Fluoro-Jade C染色在小鼠匹罗卡品癫痫模型中的应用

目的应用Fluoro-Jade C(FJC)染色技术在小鼠匹罗卡品癫痫模型中探测神经元变性,以了解FJC应用价值及其细胞死亡模式。方法雄性昆明小鼠6只:对照组3只;匹罗卡品处理组3只。处理组小鼠在癫痫持续状态后12 h处死,在海马水平切制冠状切片,先行FJC染色,用荧光显微镜观察;之后在切片上行FJC与Hoechst双标。结果处理组,FJC阳性细胞清晰显示,呈神经元形态,对照组未见。双标资料显示FJC(100%)与Hoechst33342双标记。结论小鼠匹罗卡品癫痫模型成功应用FJC染色技术探测了神经元变性,证实此技术在探测神经元变性方面敏感、可靠且简单,并显示此模型中FJC阳性细胞也许主要是凋亡性的。     

鼻咽癌细胞株侧群细胞染色条件的优化及其表面标记物的测定

目的探讨鼻咽癌细胞株侧群细胞所需荧光染料Hoechst33342最佳的孵育时间和浓度;研究细胞密度对侧群细胞比例的影响;测定其表面标记物CD133和ABCG2蛋白的表达。方法荧光染料Hoechst33342孵育细胞,选择不同的时间(30、60、90、120、150、180min)及不同的浓度(3、4、5、6、7、8mg/L),用流式细胞仪检测,倒置荧光显微镜观察选择合适的孵育时间和浓度。选取不同生长密度(70%、100%)的细胞,流式细胞仪检测侧群细胞比例的变化。荧光抗体CD133和ABCG2共孵育Hoechst33342,流式细胞仪测定细胞表面标记物CD133和ABCG2蛋白的表达。结果Hoechst33342孵育时间为70min,侧群细胞染色效果较佳。不同的鼻咽癌细胞株,合适孵育终浓度不同,如CNE2为6mg/L,而CNE1为2mg/L。另外,侧群细胞的比例呈生长密度依赖性。CD133蛋白总含量恒定在0.1%～0.2%,侧群细胞和主群细胞表达量没有显著差异,CD133并不富集于侧群细胞中。而侧群细胞ABCG2蛋白较主群细胞优势表达,比例达80%以上。结论鼻咽癌细胞株侧群细胞最佳的荧光染料染色时间为70mi...     

Out of the blue: a comparison of Hoechst side population (SP) analysis of murine bone marrow using 325, 363 and 407 nm excitation sources

BACKGROUND: The discrimination of side population (SP) cells is a useful flow cytometric tool for the study of putative stem cells in many tissue types. Previous studies on murine bone marrow (BM) SP cells and concurrent surface immunophenotyping provide a good model for comparing Hoechst bivariate profiles. Here we compare the results of SP cells from the same specimens generated on three different instruments. METHODS: Concurrent SP analysis and immunophenotyping was performed on murine BM. The data acquisition was performed on a DakoCytomation MoFlo, a Becton Dickinson LSR and a Becton Dickinson FACSAria to compare the ability of each instrument to discriminate putative stem cells. Further experiments examined the effect of apoptotic changes, assessed by Annexin V binding, and the presence of nucleated erythroid precursors on the Hoechst profile. RESULTS: Identifiable and equivalent side populations were generated on all three instruments despite differences in appearance of the Hoechst profiles. Differences in laser type, collection filter combinations and the presence of erythroid precursors and apoptotic cells all contributed to this effect. CONCLUSIONS: In our hands, murine SP cells with the expected surface immunophenotype can be identified on a MoFlo, LSR and a factory standard FACSAria.     

黄癸素诱导小鼠黑色素瘤B16细胞凋亡的研究

目的探讨黄癸素诱导小鼠黑色素瘤B16细胞凋亡的作用。方法应用MTT法检测黄癸素对体外培养的B16细胞增殖的抑制作用,观察量效及时效关系;通过Rhodamine123染色、琼脂糖凝胶电泳、Hoechst33258染色、caspase-3和caspase-8活性检测,观察黄癸素诱导细胞凋亡的作用和机制。结果黄癸素抑制B16细胞的增殖,具有明显的时间依赖性和浓度依赖性,作用24、48、72h的IC50分别为16.59、9.29和6.22mg·mL-1;B16细胞经黄癸素处理后,出现染色质固缩、DNALadder等凋亡表现,Rhodamine123染色荧光降低和caspase-3、caspase-8活性增强,提示黄癸素引起线粒体膜电位降低可能触发caspases级联反应而导致细胞凋亡。结论黄癸素可抑制B16细胞增殖,诱导B16细胞凋亡。     

Activation of distinct P2Y receptor subtypes stimulates insulin secretion in MIN6 mouse pancreatic β cells

Extracellular nucleotides and their receptor antagonists have therapeutic potential in disorders such as inflammation, brain disorders, and cardiovascular diseases. Pancreatic β cells express several purinergic receptors, and reported nucleotide effects on insulin secretion are contradictory. We studied the effect of P2Y receptors on insulin secretion and cell death in MIN6, mouse pancreatic β cells. Expression of P2Y₁ and P2Y₆ receptors was revealed by total mRNA analysis using RT-PCR. MIN6 cells were stimulated in the presence of 16.7 mM glucose with or without P2Y₁ and P2Y₆ agonists, 2-MeSADP and Up₃U, respectively. Both the agonists increased insulin secretion with EC₅₀ values of 44.6 ± 7.0 nM and 30.7 ± 12.7 nM respectively. The insulin secretion by P2Y₁ and P2Y₆ agonists was blocked by their selective antagonists MRS2179 and MRS2578, respectively. Binding of the selective P2Y₁ receptor antagonist radioligand [¹²⁵I]MRS2500 in MIN6 cell membranes was saturable (K_D 4.74 ± 0.47 nM), and known P2Y₁ ligands competed with high affinities. Inflammation and glucose toxicity lead to pancreatic β cell death in diabetes. Flow cytometric analysis revealed that Up₃U but not 2-MeSADP protected MIN6 cells against TNF-α induced apoptosis. Overall, the results demonstrate that selective stimulation of P2Y₁ and P2Y₆ receptors increases insulin secretion that accompanies intracellular calcium release, suggesting potential application of P2Y receptor ligands in the treatment of diabetes.     

Relationship between tumour endothelial cell apoptosis and tumour blood flow shutdown following treatment with the antivascular agent DMXAA in mice

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is currently undergoing clinical evaluation as an antivascular agent for the treatment of cancer. We have previously demonstrated that DMXAA induces apoptosis of vascular endothelial cells in murine tumour sections and in a breast carcinoma biopsy from one patient in a Phase I trial. We wished to determine the tissue selectivity of this effect and its relationship to induced blood flow changes. Mice with Colon 38 tumours were treated with DMXAA and tissues were examined for apoptosis by TdT-mediated dUTP nick-end labelling (TUNEL). Hoechst 33342 was used to stain functional vessels, with the loss of stained vessels used as a measure of tumour vascular collapse. Treatment with DMXAA at 25 mg kg(-1), its maximum tolerated dose (MTD), showed, after 3 h, a 12-fold increase in TUNEL staining of tumour vascular endothelial cells. In contrast, tissue from the heart, brain, liver and spleen showed no increase. Induction of apoptosis in tumour tissue was both dose-dependent, observable at doses as low as 5 mg kg(-1), and time-dependent. Apoptosis was significantly lower in Colon 38 tumours of mice, with a targeted disruption in the TNF gene (TNF(-/-)), or in the TNF receptor 1 gene (TNFR(-/-)), as compared with that in wild-type mice. Increasing the DMXAA dose to 50 mg kg(-1) in these knockout mice raised tumour apoptosis to a level comparable to that induced in wild-type mice given DMXAA at the MTD. For all the data, a significant correlation (r=0.94; P<0.001) was found between logarithmic percentage apoptosis induction and the logarithmic density of Hoechst-stained vessels. These results suggest that blood flow inhibition caused by DMXAA is tumour tissue-specific and is a consequence of induction of apoptosis in tumour vascular endothelial cells.     

Effect of antivascular endothelial growth factor treatment on the intratumoral uptake of CPT-11

Promising preclinical activity with agents blocking the function of vascular endothelial growth factor (VEGF) has been observed in various cancer types, especially with combination therapy. However, these drugs decrease microvessel density, and it is not known whether this reduced vessel density (VD) results in decreased delivery of concomitantly administered classical anticancer drugs. We designed an in vivo study to investigate the relation between VEGF-blocking therapy, tumoral blood vessels, and intratumoral uptake of anticancer drugs. Nude NMRI mice bearing colon adenocarcinoma (HT29) were treated with the anti-VEGFmAb A4.6.1 or placebo. After 1 week, CPT-11 was administered 1 h prior to killing the animals. In A4.6.1 treated tumours, there was a significant decrease in VD, more pronounced with potentially functional large vessels than endothelial cords. Interestingly, a trend to increased intratumoral CPT-11 concentration was observed (P=0.09). In parallel, we measured an increase in tumour perfusion, as estimated by high-performance liquid chromatography determination of intratumoural Hoechst 33342 concentration. In the growth delay study, CPT-11 was at least equally effective with or without pretreatment with A4.6.1. These data suggest that tumour vascular function and tumour uptake of anticancer drugs improve with VEGF-blocking therapy, and indicate the relevance for further investigations.     

Hochest染料-荧光分光光度法测定基因治疗非病毒载体中DNA的含量

目的　利用荧光染料Hoechst 332 5 8与DNA结合后 ,荧光强度显著增强的特点 ,建立了基因治疗非病毒载体中DNA的含量测定方法。方法　对Hoechst 332 5 8-DNA溶液进行荧光扫描 ,并建立了标准曲线。分别测定了纳米粒胶体溶液超速冷冻离心后的上清液和用酶消化后的脂质体提取液中DNA的含量。结果　Hoechst332 5 8-DNA溶液的最佳激发波长为 35 3.6nm ,最佳发射波长为 4 5 4 .4nm ,标准曲线的线性范围为 0 .2～ 1.0 μg·ml-1。测得包载DNA纳米粒的平均包封率为 75 .1%± 8.6 %(n =5 ) ,DNA阳离子脂质体的平均抗核酸酶能力为 84 .9%± 7.8% (n =5 )。结论　荧光染料Hoechst332 5 8可用于测定基因治疗非病毒载体中DNA的含量 ,所建立的荧光分光光度法为载基因纳米粒、脂质体制备工艺和质量标准的建立提供了依据。     

The Mammary Gland “Side Population”: A Putative Stem/Progenitor Cell Marker?

Hematopoietic Stem Cells have been isolated by their ability to pump out Hoechst 33342 dye and form a distinct population definable by flow cytometry—the Side Population (SP). The membrane pump Bcrp has been identified as the molecular determinant of the SP phenotype. An SP population with Bcrp activity has been defined in a number of tissues, including mouse mammary and human breast epithelium, and it has been proposed that the SP phenotype is a universal stem cell marker. Studies of mouse and human breast SP suggest that the population is undifferentiated but capable of differentiating into epithelial structures of both luminal and myoepithelial lineages both in vitro and in vivo. However, evidence that the SP is enriched for stem cells is, at the moment, only correlative, and there are potentially confounding technical issues. We still await formal proof that the SP contains a stem cell population.     

A sensitive assay of cytotoxicity applicable to mixed cell populations

The fluorescent vital dye, Hoechst 33342, was used to stain cultured cells prior to assay of antibody dependent complement mediated cytotoxicity. The fluorescence of nonviable dye stained cells is quenched by cellular uptake of trypan blue, but trypan blue excluding cells remain intensely fluorescent. Detection by fluorescence microscopy of one viable prestained cell per 10(5) unstained cells was accurate and reliable. The technique was found to have sensitivity equal to a clonogenic assay for measuring cytotoxicity. The dye stained cell assay may be used to measure depletion of a selected cell type, when those cells are stained prior to mixing with another cell population. This technique may prove useful to study model systems for depletion of tumor cells or T-cells from bone marrow.