3-MA 对阿霉素诱导 K562、K562 / ADM 细胞凋亡及其相关基因 mRNA 表达的影响

目的：通过自噬抑制剂3-甲基腺嘌呤（3-MA）对阿霉素（ADM）诱导白血病K562细胞及K562／ADM细胞的细胞效应和自噬基因Beclinl、凋亡抑制基因SurvivinmR．NA表达的变化观察,探讨自噬在细胞凋亡中的作用和机制．方法：体外培养K562和K562／ADM细胞,采用MTT法分别检测ADM及3-MA预处理对K562、K562／ADM细胞增殖的影响,流式细胞仪检测细胞凋亡率,实时RT—PCR法检测细胞自噬及凋亡相关基因（Beclinl、Survivin）mRNA表达的变化．结果：ADM可抑制K562与K562／ADM细胞增殖,且抑制作用呈现浓度与时间依赖性．ADM诱导组K562与K562／ADM细胞在24h、48h、72h细胞凋亡率均较空白对照组明显提高（P〈0．05）．在ADM诱导前,经3-MA预处理可使ADM诱导的K562与K562／ADM细胞抑制率和细胞凋亡率均较单用ADM显著提高（尸〈0．05）,Bedin1、SurvivinmRNA相对表达量均较单用ADM明显下降（P〈0．05）,呈正相关（r=0．827,P〈0．01）．结论：ADM可抑制K562、K562／ADM细胞的生长,并诱导细胞凋亡．3-MA通过抑制细胞的自噬可增强ADM诱导白血病细胞K562、K562／ADM的凋亡,其机制可能与下调BeclinlmRNA表达,而使Survivin表达受抑制有关．     

羟基喜树碱联合丝裂霉素诱导人白血病K562细胞凋亡

观察羟基喜树碱与丝裂霉索联合应用对人白血病细胞K562的作用效果,并探讨其机制。不同浓度羟基喜树碱与丝裂霉素单独及联合作用于人白血病细胞K562后,应用台盼蓝拒染法检测细胞生长抑制率,计算合用指数（CI）,流式细胞仪（FCM）检测K562细胞凋亡率,吖啶橙（AO）荧光染色和透射电镜观察凋亡形态学变化。结果表明：单独应用时羟基喜树碱和丝裂霉素的IC50分别是8μ/mL和12．5μg/mL,联合应用时IC50下降为4μg/mL（HCPT）和3．6μg/mL（MMC）,CI=0．78,为协同效应。羟基喜树碱与丝裂霉素单独及联合应用均可诱导K562细胞凋亡。2种药物联合应用时的凋亡率高于各自单独用药。羟基喜树碱与丝裂霉素联合应用可以通过共同诱导细胞凋亡。协同抑制人白血病细胞生长。     

灵芪胶囊含药血清对K562白血病细胞影响

观察灵芪胶囊含药血清在体外对K562白血病细胞增殖的影响.采用血清药理学方法制备灵芪胶囊含药血清,以不同浓度的含药血清处理体外培养的K562白血病细胞,采用MTT比色法观察灵芪胶囊含药血清对K562细胞增殖的影响,采用Wright-Giemsa染色观察肿瘤细胞形态学变化.不同浓度灵芪胶囊含药血清对K562细胞增殖具有抑制作用,并呈剂量依赖关系.当含药血清作用96h后,其抑制作用开始减弱.高剂量LQC含药血清对K562细胞形态学有明显的影响.灵芪胶囊含药血清具有抑制K562白血病细胞增殖作用,其作用强度与时间-浓度呈正相关,其作用机理可能与其直接细胞毒作用有关.     

利用体细胞核移植技术生产黄牛和水牛同种、异种转基因克隆囊胚

以含neo和GFP基因双标记的水牛、黄牛胎儿成纤维细胞进行牛转基因体细胞核移植,结果发现：阿菲迪霉素可有效将胎儿成纤维细胞同步于G0／G1期;以GFP阳性细胞进行核移植,其卵裂率、囊胚率与对照组间无显著差异（P〉0．05）,所构建的重组胚,2-细胞阶段观察不到GFP的表达,4-细胞以后GFP的表达逐渐增强,在囊胚的内细胞团和滋养层细胞均可检测到GFP的表达,将黄牛同种转基因克隆囊胚进行移植,获得1例妊娠;黄牛卵母细胞-水牛供核细胞构建异种重组胚囊胚率显著高于黄牛供核细胞-水牛卵母细胞构建的异种重组胚（P〈0．05）,并且异种核移植重组胚中可检测到GFP表达,GFP可作为异种核移植胚胎来源的一种新标记．     

腺病毒载体介导的GFP在鸡胚成纤维细胞中的表达及RNA干涉

鸡胚成纤维细胞（CEF）是多种动物及人类病毒的易感细胞;也是研究鸡的基因组功能的理想材料．本试验的目的是研究非复制型腺病毒载体对CEF的转染效率及安全性;并建立在CEF细胞中进行RNAi的技术平台．采用GFP重组非复制型腺病毒载体（Ade-GFP）转染CEF细胞：结果证明0．1．2000 MOI Adv-GFP均可转染CEF细胞并表达GFP;转染后16h开始出现GFP阳性细胞：胞核与胞浆可见明亮的荧光：荧光细胞数量及荧光强度在24～36h达到高峰：以后逐渐衰减;约180h全部消失;转染效率最高为22.3％;形态学观察及流式细胞仪测定结果显示：Adv-GFP转染的CEF细胞未见细胞病变;多数转染组细胞活力不受影响：说明用Adv-GFP携带外源转染CEF细胞是安全可行的;用脂质体转染试剂转染化学合成的GFP-siRNA;成功地干涉了腺病毒载体介导的GFP基因在鸡胚成纤维细胞的表达：干涉效率为85％：证明了鸡胚成纤维细胞中存在RNAi机制：为进一步利用非复制型腺病毒载体递送siRNA在CEF细胞内进行RNAi的研究奠定了基础．     

转染野生型p53基因对人白血病细胞系生长的影响

目的研究转染野生型p53基因对人白血病细胞的生长影响作用．方法将克隆有野生型p53基因的pUHD10-3质粒,通过Lipofectin转染人白血病细胞株U937、K562、HL60,用四甲基偶氮唑（Mlvr）法观察细胞的生长情况．结果转染野生型p53基因,可使人白血病细胞U937、K562、HL60的生长明显受到抑制,第4天生长抑制率分别是28．6％、44．9％及49．0％．结论转染野生型p53基因能有效抑制人白血病细胞U937、K562、HL60的生长．     

石蒜碱诱导人白血病K562细胞凋亡的研究

为研究石蒜碱诱导白血病K562细胞凋亡机制,采用体外细胞培养方式,通过CCK-8法描绘细胞生长曲线,测定细胞增殖活性,CCK-8结果显示石蒜碱能够抑制K562细胞生长,且呈现剂量依赖性,其IC50=4.13μmol/L.倒置显微镜进行细胞形态学观察,石蒜碱作用细胞后,细胞形态学发生改变.PI单染法检测细胞凋亡率,数据显示,石蒜碱诱导K562细胞的凋亡率与给药剂量呈正相关.Western-blot法检测P53蛋白表达情况,随着石蒜碱浓度的增加,P53活性增强(P0.05).结果显示,石蒜碱抑制白血病K562细胞增殖,诱导细胞凋亡,其凋亡诱导作用可能与细胞内P53基因的表达有关.     

天蓝苜蓿含药血清抗白血病K562细胞的体外实验研究

探讨天蓝苜蓿含药血清对白血病细胞株K562细胞增殖抑制的影响,为探索中药治疗白血病提供一定理论依据。方法用不同浓度天蓝苜蓿含药血清处理白血病细胞株K562,24、48、72h后,采用MTT法检测细胞抑制率。结果天蓝苜蓿含药血清能有效的抑制K562细胞增殖,呈时间和剂量依赖关系。细胞发生一系列形态学改变。结论天蓝苜蓿含药血清对K562细胞增殖有一定的抑制作用。     

氮芥新衍生物抗白血病细胞活性研究

对合成的氮芥新衍生物(化合物Y-65)进行了白血病细胞的抗增殖活性研究。采用MTT法检测化合物Y-65对3种白血病细胞(慢性粒细胞白血病细胞(K562)、人急性白血病细胞(CCRF-CEM)、人早幼粒急性白血病细胞(HL-60))及人正常肝上皮细胞(L02)的抗增殖效果,并通过测定时间浓度依赖曲线、观察白血病细胞的形态、Hoechst33258染色、Annexin V-FITC和PI染色以及JC-1线粒体染色一系列实验,研究化合物Y-65诱导白血病细胞CCRF-CEM的初步凋亡机制。结果表明：化合物Y-65对3种白血病细胞(K562,CCRF-CEM,HL-60)均有较好抗增殖活性(IC₅₀分别为：25.9±1.7,4.3±0.3,12.4±6.2μmol/L),特别是对K562细胞的抗增殖活性明显高于苯丁酸氮芥(CLB)对K562细胞的抗增殖活性。研究其凋亡机制发现,经过化合物Y-65作用的CCRF-CEM细胞,可能是通过改变线粒体膜电位来诱导白血病细胞发生凋亡,且化合物Y-65诱导细胞凋亡呈现时间和浓度依赖。设计合成的氮芥新衍生物Y-65对白血病细胞有较强的抗...     

用基因芯片研究苦丁茶甙对K562细胞基因表达的影响

以人红白血病K562细胞株为材料，应用基因芯片技术研究苦丁茶甙对人红白血病K562细胞作用前后基因表达的差异，以诱导分化药物羟基脲处理作为正对照，分别提取药物处理前后细胞RNA进行逆转录cDNA，使获得的cDNA分别标记上Cy3和Cy5两种荧光物质；然后与由1632条cDNA片段制作的基因表达谱芯片杂交，经扫描及对获得的数据用相关软件分析，确定K562细胞经苦丁茶甙处理前后差异表达基因48条，有41条与羧基脲处理相同，其中上调表达的基因有32条，下调表达的基因有16条，这将为进一步建立基因芯片技术药物筛选模型奠定基础。     

Green fluorescent protein （GFP） as a vital marker for studying the interaction of Phytophthora sojae and soybean

Transgenic Phytophthora sojae strains that produce green fluorescent protein （GFP） were obtained after stable DNA integration using the Hsp70 promoter and the Ham34 terminator of Bremia lactucae. The expression of GFP during different developmental stages of P. sojae was observed using fluorescent microscopy. Based on this reporter system, the histopathologic events caused by the pathogen in soybean leaves, hypocotyls and roots were monitored. Meanwhile, the difference in resistance between different soybean cultivars against P. sojae was analyzed microscopically in roots. The results indicate that GFP can be stably expressed in zoosporangia, zoospores, cysts, hyphae and oospores of P. sojae. Using the GFP marker, the infecting pathogens in leaves, hypocotyls and roots of host could be distinctly visualized. The germ tube length of cysts germinating on the roots of resistant cultivar Nannong 8848 was longer than that on the roots of susceptible cultivar Hefeng 35. These results show for the first time that this eukaryotic reporter can be used in P. sojae as a stable and vital marker, allowing the study of genetics of this hemibiotrophic pathogen.     

改进的鸡胚绒毛尿囊膜技术--无气室孵育法

对传统的鸡胚绒毛尿囊膜(CAM)方法进行改进。将孵育6d的鸡胚去掉气室后，将待测样品加入到CAM上，用无菌透明胶封口，继续孵育2—3d，去掉封口暴露CAM，用甲醇／丙酮等体积混合液室温固定10min后进行观察，结果鸡胚血管网完好无损，样品作用效果显示明显，表明该方法相对于传统方法而言，具有操作简单，鸡胚存活率高，样品效果显示更直观等优点。     

27种中药提取物抑制血管生成活性的评价

对27种中药分别采用石油醚、乙酸乙酯、水进行系统提取,利用转基因斑马鱼对提取物进行血管生成抑制活性检测。提取物处理斑马鱼胚胎24 h后,统计斑马鱼节间血管(ISV)的生长情况,并计算抑制率;部分有活性的提取物进一步采用鸡胚尿囊膜（CAM）实验进行验证。实验结果显示,川芎水提物、白芍乙酸乙酯提取物、竹黄水提物、鬼箭羽乙酸乙酯提取物和水提物、乳香水提取物、大青叶乙酸乙酯提取物显示出抑制血管活性现象;其中川芎水提取物、鬼箭羽乙酸乙酯和水提物、乳香水提取物对鸡胚尿囊膜血管生成具有明显的抑制作用。从27种中药中发现了7种提取物具有抑制转基因斑马鱼节间血管生长的作用,这种作用与血管内建生长因子受体2(VEGFR2)表达有关。     

Expression of green fluorescent protein gene with baculovirus vector in insect cells

The green fluorescence of bioluminescent jellyfishAequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10³ dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus-insect cell expression system. Supported by the National Natural Science Foundation of China Hu Jianhong: born in July, 1972, Master graduate student     

Foreign gene transfer into Chinese shrimps (Penaeus chinensis) with gene gun

Plasmids pG DNA-RZ1 with a GFP (green fluorescent protein) reporter gene and a ribozyme gene incising penaeid white spot baculovirus (WSBV) were first introduced into the fertilized eggs of Chinese shrimps by gene gun. The treated and control samples of different development stages were observed with a fluorescent microscope. The transient expression of GFP gene was high in nauplius and zoea larvae. Results from RT-PCR and PCR for adults showed that the foreign genes had been transferred into the shrimps and had expressed the corresponding proteins. This work has established a transgenic method for penaeid shrimps, which will set base for the application of genetic engineering breeding into industry.     

Knockdown of <Emphasis Type="Italic">MRP4</Emphasis> by lentivirus-mediated siRNA improves sensitivity to adriamycin in adriamycin-resistant acute myeloid leukemia cells

Chemotherapy remains the standard treatment for acute myeloid leukemia;however,the emergence of drug resistance is a major hurdle in the successful treatment of leukemia.The expression of multidrug resistance-associated protein 4(MRP4)induces re- sistance in the adriamycin-resistant acute myeloid leukemia cell line,K562/ADR.The aim of this study was to investigate whether knockdown of MRP4 by lentivirus-mediated siRNA could improve the sensitivity of K562/ADR cells to adriamycin.Five lenti- virus-mediated short hairpin RNAs(lv-shRNAs-MRP4)were designed to trigger the gene silencing RNA interference(RNAi) pathway.The efficiency of lentivirus-mediated siRNA infection into K562/ADR cells was determined using fluorescence mi- croscopy to observe lentivirus-mediated GFP expression.MRP4 expression in infected K562/ADR cells was evaluated by real- time PCR and Western blot analysis.The MTS assay was used to measure cell viability and flow cytometry was used to measure apoptosis.The transfection efficiency of K562/ADR cells was over 80 percent.The gene silencing efficacy of lv-shRNA1-MRP4 was superior to the other constructs.Infection of K562/ADR cells with lv-shRNA1-MRP4 led to strong inhibition of MRP4 mRNA and protein expression.Combined treatment with lv-shRNA1-MRP4 and adriamycin decreased cell growth and increased apoptosis compared to treatment with lv-shRNA1-MRP4 or adriamycin alone.These data indicate that in K562/ADR cells MRP4 is involved in drug resistance mechanisms and that lentivirus-mediated knockdown of MRP4 may enhance sensitivity to adriamycin.     

鸟类胚胎消化道原基细胞分化的研究

本文用不同发育时期鹌鹑胚与鸡胚的种间移植实验结果表明,相当于鸡胚H.＆ H.17—23期的鹌鹑胚后肠原基尚不具备自主分化的潜能,只能并入寄主大肠发育,但不同区域的消化道中胚层间质细胞对内胚层细胞具有不同的诱导能力.相当于鸡胚H.＆ H.28—23期的鹌鹑胚后肠原基已具有自主分化的潜能,在寄主鸡胚体腔内能定向发育成鹌鹑大肠.本文还对移植系统中消化道原基细胞的分化进行了分析.     

PTD-Tat之C端融合在活体体内的跨膜递送作用

报道了Tat蛋白转导区域(PTD-Tat)的C端融合蛋白在线虫和小鼠体内的跨膜递送作用.将重组表达质粒pGEX-GFP-Tat在大肠杆菌中高效表达出的融合蛋白GST-GFP-Tat对小鼠腹腔注射(ip)17h后,荧光显微镜观察其心、肝和肾组织,均检测到强烈的绿色荧光,甚至该蛋白跨越了血脑屏障(BBB).此外用融合蛋白喂食线虫发现蛋白分布于线虫消化管道及其原体腔,且表现出的跨膜递送活性与喂食时间和蛋白浓度呈现正相关.该研究结果拓宽了PTD-Tat在蛋白药物递送方面的应用范围,为蛋白质疗法开辟了新的视野,并为其有效应用提供理论指导.     

Production of transgenic mice carrying green fluorescence proteingene by a lentiviral vector-mediated approach

A pseudo-lentivirus, which carries green fluorescence protein (GFP) expressing cassette, was injected into the perivitelline space of murine fertilized oocytes before transplanting into the oviducts of the foster mothers. The GFP transgenic pups were then obtained. By PCR amplification, fluorescent microscopy and flow assisted cytometry sorting analysis, we found that the integration rate of the transgene was estimated at above 40%. Real-time PCR analysis indicated that the copy number of the integrated GFP cassette was around 40. Fluorescent in situ hybridization analysis demonstrated that the integration pattern was random but inheritable. The transgenic mice with multi-integration sites and various expression levels possessed a great value in practice as well as research. The approach reported herein provides an efficient way to generate and screen the transgenic mouse strains.     

利用有机溶剂法快速纯化绿色荧光蛋白

介绍非色谱GFP纯化方法——有机溶剂纯化法．该方法在短时间内（30min）即可获得大量较高纯度（91．7％）,且荧光光谱特征不改变的GFP蛋白．纯化过程简单,无需昂贵的仪器和设备,适合绝大多数中小实验室使用．