黄鱼鱼鳔肽分离及其诱导前列腺癌DU-145细胞凋亡的机制

为探讨黄鱼鱼鳔肽诱导前列腺癌DU-145细胞凋亡的作用机制,从黄鱼鱼鳔酶解产物中分离纯化得到鱼鳔肽,鉴定其氨基酸序列。采用CCK-8法检测鱼鳔肽作用后DU-145细胞抑制率的变化;AO/EB法检测DU-145细胞凋亡情况;流式细胞术检测DU-145细胞凋亡率和周期;免疫印记法检测DU-145细胞凋亡蛋白表达。结果表明,分离得到的鱼鳔肽YCSB-1c中包含Ser-Pro-Ser-Pro和Gly-Pro-Ala-Arg两条寡肽。与空白组相比,YCSB-1c组中凋亡细胞明显增多,且呈质量浓度依赖性;流式细胞仪检测得出YCSB-1c组中存活细胞明显减少,晚期凋亡细胞明显增多。YCSB-1c将DU-145细胞的细胞周期阻滞在G0/G1期,上调Bax、Caspase-3及Caspase-9蛋白表达,诱导DU-145细胞凋亡。YCSB-1c能有效诱导DU-145细胞凋亡,其作用机制与细胞周期停滞和线粒体介导的凋亡途径有关。     

松口蘑菌丝体蛋白质诱导细胞凋亡

采用深层发酵技术培养菌丝体并提取分离出的蛋白质,进行体外抗人子宫颈癌HeLa细胞增殖及诱导细胞凋亡机理的研究.试验发现松口蘑菌丝体水提液中活性蛋白质TMP在体外具有抑制肿瘤细胞增殖的作用,扫描电镜观察到TMP处理细胞产生明显的凋亡小体,TMP对细胞周期的影响是通过抑制细胞从S到G2M期的转化来抑制HeLa细胞增殖,诱导细胞发生凋亡的.DNA电泳出现以200bp左右为单位的DNA碎片.结果表明采用液体培养方法生产的松口蘑菌丝体蛋白质,具有显著的抑制细胞增殖和诱导细胞凋亡作用.     

三棱黄酮抗HeLa宫颈癌:降低分裂期细胞比率诱导细胞凋亡

目的:研究三棱的总黄酮(Rhizoma sparganii flavonoids,RSF)对HeLa宫颈癌细胞的毒理作用及其作用机制。方法:分别检测用含有质量浓度11.7～750μg/mL RSF的DMEM培养基培养时,组间HeLa细胞的增殖活性差异,通过免疫荧光技术、流式细胞术和核型分析方法分析RSF诱导HeLa细胞毒理的机制。结果:RSF含量高于188μg/mL的DMEM培养基可呈剂量性抑制HeLa细胞的增殖活性(P<0.01)、增加细胞凋亡小体的比率(P<0.01);与1%甲醇对照组相比,RSF-375(RSF含量为375μg/mL)与RSF-750(RSF含量为750μg/mL)药物组HeLa细胞的分裂(M)期细胞比率显著下降(P<0.01),细胞间的接触生长状态消失,体积增大;在本次实验中,RSF-375与RSF-750药物组HeLa细胞微管(a-tublin)细胞骨架形态的特异性免疫结果与对照组相比,细胞形态呈显著不规则状改变,微丝突触增多,表现为较高的迁移运动趋势。结论:RSF是三棱抗HeLa宫颈癌的有效成分,在本实验中,RSF可以通过干扰细胞有丝分裂相关的机制显著抑制HeLa细胞的增殖活性(P...     

珠母贝糖胺聚糖PG-Ⅱ诱导人宫颈癌HeLa细胞凋亡的研究

目的:探讨珠母贝糖胺聚糖PG-II诱导人宫颈癌HeLa细胞凋亡,从而抑制其增殖。方法:MTT法检测珠母贝糖胺聚糖PG-II对人宫颈癌HeLa细胞生长的抑制作用。倒置显微镜、荧光显微镜观察HeLa细胞凋亡的形态学特征,琼脂糖凝胶电泳检测DNA断裂片段,流式细胞仪定量检测细胞凋亡的百分率及对其细胞周期的影响。结果:MTT法显示PG-II可抑制人宫颈癌HeLa细胞的增殖。倒置显微镜和荧光显微镜形态学观察可见HeLa细胞凋亡的形态明显变化。琼脂糖凝胶电泳显示PG-II作用HeLa细胞24h,出现分子质量为200bp到2000bp不等的细胞凋亡的特征性DNA梯状条带。流式细胞术测试表明:PG-II可使HeLa细胞阻滞于G1期,剂量为100mg/L作用24h阻滞作用最明显,凋亡指数为42.6%。结论:珠母贝糖胺聚糖PG-II可能通过诱导人宫颈癌HeLa细胞凋亡而抑制其增殖。     

广西牡蛎活性肽抑制卵巢癌淋巴结定向高转移细胞增殖及对运动迁移能力的调控作用

目的：研究广西牡蛎活性肽对卵巢癌淋巴结定向高转移细胞（SKOV3-PM4）的增殖及运动迁移能力的抑制作用。方法：采用四甲基偶氮唑蓝（3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MTT）法测定广西牡蛎活性肽作用24 h后对细胞增殖的抑制作用。细胞经不同质量浓度的广西牡蛎活性肽处理后,采用细胞计数法和集落形成实验测定细胞增殖能力;细胞划痕实验和Transwell小室测定细胞体外运动和侵袭迁移能力。结果：MTT实验结果显示牡蛎活性肽质量浓度在5～80 mg/L范围内抑制SKOV3-PM4的增殖,24 h的半数抑制浓度（inhibitory concentration 50%,IC50）为35.36 mg/L。细胞划痕实验表明无细胞毒性的质量浓度为4 mg/L的牡蛎活性肽能抑制SKOV3-PM4的增殖能力,牡蛎活性肽作用SKOV3-PM4细胞后其侵袭、迁移能力降低,与空白对照组比较差异显著（P＜0.05）。结论：从牡蛎中分离出的小分子多肽在一定质量浓度下对SKOV3-PM4细胞的生长增殖、运动迁移具有抑制作用。     

牡蛎蛋白活性肽的分离及生物活性研究进展

牡蛎活性肽是海洋天然产物研究的重要组成部分,现代医学研究表明,牡蛎活性肽在抗肿瘤、抗氧化、降血压、降血糖等方面具有特殊的生理活性。就牡蛎蛋白质酶解法制备活性肽的分离纯化技术、牡蛎活性肽的生物活性作用等研究进展进行概述,为牡蛎活性肽的深入研究提供参考。     

沙蚕活性蛋白酶诱导人肺癌SPC-A-1细胞凋亡的机制研究

本文探讨了沙蚕活性蛋白酶(Nereis active protease,NAP)诱导SPC-A-1细胞凋亡机制。采用MTT法检测NAP对SPC-A-1细胞的抑制作用,倒置显微镜及AO/EB染色观察SPC-A-1细胞的形态学的变化,采用流式细胞术检测细胞早期凋亡率和细胞膜电位的变化;并通过Western Blotting检测细胞中凋亡相关蛋白的表达变化。NAP对SPC-A-1细胞活性具有明显的抑制作用且呈现剂量和时间的依赖性;NAP作用后细胞出现凋亡的形态学特征;经流式细胞术检测结果显示,随着NAP作用浓度的增加,SPC-A-1细胞的早期凋亡率从13.50%提高到22.98%,且线粒体膜电位下降所占的百分率从12.95%提高到25.28%。Western Blotting结果显示,50μg/mL NAP作用24h后,Bax/Bcl-2的比率相对对照组明显增加了6.05倍;Cyt-C、Cleaved-Caspase 9、Cleaved-Caspase 3、Cleaved-PARP等含量显著上调,相对表达量分别达到对照组的2.32、3.07、3.68、1.36倍。NAP诱导SPC-A-1细胞凋亡的作用机理有可能是通过下调Bcl-2蛋白的表达、上调Bax蛋白的表达,进而诱导线粒体膜电位的下降,促使Cyt-C的转移以及激发Caspase家族发生级联反应最终导致SPC-A-1细胞的凋亡。     

凋亡素融合蛋白质高表达菌株的筛选及其产物活性测定

目的筛选凋亡素-HBD融合蛋白质高表达的工程菌株,验证其目标产物具有抑制肿瘤细胞增值的活性。方法在不同大肠杆菌宿主中凋亡素融合蛋白质表达,筛选出高表达工程菌,随后诱导表达和纯化凋亡素融合蛋白质,并通过噻唑蓝法（MTT法）检测目标产物对肿瘤细胞的抑制活性。结果凋亡素-HBD融合蛋白质在SG工程菌的表达量较好。MTT法检测表明,凋亡素融合蛋白质有抑制HeLa细胞增值的活性。结论获得一种凋亡素-HBD融合蛋白质的高表达工程菌株,并证实其产物能够进入HeLa细胞并诱导细胞凋亡。     

卵叶娃儿藤生物碱对宫颈癌细胞株HeLa的体外作用

为了观察卵叶娃儿藤总生物碱[Tylophora ovata(Lindl.)Hook.ex Steud,alkaloids TOHa]体外抑制人宫颈癌细胞株HeLa增殖、诱导细胞凋亡的作用,将不同浓度的TOHa与HeLa细胞在体外培养,用MTT法观察TOHa对HeLa细胞的生长抑制作用,测定其在490nm的OD值;用细胞形态学、AnnexinV/PI双标记检测细胞凋亡。结果表明,TOHa能抑制HeLa细胞的增殖和活力,呈现作用时间和剂量的依赖关系。HeLa细胞经TOHa作用后,Annexin V /PI-表达升高,Giemsa染色后出现凋亡细胞的特征性改变。     

野生笃斯越桔总花青素对SPCA-1细胞活力抑制及机理

从中国野生笃斯越桔中提取总花青素,并经过大孔树脂XAD-7的分离纯化,探究总花青素提取物对SPCA-1细胞增殖、周期、凋亡和细胞内活性氧释放的作用情况,采用酶联免疫Elisa检测与凋亡相关的蛋白活性进而初步探讨其作用机理。实验结果发现,中国野生笃斯越桔总花青素对SPCA-1细胞活力有显著抑制作用,抑制增值IC50值为107.399μg/mL,总花青素显著影响了细胞周期并引起早期凋亡和细胞内活性氧的产生,且细胞各凋亡蛋白的表达量变化都表明了总花青素诱导SPCA-1细胞的凋亡。     

十二烷醇对酵母细胞影响的研究

以残糖和细胞生长OD值为分析指标,探讨了十二烷醇对酵母游离细胞和固定化细胞的影响.结果表明:萃取剂对游离细胞毒害性比较大,发酵残糖含量高；固定化细胞采用外部随程萃取发酵则可以忽略其对细胞的毒害作用,发酵残糖几乎没有.     

Cell microarray for screening feeder cells for differentiation of embryonic stem cells

Microarrays are currently recognized as one of major tools in the assessment of gene expression via cDNA or RNA analysis and are now accepted as a powerful experimental tool for high-throughput screening of a large number of samples, such as cDNA and siRNAs. In this study, we examined the potential of the microarray methodology for high-throughput screening of candidate cells as feeder cells which effectively differentiate embryonic stem (ES) cells to the specific lineage. Cell arrays were prepared by applying three kinds of cells, PA6, human umbilical vein endothelial, and COS-1 cells, to circular spots, 2 mm in diameter, on a glass plate, followed by the application of mouse ES cells to the cell microarray. After 8 d in culture, TuJ1 (neuron-specific class III beta-tubulin) immunocytochemical staining clearly demonstrated that only PA6 cell spots had the capability to induce ES cells to neuronal differentiation. Although this is a model experiment, these findings clearly indicate that the cell microarray will become a powerful tool for high-throughput screening large numbers of candidate feeder cells for specific differentiation.     

亚硒酸钠在SGC-7901细胞生长中的抑制作用

目的：研究亚硒酸钠对SGC-7901细胞生长的影响。方法：应用细胞培养和SRB实验探讨了亚硒酸钠对SGC-7901细胞生长曲线的影响：应用集落形成试验、分裂指数试验研究了亚硒酸钠对SGC-7901细胞集落形成和分裂指数的影响；应用流式细胞仪检测了亚硒酸钠对SGC-7901细胞生长周期的影响；应用电镜、TUNEL染色及流式细胞仪观察了亚硒酸钠诱导SGC-7901细胞凋亡的作用。结果：亚硒酸钠溶液对SGC-7901细胞的生长曲线、集落形成、分裂指数有明显的抑制作用，其抑制作用与其作用浓度和作用时间呈正相关。流式细胞仪检测显示亚硒酸钠作用SGC-7901细胞24h后，细胞周期发生改变，G1期细胞百分率减少，S期细胞百分率增加，与对照组相比有显著性差异（p〈0．05）：电镜下，细胞核固缩，染色质凝集呈新月形紧贴于核膜周边，核膜扭曲；DNA直方图上出现典型的亚二倍体的“凋亡峰”；TUNEL染色法检测细胞凋亡指数在10．4％～33．4％。结论：亚硒酸钠溶液对SGC-7901细胞的生长具有抑制作用，其抑制作用程度与其作用浓度和时间呈正相关：亚硒酸钠通过阻滞细胞S期抑制细胞增殖及诱导细胞凋亡可能是其抑制SGC-7901细胞生长的机理之一。     

菱灵颗粒有效成分的抗肿瘤作用研究

目的:研究菱灵颗粒有效成分-化合物Ⅰ在体外对Hela细胞的作用及其作用机制。方法:采用四甲基偶氮唑盐(MTT)还原法检测化合物Ⅰ对肿瘤生长抑制作用,应用光学显微镜、透射电子显微镜观察细胞形态,流式细胞仪检测细胞周期及细胞凋亡情况。结果:化合物Ⅰ对人宫颈癌细胞生长具有明显的抑制作用及诱导细胞凋亡作用,且这种作用有剂量依赖关系,化合物Ⅰ25、12.5、6.25mg/L剂量组30h抑瘤率为52.04%、34.44%、23.72%,100、50、25、12.5、6.25、3.125mg/L剂量组30h抑瘤率显著正相关,相关系数r=0.9860(p<0.01),IC50值为10.9mg/L。透射电子显微镜对化合物Ⅰ25、12.5、6.25mg/L剂量组受试细胞观察,均出现不同程度细胞凋亡现象,细胞核明显皱缩、染色质趋边凝聚、线粒体空化等特征。流式细胞仪检测发现凋亡峰。结论:化合物Ⅰ对Hela细胞增殖抑制作用明显并且有诱导细胞凋亡作用。     

参数化单胞计算三维编织预制件纤维体积含量

在Pro/E中建立了三维编织预制件的内部单胞、表面单胞和棱角单胞的3D实体模型,并对模型进行了参数化设计。当编织工艺参数改变时能自动生成新的单胞模型,单胞纤维体积由Pro/E的模型分析功能直接输出。提出了基于参数化单胞计算预制件纤维体积含量的公式。计算了长方体形预制件的纤维体积含量,并与实测值进行比较,理论计算与实验结果吻合良好。计算了扇环形预制件的纤维体积含量,为复杂外形预制件纤维体积含量的计算提供了新方法。     

Selenomethionine increases proliferation and reduces apoptosis in bovine mammary epithelial cells under oxidative stress

The decline in mammary epithelial cell number as lactation progresses may be due, in part, to oxidative stress. Selenium is an integral component of several antioxidant enzymes. The present study was conducted to examine the effect of oxidative stress and selenomethionine (SeMet) on morphology, viability, apoptosis, and proliferation of bovine mammary epithelial cells (BMEC) in primary culture. Cells were isolated from mammary glands of lactating dairy cows and grown for 3 d in a low-serum gel system containing lactogenic hormones and 0 or 100 μM H₂O₂ with 0, 10, 20, or 50 nM SeMet. Hydrogen peroxide stress increased intracellular H₂O₂ to 3 times control concentrations and induced a loss of cuboidal morphology, cell-cell contact, and viability of BMEC by 25%. Apoptotic cell number more than doubled during oxidative stress, but proliferating cell number was not affected. Supplementation with SeMet increased glutathione peroxidase activity 2-fold and restored intracellular H₂O₂ to control levels with a concomitant return of morphology and viability to normal. Apoptotic BMEC number was decreased 76% below control levels by SeMet and proliferating cell number was increased 4.2-fold. These findings suggest that SeMet modulated apoptosis and proliferation independently of a selenoprotein-mediated reduction of H₂O₂. In conclusion, SeMet supplementation protects BMEC from H₂O₂-induced apoptosis and increased proliferation and cell viability under conditions of oxidative stress.     

芹菜素对甲状腺癌BCPAP细胞生长及细胞周期的影响

研究芹菜素对人乳头状甲状腺癌BCPAP细胞生长的抑制作用及对细胞周期的影响。采用MTT法检测不同浓度芹菜素在24h对BCPAP细胞的抑制作用,以及12.5,25.0,50.0μmol/L芹菜素分别在24,48,72h对BCPAP细胞的抑制作用;通过明场细胞形态学照片分析,对比不同浓度芹菜素对BCPAP细胞形态的影响,评价其对细胞生长的抑制作用。利用流式细胞仪检测BCPAP细胞周期和凋亡。结果表明,不同浓度(12.5~100.0μmol/L)芹菜素对BCPAP细胞的毒性有明显剂量和时间依赖性,其24h的IC50值为40.65μmol/L。芹菜素对BCPAP细胞的形态学变化有显著影响,高浓度芹菜素强烈抑制BCPAP细胞数目的增长。芹菜素可使BCPAP细胞周期的构成发生明显的变化并诱导细胞凋亡。芹菜素对BCPAP细胞有较明显的细胞毒性和生长抑制作用,其抑制机制可能是使BCPAP细胞生长停滞在G2/M期并诱导细胞凋亡,使得细胞生存率下降,从而抑制细胞活性和数目增长。     

Cell surface hydrophobicity and colony morphology of Trichosporon asahii clinical isolates

Trichosporon asahii is a pathogenic basidiomycetous yeast. Individual strains of T. asahii have different colony morphologies. However, it is not clear whether cell surface phenotypes differ among the colony morphologies. Here we characterized the cell surface hydrophobicity and analysed the carbohydrate contents of the cell surface polysaccharides in T. asahii clinical isolates with various colony morphologies. Among the three distinctive colony morphologies obtained from one clinical isolate, the white‐type morphology exhibited higher hydrophobicity. The hydrophobicity of heat‐killed T. asahii cells was greatly reduced after periodate oxidation of the cell surface carbohydrates. Furthermore, the cell wall and extracellular polysaccharide components differed among the morphologies. Our results suggest that T. asahii cell surface hydrophobicity is affected by cell surface carbohydrate composition. Copyright © 2016 John Wiley & Sons, Ltd.     

水凝胶封装细胞研究进展

近年来,活细胞广泛应用于组织工程、细胞治疗等领域。为了保证活细胞在应用过程中的活性与功能完整性,在细胞表面构建保护性外壳的细胞封装策略应运而生。细胞封装可以避免免疫细胞、抗体、酶等物质与细胞的直接接触,同时保证氧气、营养物质、代谢物等一些小尺寸物质的自由交换。水凝胶因其与细胞基质相似且可形成3D结构模拟细胞微环境而被广泛用于细胞封装。本文首先对细胞封装进行了简单介绍,随后在多细胞封装中重点介绍了木质纤维素基水凝胶在细胞封装中的应用;在单细胞封装中从层层（LbL）自组装、接枝到表面（grafting to）和从表面接枝（grafting from）的3种方法,分别阐述了水凝胶壳层对细胞活性与功能特性的影响。最后讨论了水凝胶封装细胞未来发展方向与前景,希望能为医药食品等领域活细胞的储存及应用提供参考。     

Saccharomyces cerevisiae YCRO17c/CWH43 encodes a putative sensor/transporter protein upstream of the BCK2 branch of the PKC1-dependent cell wall integrity pathway.

The Saccharomyces cerevisiae cwh43-2 mutant, originally isolated for its Calcofluor white hypersensitivity, displays several cell wall defects similar to mutants in the PKC1-MPK1 pathway, including a growth defect and increased release of beta-1,6-glucan and beta-glucosylated proteins into the growth medium at increased temperatures. The cloning of CWH43 showed that it corresponds to YCR017c and encodes a protein with 14-16 transmembrane segments containing several putative phosphorylation and glycosylation sites. The N-terminal part of the amino acid sequence of Cwh43p shows 40% similarity with the mammalian FRAG1, a membrane protein that activates the fibroblast growth factor receptor of rat osteosarcoma (FGFR2-ROS) and with protein sequences of four uncharacterized ORFs from Caenorhabditis elegans and one from Drosophila melanogaster. The C-terminus of Cwh43p shows low similarities with a xylose permease of Bacillus megaterium and with putative sugar transporter from D. melanogaster, and has 52% similarity with a protein sequence from a Schizosaccharomyces pombe cDNA. A Cwh43-GFP fusion protein suggested a plasma membrane localization, although localization to the internal structure of the cells could not be excluded, and it concentrates to the bud tip of small budded cells and to the neck of dividing cells. Deletion of CWH43 resulted in cell wall defects less pronounced than those of the cwh43-2 mutant. This allele-specific phenotype appears to be due to a G-R substitution at position 57 in a highly conserved region of the protein. Genetic analysis places CWH43 upstream of the BCK2 branch of the PKC1 signalling pathway, since cwh43 mutations were synthetic lethal with pkc1 deletion, whereas the cwh43 defects could be rescued by overexpression of BCK2 and not by high-copy-number expression of genes encoding downstream proteins of the PKC1 pathway However, unlike BCK2, whose disruption in a cln3 mutant resulted in growth arrest in G(1), no growth defect was observed in a double cwh43 cln3 mutants. Taken together, it is proposed that CWH43 encodes a protein with putative sensor and transporter domains acting in parallel to the main PKC1-dependent cell wall integrity pathway, and that this gene has evolved into two distinct genes in higher eukaryotes.