastic leukemia and acute nonlymphoblastic leukemia cells in 90% of the cases.     

胃癌组织幽门螺杆菌感染与EB病毒潜伏膜蛋白表达的相关性

目的：观察胃癌组织中幽门螺杆菌（H．pylori）感染和EB病毒潜伏膜蛋白（EBV—LMP）的同步表达情况，探讨两者在胃癌中的相互关系。方法：80例正常胃黏膜组织和97例胃癌组织，利用HE染色对所取标本进行组织病理学诊断，EBV—LMP测定采用免疫组化sP法，H．pylori感染判定采用HE，H．pylofi—DNA PCR和血清ELISA法测定IgG抗体三种方法。结果：正常胃黏膜组织未见EBV—IMP的表达，胃癌黏膜组织EBV—LMP表达阳性率为7．2％（7／97），胃癌组织EBV—LMP表达明显高于正常胃黏膜组织，差异有统计学意义（P〈0．05）。本组病例中H．pylofi感染的胃癌黏膜组织中EBV—LMP的阳性表达率为13．5％（7／52），无H．pylori感染的胃癌组织EBV—LMP的阳性表达率为0，H．pylori感染的胃癌组织EBV—LMP表达明显高于无H．pylofi感染的胃癌组织，差异有统计学意义（P〈0．05）。结论：正常胃黏膜组织无EBV—LMP的表达，胃癌组织中EBV—LMP表达明显高于正常胃黏膜组织，H．pylofi感染胃癌组织比无H．pylori感染胃癌组织EBV—LMP呈现高表达，H．pylofi感染和EB病毒感染在胃癌的发生和发展过程中可能具有更加深在的相互关系。     

5-bromodeoxyuridine (BUdR) quenching of acridine orange fluorescence distinguishes cycling and non-cycling normal and malignant bone marrow cells in vitro

Suppression of the green fluorescence of acridine orange by 5-bromodeoxyuridine incorporation into cellular DNA was measured by flow cytometry. Bone marrow cells from normal volunteers and patients with chronic myelogenous leukemia acute lymphocytic leukemia acute myelogenous leukemia and multiple myeloma were incubated with BUdR in vitro. By 24 h acridine orange stained cycling cells that had synthesized DNA in the presence of BUdR were differentiated from quiescent cells as a second peak with quenched green fluorescence (DNA). After 72 h in culture 11-65% of the G0/G1 cells from normal bone marrows and bone marrows with myeloid leukemia were identified as cycling in culture by the presence of a second peak with quenched green fluorescence. A greater percentage of cells with BUdR quenched AO fluorescence was associated of acridine orange was higher in the cycling cells that had synthesized DNA in the presence of BUdR than in the non-cycling G0/G1 cells. In one patient with AML there was quenching of the DNA fluorescence of the aneuploid population but not the diploid population indicating that the aneuploid leukemia cells were proliferating. In contrast in patients with multiple myeloma, the quenched fluorescence of the diploid cell population and not the aneuploid cells, indicated that the diploid cells were proliferating. The cells from patients with untreated ALL failed to proliferation prohibiting an in vivo assessment of growth. Although measurements of proliferation obtained by this method are clearly influenced by the cell's adaptation to culture, measurement of BUdR quenching of acridine orange fluorescence is technically feasible and can identify and allow characterization of the cycling population of cells.     

Aloe-Emodin Influence on the Lysosomal Compartment of Hela Cells

Background: Aloe-emodin belongs to the group of anthraquinones having extremely high biological activity. The aim of this study was to evaluate the range of morphological and biochemical changes in HeLa cells treated with aloe-emodin, especially with regard to the lysosomal compartment. Methods: Marking of lysosomes was performed with neutral red staining for conventional light microscopy and acridine orange staining for confocal microscopy. To evaluate ctivity of lysosomal enzymes and permeability of the lysosomal membrane, spectrophotometric techniques were employed. Results: Aloe-emodin caused increased permeability of lysosomal membranes in HeLa cells, expressed inter alia by extinction of the orange color of acridine orange (lysosomal marker) and in reduction of neutral red uptake by lysosomes. These changes are accompanied by release of cathepsins from the interior of the lysosomes with a simultaneous highly significant increase in their activity in the cytoplasm. Conclusion: The results indicate that aloe-emodin can activate lysosomal pathway-dependent apoptosis in HeLa cells.     

Significance of serum and tissue carcinoembryonic antigen for the prognosis of gastric carcinoma patients

BACKGROUND AND OBJECTIVES: Carcinoembryonic antigen (CEA) has been widely accepted as a tumor marker useful in the diagnosis and management of colorectal carcinoma. When CEA levels are positive in patients with gastric carcinoma, they could be useful prognostic indicators. The value of CEA as a tumor marker for gastric carcinoma, however, remains a matter of controversy. The purpose of this study was to determine whether preoperative serum CEA value and tissue CEA staining are useful prognostic indicators for gastric carcinoma. METHODS: We measured preoperative serum CEA levels by radioimmunoassay and stained tissue CEA production by tumor cells from gastric carcinomas using immunohistochemical staining in patients with gastric carcinoma. RESULTS: The patients with preoperative serum CEA levels >10.0 ng/mL had a more prominent serosal invasion, much more lymph node involvement, more advanced stage and more poorly differentiated than did the patients with preoperative serum CEA levels <5.0 ng/mL. The survival rate of patients with serum CEA levels >10.0 ng/mL was poorer than those of patients with serum CEA levels between 5.0 and 10.0 ng/mL, and those of patients with serum CEA levels <5.0 ng/mL (P < 0.05). The preoperative serum CEA levels and tumor CEA-positivity were correlated (P < 0.05). In patients with lymph node metastases, the CEA-positivity (78.0%) was higher than in patients without lymph node metastasis (63.2%) (P < 0.05). A correlation was also found between the depth of tumor invasion and tissue CEA-positivity (P < 0.001). The postoperative survival rate was significantly better in the CEA-negative staining group (78.0%) than in the CEA-positive staining group (60.0%). CONCLUSIONS: These data suggest that preoperative serum CEA levels and staining for CEA in gastric carcinoma tissue sections may have a predictive value in determining prognostic information for patients with resectable gastric carcinoma.     

The Apoptotic Properties of Leaf Extracts of Simarouba glauca against Human Leukemic Cancer Cells

Background and objective: Simarouba glauca is a plant belonging to the family of Simaroubaceae. It is a potent source of secondary metabolites. The aim of this study was to evaluate the apoptotic properties of leaf extracts of Simarouba glauca against human leukemic cancer cells. Materials and Methods: Cytotoxicity of Simarouba glauca was assessed in the leaf extract of petroleum ether against leukemic cells by MTT assay. To detect the apoptotic features, fluorescence microscopy analysis was done with dual acridine orange/ethidium bromide fluorescent staining and Hoechst staining. To determine the externalization of phosphatidylserine, annexin v staining was done. Mitochondrial or death receptor activation was confirmed by caspase 3 analysis by flow cytometry. Results: This study revealed that Simarouba glauca was able to treat leukemia. Among the four extracts, petroleum ether extract showed a higher order of in vitro anticancer activity. The petroleum ether extract strongly inhibited the proliferation of K562 cell lines with IC50 values of 186 µg/ml. Dual acridine orange/ethidium bromide fluorescent staining and Hoechst staining revealed the characteristic features of apoptosis. Annexin V confirmed early and late stage apoptosis. Caspase-3 analysis revealed that cell death was due to mitochondrial or death receptor activation in mitochondrial pathway. Conclusion: These findings suggested that Simarouba glauca leaf extracts inhibited leukemic cells in a time- and dose-dependent manner either through mitochondrial or death receptor activation. The leaf extracts of Simarouba glauca was found to be nontoxic to lymphocytes. It can be concluded that Simarouba glauca is an important source of phytochemicals posing efficacy against leukemic cancer cells.     

Bicalutamide dose-dependently inhibits proliferation in human prostatic carcinoma cell lines and primary cultures

OBJECTIVE: Androgen antagonists inhibit prostatic cell proliferation in normal and pathological conditions and are useful antitumor agents in prostatic carcinoma (PCa). Bicalutamide (BCLT) is a well-known non-steroidal antiandrogenic agent able to interfere with androgen receptor (AR). We tested the efficacy of BCLT in inhibiting proliferation of human PCa cell lines and of primary cultures from biopsies of PCa patients. MATERIALS AND METHODS: Human prostatic carcinoma cell lines (PC3, DU145, LNCaP ALVA 31 and ND1) and short-term primary tissue cultures from PCa patients were treated with BCLT. Cell proliferation and orange acridine and ethidium bromide fluorescence staining studies were performed. RESULTS: BCLT was able to inhibit, significantly and dose-dependently, cell proliferation in AR-positive human PCa cell lines and in 10 cases of primary cultures with Gleason grades 4 to 8. Its action appears to be mainly apoptotic in AR-positive cells and cytotoxic in AR-negative cells. CONCLUSION: BCLT, which inhibits growth in both human PCa cell lines and PCa primary cultures from patients with medium and low-grade tumors, deserves attention as a potential widely effective antiandrogenic monotherapy in prostatic carcinoma. However, its efficacy in AR-negative cells requires further research.