以胰岛素样生长因子受体1基因为靶的反义寡核苷酸体内外抗肿瘤研究

以胰岛素样生长因子受体－1基因为靶筛选抗肿瘤药物.根据IGF1RmRNA的二级结构设计了9条反义寡核苷酸药物,以脂质体介导进行转染,MTT染色计算细胞生长抑制率,从中筛选出一条序列并对之进行优化,最后以最佳序列进行体外作用持续时间及体内细胞生长抑制率分析.结果表明该序列在体内外具有良好的抗肿瘤活性,具有剂量依赖性关系,且对荷瘤裸鼠无明显的毒性.IGF1R可作为肿瘤治疗的新靶点.     

In vitro expression and analysis of the 826 human G protein-coupled receptors

G protein-coupled receptors (GPCRs) are involved in all humanphysiological systems where they are responsible for transducing extracellular signals into cells. GPCRs signal in response to a diverse array of stimuli including light, hormones, and lipids, where these signals affect downstream cascades to impact both health and disease states. Yet, despite their importance as therapeutic targets, detailed molecular structures of only 30 GPCRs have been determined to date. A key challenge to their structure determination is adequate protein expression. Here we report the quantification of protein expression in an insect cell expression system for all 826humanGPCRs using two different fusion constructs. Expression characteristics are analyzed in aggregate and among each of the five distinct subfamilies. These data can be used to identify trends related to GPCR expression between different fusion constructs and between different GPCR families, and to prioritize lead candidates for future structure determination feasibility.     

In vitro effects of levamisole and ivermectin against Echinococcus granulosus protoscoleces

The in vitro effects of levamisole and ivermectin against Echinococcus granulosus protoscoleces were studied by means of light and electron microscopy. Both drugs had a protoscolicidal activity that increased proportionally with increasing concentrations of the drugs. Ivermectin showed the more rapid effects and caused contraction and paralysis of protoscoleces. A paralyzing effect was also observed with levamisole, followed by irreversible tissue vacuolation leading to death.     

In vitro effects of fluor-hydroxyapatite, fluorapatite and hydroxyapatite on colony formation, DNA damage and mutagenicity

The number of biomaterials used in biomedical applications has rapidly increased in the past two decades. Fluorapatite (FA) is one of the inorganic constituents of bone or teeth used for hard-tissue repairs and replacements. Fluor-hydroxyapatite (FHA) is a new synthetically prepared composite that in its structure contains the same molecular concentration of OH⁻ groups and F⁻ ions. The aim of this experimental investigation was to evaluate cytotoxic, genotoxic and mutagenic effects of FHA and FA eluates on Chinese hamster V79 cells and to compare them with the effects of hydroxyapatite (HA) eluate. Cytotoxicity of the biomaterials tested was evaluated by use of the cell colony-formation assay and by direct counting of the cells in each colony. Genotoxicity was assessed by single-cell gel electrophoresis (comet assay) and mutagenicity was evaluated by the Hprt gene-mutation assay and in bacterial mutagenicity tests using Salmonella typhimurium TA100. The results show that the highest test concentrations of the biomaterials (100% and 75% eluates) induced very weak inhibition of colony growth (about 10%). On the other hand, the reduction of cell number per colony induced by these concentrations was in the range from 43% to 31%. The comet assay showed that biomaterials induced DNA breaks, which increased with increasing test concentrations in the order HA < FHA < FA. None of the biomaterials induced mutagenic effects compared with the positive control (N-methyl-N′-nitro-N-nitrosoguanidine), and DNA breakage was probably the reason for the inhibition of cell division in V79 cell colonies.     

In vitro cultivation of Diplostomum spathaceum and Diplostomum phoxini metacercariae

Using a new egg macerate medium Diplostomum spathaceum metacercariae were cultured to the stage of egg production for the first time, and an improved vitelline and growth response was obtained in Diplostomum phoxini. The importance of physical factors in the cultivation of strigeoid trematodes was demonstrated. Methods involving a continuous circulating system, diphasic media, a double culture tube technique or the chorio-allantoic membrane proved to be of no value in the cultivation of Diplostomum spp. The addition of selected hormones with known growth promoting properties did not have an appreciable effect on D. phoxini cultures. Supplementation of egg macerate cultures with various nutrients had only slight or no beneficial effect. The ability to use stored metacercariae for cultures was demonstrated.     

甜叶菊同源四倍体离体诱导及鉴定

用不同浓度秋水仙素溶液处理甜叶菊不定芽,诱导同源四倍体,并进行解剖学、染色体鉴定和流式细胞仪鉴定倍性。结果表明:(1)用0.20%的秋水仙素溶液浸泡甜叶菊不定芽12h,同源四倍体诱导率最高,可达32.14%。(2)同源四倍体植株与二倍体(对照)相比,其气孔、叶片等均表现巨大性,且叶片变厚、叶色浓绿、叶片皱缩。(3)对照植株染色体2n=2x=22,四倍体植株染色体2n=4x=44;流式细胞仪倍性鉴定结果显示,对照DNA相对含量为100,四倍体DNA相对含量为200。(4)该研究共鉴定出48株甜叶菊同源四倍体植株,为进行倍性植株的诱导奠定了技术基础,为进一步开展甜叶菊同源四倍体新品种的选育提供了实验材料。     

猕猴脑胱天蛋白酶-3活化及其靶蛋白的体外研究(英)

凋亡的主要生化过程包括胱天蛋白酶的活化及其对细胞内蛋白质的选择性切割.在已知的胱天蛋白酶中,可被多种凋亡刺激信号激活的胱天蛋白酶-3备受注目.为进一步揭示灵长类动物神经组织中未知的胱天蛋白酶-3靶蛋白,采用成年猕猴脑组织粗提物作为无细胞体系,通过加入granzyme B引发凋亡途径的部分反应,如胱天蛋白酶-3的活化及随后发生的蛋白质水解.经蛋白质印迹分析发现,与granzyme B共孵育后,猕猴脑胱天蛋白酶-3以两步方式从酶原转化为活性酶.对猕猴脑组织自身蛋白质的进一步分析显示,多聚ADP-核糖聚合酶（PARP）被水解为长85 ku的片段,此片段提示胱天蛋白酶-3的特异切割活性.此外,神经元凋亡抑制蛋白（NAIP）也被切割,产生长约40 ku的小片段,但是它的出现不被胱天蛋白酶-3特异性抑制剂Ac-DEVD-CHO阻断,因此可能是granzyme B直接作用于NAIP所致.以上结果提示,凋亡相关酶切反应可在成年猕猴脑组织提取物中得到重现;NAIP可能是granzyme B而非胱天蛋白酶-3的作用靶点.     

(S)-雌马酚对肠道菌群的体外调控作用及其可代谢性研究

[目的]研究(S)-雌马酚对人体肠道菌群的体外调控作用和人体肠道菌群对(S)-雌马酚的代谢衍生作用。[方法]采用人体肠道菌群体外批量发酵、细菌16S rRNA基因高通量测序、气相色谱、液相色谱和质谱等检测(S)-雌马酚与人体肠道菌群体外相互作用。[结果]体外添加(S)-雌马酚对总体人肠道菌群结构和短链脂肪酸产量影响不明显。添加0.45 mmol/L (S)-雌马酚组与对照组相比,未检测到相对丰度发生显著变化的细菌;添加0.90 mmol/L (S)-雌马酚组与对照组相比,显著增加了肠杆菌科(Enterobacteriaceae)等条件致病菌的相对丰度,减少了潜在益生菌粪球菌属(Coprococcus)的比例。代谢分析发现,发酵培养液中(S)-雌马酚的浓度降低了约15%−30%,推测可能被微生物进一步降解或衍生修饰。[结论]从体外调控肠道菌群的角度判断,0.45 mmol/L (S)-雌马酚相对较安全,而0.90 mmol/L (S)-雌马酚可能会破坏肠道菌群平衡。(S)-雌马酚可以被人体肠道菌群进一步代谢,其特定代谢产物的结构与功能及其体内生物安全性有待进一步研究。     

嗜肺军团菌mip/ctxB融合基因体外表达与动物免疫试验的免疫原性研究

ＰＣＲ扩增嗜肺军团菌mip基因和霍乱弧菌ctxB基因,克隆入载体pcDNA3.1(+),重组子经限制性酶切分析、PCR、序列分析鉴定正确后,命名为pcDNA3.1-mip/ctxB.脂质体法将重组质粒pcDNA3.1-mip、pcDNA3.1-mip/ctxB转染NIH3T3细胞,用免疫荧光法和蛋白质印迹鉴定瞬时表达和稳定表达产物,结果发现：重组质粒成功转入细胞并获得短暂表达,稳定转染细胞分别在24 ku和35 ku处检测到阳性杂交信号.将pcDNA3.1-mip、pcDNA3.1-mip/ctxB作为DNA疫苗免疫BALB/c小鼠,检测免疫小鼠体内抗原特异性抗体水平、脾淋巴细胞增殖活性、IFN-γ产生水平、细胞毒性T淋巴细胞(CTL)杀伤活性等体液免疫和细胞免疫反应的指标,评价疫苗的免疫原性.结果发现：各实验组均检测到免疫原性,pcDNA3.1-mip/ctxB免疫组的免疫原性高于pcDNA3.1-mip免疫组,有显著性差异(P＜0.01).研究结果为mip/ctxB融合基因DNA疫苗的研制提供了初步的实验依据.     

催吐萝芙木离体培养和植株再生体系的建立

为建立催吐萝芙木(Rauvolfia vomitoria Afzel.)的快繁再生体系,以茎段为外植体,比较了植物生长调节剂对其愈伤组织诱导、分化及生根的影响。结果表明,诱导愈伤组织的适宜培养基为MS+2,4-D 1.0 mg L~(–1)+TDZ 0.5 mg L~(–1)或MS+2,4-D2.0 mg L~(–1)+TDZ 0.5 mg L~(–1),出愈率达100%且生长状况良好;诱导丛生芽的最佳培养基为MS+6-BA 3.0 mg L~(–1)+NAA 0.1 mg L~(–1),出芽率为46.6%,平均出芽数为3.04。这为催吐萝芙木的快速繁殖和遗传转化研究奠定了基础。     

Caveolin-1抑制胰腺癌细胞PANC1的体内外生长和增殖

窖蛋白-1(caveolin-1)是胞膜窖(caveolae)中重要的结构和功能蛋白.Caveolin-1参与细胞的多种生命活动并与恶性肿瘤的发生相关.为探讨caveolin-1对胰腺癌细胞PANC1的体外增殖、迁移、侵袭以及裸鼠体内成瘤能力的影响,通过基因转染技术培育caveolin-1过表达细胞株PANC1/cav-1作为实验组,转染空载体细胞株PANC1/vector作为对照组,采用RT-PCR及Western blot方法检测caveolin-1的表达量,流式细胞术分析细胞周期,软琼脂细胞克隆实验检测细胞增殖能力,侵袭小室实验检测癌细胞迁移和侵袭的能力,建立裸鼠皮下种植瘤模型并检测肿瘤组织的增殖与凋亡.PANC1/cav-1中的caveolin-1表达稳定,表达量明显高于对照组细胞株和亲本细胞株(P<0.01),细胞周期检测显示大量PANC1/cav-1细胞被抑制于G0/G1期,caveolin-1抑制PANC1的增殖,迁移和侵袭能力.在裸鼠的体内实验中,caveolin-1显著抑制PANC1细胞在裸鼠体内的生长,Ki-67染色和TUNEL染色表明在PANC1细胞中过表达caveolin-1,可以抑制肿瘤增殖并诱导肿瘤凋亡.上述结果表明,caveolin-1可能通过对胰腺癌细胞周期的影响(抑制于G0/G1期),抑制胰腺癌PANC1细胞在体内外的增殖、迁移和侵袭,并导致肿瘤凋亡.     

维拉帕米对铜绿假单胞菌群体感应抑制作用的体外研究

考察维拉帕米对铜绿假单胞菌（Pseudomonas aeruginosa, PA）群体感应（Quorum sensing, QS）的抑制作用。测定维拉帕米最小抑菌浓度(Minimal inhibit concentration,MIC) ;构建培养环境,考察维拉帕米对PA生长的影响,绘制生长曲线;毒力因子表达实验中,分别考察维拉帕米对PA弹性蛋白酶表达、蛋白水解酶表达和绿脓毒素表达的影响。结果表明,维拉帕米MIC₅₀为128 μg/mL,MIC₉₀为512 μg/mL,最低抑菌质量浓度范围为128~512 μg/mL时,具有较好抑菌活性;生长曲线表明,维拉帕米质量浓度为16 μg/mL时,开始抑制PA的生长,随着浓度的增加,抑制作用逐渐增大;当维拉帕米质量浓度为512、256、128、64、32、16 μg/mL时,明显抑制弹性蛋白酶表达（P<0.01）,质量浓度为8 μg/mL时,对弹性蛋白酶有一定抑制作用（P<0.05）,维拉帕米质量浓度为512、256、128、64、32 μg/mL时,明显抑制蛋白水解酶的活性和绿脓毒素的表达（P<0.01）,当质量浓度为16 μg/mL时,对蛋白水解酶活性和绿脓毒素的表达有一定抑制作用（P<0.05）;维拉帕米对QS的抑制有浓度依赖。维拉帕米对PA的QS有明显抑制作用,体外可明显抑制PA生长,可作为抗菌增效的潜在开发药物。     

连香树水提物和乙醇提取物的体外抗氧化研究

为了探究连香树水提物和乙醇提取物的主要成分和抗氧化作用,该研究采用水提和醇提两种方法提取连香树叶片中的代谢物并测定其主要成分,通过体外抗氧化实验,即清除羟自由基(·OH)、DPPH自由基(DPPH·)、超氧阴离子(O_2~-·)和还原铁离子(Fe~(3+))的能力等四个指标来评价其抗氧化作用。结果表明:连香树水提物和乙醇提取物中均含有山萘酚。此外,水提物中还含有苜蓿素和异槲皮苷等黄酮类物质;乙醇提取物中还含有柚皮素和槲皮素3-O-β-D-葡萄糖苷等黄酮类物质。水提物和乙醇提取物均有清除羟自由基、DPPH自由基、超氧阴离子及还原三价铁离子的能力。抗氧化的作用随提取物浓度的增大而增强,其中清除超氧阴离子(IC50值分别为0.092、0.002 mg·mL~(-1))的能力强于阳性对照Vc(IC_(50)值为0.241 mg·mL~(-1))且铁离子还原力的IC_(50)值(水提物为0.014 mg·mL~(-1),乙醇提取物为0.001 mg·mL~(-1))相对较小,说明其总抗氧化活性较强。由此可见,连香树水提物和乙醇提取物均具有良好的抗氧化作用,可作为一种潜在的天然抗氧化剂。     

In vitro organogenesis and plant regeneration from unpollinated ovary cultures of Azadirachta indica

A novel method of organogenesis in neem (Azadirachta indica A. Juss.) from unfertilized ovaries is described. The Murashige and Skoog’s (MS) medium with 9 % sucrose, 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5 μM 6-benzylaminopurine (BAP) was the best for callus induction from unfertilized ovaries. However, further proliferation of callus occurred better on MS medium supplemented with 0.5 μM 2,4-D either alone or in combination with 4.5 μM kinetin. Maximum shoot regeneration (78 %) was observed when calli, induced from ovaries of 4 mm size flower buds and proliferating on MS + 0.5 μM 2,4-D, were subcultured to MS medium containing 5 μM BAP. Histological analysis revealed that 4 mm sized flower bud corresponds to a 2-nucleate stage of embryo sac. The shoots were then multiplied by forced axillary branching on MS medium supplemented with 1.0 μM BAP and 250 mg dm⁻³ casein hydrolysate. The shoots could be rooted on 1/4 strength MS medium supplemented with 0.5 μM indole-3-butyric acid (IBA) at a frequency of 79 %. Cytological analysis by root tip squash preparations revealed that all the plantlets were diploids. These plants were subsequently hardened and established in soil with transplantation rate of 81.8 %.     

在大肠杆菌中表达有活性的人STK11蛋白

STK11蛋白(serine/threonine kinase11)是近年来发现的具有多种重要功能的蛋白,可参与调控细胞周期、p53介导的细胞凋亡、ras诱导的细胞转化、细胞极化等多种生物学过程。利用大肠杆菌高效表达有活性的人STK11蛋白,可为其结构和功能的深入研究打下良好基础。利用本室克隆的人STK11 cDNA和原核表达载体pET-44a( )构建带有Nus融合标签的诱导型表达载体pET-Nus-STK11,在不同的大肠杆菌宿主中诱导表达。SDS-PAGE和Western blot检测表明,在BL21(DE3)宿主中表达的融合蛋白主要以包涵体形式存在,占菌体总蛋白的8.9%;在Rosetta-gami(DE3)pLysS宿主中主要表达为可溶性蛋白,占菌体总蛋白的16.7%。而经纯化和包涵体蛋白复性处理后,以Chariot介导重组融合蛋白进入人肝癌细胞SMMC-7721检测其对细胞生长和细胞周期的影响。与对照组相比,BL21(DE3)中表达的Nus-STK11蛋白几乎无抑制活性;而Rosetta-gami(DE3)pLysS中表达的Nus-STK11蛋白可以显著抑制SMMC-7721细胞的生长,抑制率达47.05%,并导致细胞周期的G0/G1期阻滞,证实表达的重组融合蛋白具有明显的生物学活性。上述结果为在大肠杆菌中成功表达有活性的重组STK11蛋白的首次报道。     

体外法探究猪空肠和回肠黏膜微生物对低聚半乳糖和低聚甘露糖的发酵特性

【背景】小肠黏膜微生物是肠道菌群的重要组成部分,大量研究表明日粮添加低聚半乳糖（galacto-oligosaccharides,GOS）和低聚甘露糖（manno-oligosaccharides,MOS）能够调控猪的大肠菌群结构,但关于其调控小肠黏膜微生物的研究较少。【目的】通过体外发酵法探究猪空肠黏膜和回肠黏膜微生物发酵GOS和MOS的规律。【方法】以生长猪的空肠黏膜微生物和回肠黏膜微生物作为接种物,以GOS和MOS作为底物进行厌氧发酵,在发酵0、6、12、24 h时采样测定总菌数量、pH、氨态氮（ammonia nitrogen,NH₃-N）、菌体蛋白（microbial crude protein,MCP）和有机酸,在24 h收集微生物提取DNA进行细菌定量分析。【结果】在24 h时,回肠黏膜组的NH₃-N浓度显著低于空肠黏膜组,而MCP浓度显著高于空肠黏膜组（P<0.05）。在发酵的前6 h各组pH无明显变化,有机酸积累较少。在12 h时,MOS组的乳酸、乙酸、丁酸和总短链脂肪酸产量显著高于GOS组（P<0.05）,此时只有回肠黏膜组有少量丙酸产生。在24 h时,MOS回肠黏膜组乳酸产量最高而pH值最低（P<0.05）。相较于MOS组,GOS组显著提高了丙酸的产量（P<0.05）。相较于GOS组,MOS组显著提高了乙酸的产量,在空肠黏膜组中显著提高了丁酸和总短链脂肪酸的产量（P<0.05）。定量结果表明,在24 h时,各处理组的厚壁菌门数量都接近总菌数量,属于优势菌门。相较于MOS组,GOS组显著提高了拟杆菌门、链球菌属、韦荣氏球菌属和普拉梭菌细菌的数量,提高了空肠黏膜组中Clostridium cluster IV和回肠黏膜组中Clostridium cluster XIVa的数量（P<0.05）。相较于GOS组,MOS组显著提高了大肠杆菌和乳酸杆菌属的数量,提高了回肠黏膜组中罗氏菌属的数量（P<0.05）。【结论】猪小肠黏膜微生物对GOS和MOS具有不同的发酵模式,主要表现在有机酸的产生和促进细菌的增殖方面。GOS具有产丙酸优势,提高了拟杆菌门和韦荣氏球菌属的数量;MOS促进了乙酸的产生,提高了大肠杆菌和乳酸杆菌的数量。     

In vitro spore formation and completion of the asexual life cycle of Neozygites parvispora, an obligate biotrophic pathogen of thrips

Capilliconidia, the asexual secondary spores of Neozygites parvispora (Zygomycetes, Entomophthorales) were produced in vitro either by entrapment of vegetative cells (hyphal bodies) in alginate pellets or after plating them onto water agar. Cultivation of the fungus for 3 days in a medium lacking hemolymph increased spore production 30 to 40-fold, and about 10% of the cells produced capilliconidia. The in vitro produced capilliconidia were infectious to Thrips tabaci and the fungus was reisolated from infected insects, thus completing its asexual life cycle under laboratory conditions. A decrease in capilliconidia production and a modification of the number of nuclei per spore were observed for isolates cultivated in vitro for more than 2 months, but subsequent host passages restored and increased sporulation efficiency without influencing the number of nuclei. Fungal cultures were stored at —80 °C for up to 7 months, and the capability to sporulate and infect T. tabaci was preserved. A bioassay procedure for infecting T. tabaci with N. parvispora is described, the first mycosed insects dying usually after 8 d of incubation.     

甜椒花药培养及再生植株技术体系的研究

为建立甜椒(Capsicum annuum var.grossum)花药培养及再生植株技术体系,对影响花药胚状体诱导和分化的因素进行了研究。结果表明,培养基组成对胚状体诱导率的影响以NAA基本培养基椰乳KT,最佳胚状体诱导培养基为NTH+0.1 mg L–1 NAA+10%椰乳+1 mg L–1 KT+50μmol L–1 Ag NO3+30 g L–1蔗糖+5 g L–1琼脂+2 g L–1活性炭。花药经过24 h低温预处理和8 d高温预培养后,胚状体诱导率可达23.38%。植物生长调节剂对胚状体出芽率的影响为6-BANAAIAA,最佳胚状体分化培养基为NTH+1 mg L–1 6-BA+0.3 mg L–1 NAA+0.1 mg L–1 IAA+30 g L–1蔗糖+5 g L–1琼脂。胚芽转入1/2MS+0.5 mg L–1 IBA+30 g L–1蔗糖+5 g L–1琼脂培养基后,生根率可达92.5%。