HMGB1在胃癌组织及细胞系中表达的研究

目的:近年来的研究表明,高迁移率族蛋白(1HMGB1)在肿瘤的发生及恶性演变过程中发挥重要作用,本研究旨在探讨HMGB1在胃癌组织、正常组织、胃癌细胞系SGC-7901、BGC-823、HGC-27、AGS及正常胃黏膜细胞系GES中表达情况。方法:免疫组织化学法检测HMGB1在32例可手术切除的胃癌患者组织标本(包括癌组织和正常组织)的表达情况;RT-PCR及Westren Blot检测HMGB1在胃癌细胞系SGC-7901、BGC-823、HGC-27、AGS及正常胃粘膜细胞系GES的m RNA及蛋白质表达。结果:胃癌组织HMGB1免疫组织化学染色评分高于正常组织(P0.05);RT-PCR结果显示SGC-7901、BGC-823、HGC-27、AGS、GES细胞系HMGB1 m RNA表达丰度均较高;Westren Blot检测发现胃癌细胞系SGC-7901、BGC-823、HGC-27中HMGB1蛋白水平显著高于胃癌细胞系AGS及正常胃粘膜细胞系GES。结论:HMGB1在胃癌组织及正常组织中的表达具有显著性差异。胃癌细胞系SGC-7901、BGC-823、HGC-27相对于其它胃细胞系存在HMGB1高表达,适合后续基因敲除分析工作。     

转移潜能不同人肝癌细胞系差异蛋白Annexin1的功能解析

解析比较蛋白质组学筛出的差异蛋白Annexin1的生物学功能,证实其是否在肝癌转移复发中发挥作用. 分别以RT-PCR、蛋白质印迹及细胞免疫化学对差异蛋白Annexin1在转移潜能不同人肝癌细胞系中的表达情况进行再验证,然后构建Annexin1反义表达质粒,转染高转移潜能人肝癌细胞系MHCC97H,通过对MHCC97H细胞的运动、侵袭、凋亡、生长周期、MMPs分泌、克隆形成等系列检测,观察目的蛋白表达降低对其生物学行为的影响,特别是转移特性的影响. 验证结果均证实Annexin1在有转移潜能人肝癌细胞系MHCC97L、MHCC97H中呈高表达. 转染Annexin1反义重组表达质粒后,MHCC97H细胞中Annexin1的表达被成功抑制. 依据MHCC97H/pcDNA3.1(+) AS Annexin1,MHCC97H/ pcDNA3.1(+),MHCC97H的检测排序,转染反义重组质粒后的MHCC97H细胞穿过上室底膜的细胞数 (运动实验) 分别为：11.13±3.31,18.88±2.03,21.86±3.38;穿过人工基底膜细胞数 (侵袭实验) 分别是：16.43±2.23,16.40±1.57,16.86±1.52;细胞平均集落形成率 (克隆形成实验) 分别为：(14.33±0.46)%,(19.35±0.49)%,(20.25±0.35)%;MHCC97H细胞凋亡比例 (FCM分析) 依次为22.2%,6.44%,6.97%;细胞周期各时相的比例依次为：G0-G1期79.5%/76.34%/80.5%,S期13.26%/14.4%/9.69% ,G2-M期7.25%/9.26%/9.81%;细胞培养上清MMP9的定量结果依次为：26.37 μg/L,28.00 μg/L,31.90 μg/L;MMP2定量结果依次为29.46 μg/L,26.37 μg/L,26.53 μg/L. 明胶酶谱分析细胞培养上清显示,转染Annexin1反义重组表达质粒的MHCC97H细胞分泌的MMP9活性与对照比变化不明显. 综合上述结果发现,转染Annexin1反义表达质粒MHCC97H细胞运动能力及集落形成率明显降低,凋亡细胞的比例增加,而侵袭潜能,细胞周期时相,细胞分泌MMP2、MMP9的量均变化不明显. 提示,差异蛋白Annexin1可能通过影响细胞凋亡和细胞运动在肝癌细胞侵袭转移过程中发挥作用.     

PC-1基因在前列腺癌细胞系LNCaP和C4-2不同细胞周期的表达

目的:检测PC-1基因在前列腺癌细胞周期中各时间点的表达变化。方法:用200 ng/mL诺可唑(nocoda-zole)处理前列腺癌细胞系LNCaP和C4-2,16 h后使细胞处于G2/M期,在不同时间点收获细胞,分别进行流式分析和Western印迹,检测PC-1基因的表达。结果:流式分析和Western印迹结果显示,在G2/M期,LNCaP和C4-2前列腺癌细胞系中PC-1基因高表达。结论:PC-1基因的表达与前列腺癌细胞的细胞周期有关,提示PC-1可能在细胞周期调控中发挥作用。     

Establishment, Growth kinetics, and Susceptibility to AcMNPV of Heat Tolerant Lepidopteran Cell Lines

Lepidopteran heat-tolerant (ht) cell lines have been obtained with sf-9, sf-21 and several Bombyx cells. They have a distinct karyotype, membrane lipid composition, morphology and growth kinetics from the parental cell lines. In this paper, we report the development of ht cell lines from other insect species and examination of their growth characteristics and virus susceptibility. Adaptation of cell lines sf-9, BTI-TN-5B1-4 (High5) and BTI-TN-MG1 (MG1) to 33℃ and 35℃ was carried out by shifting the culture ...     

Establishment, Growth kinetics, and Susceptibility to AcMNPV of Heat Tolerant Lepidop teran Cell Lines

Lepidopteran heat-tolerant(ht)cell lines have been obtained with sf-9,sf-21 and several Bombyx cells.They have a distinct karyotype,membrane lipid composition,morphology and growth kinetics from the parental cell lines.In this paper,we report the development of ht cell lines from other insect species and examination of their growth characteristics and virus susceptibility.Adaptation of cell lines sf-9,BTI-TN-5131-4(High5)and BTI-TN-MG1(MG 1)to 33℃ and 35℃ was carried out by shifting the culture temperature between 28℃ and higher temperatures by a gradual stepwise increase in temperature.The process of adaption to a higher culture temperature was accomplished over a period of 2 months.The cell lines with the temperature adaption were designated as sf9-ht33,sf9-ht35,High5-ht33,High5-ht35,MG1-ht33,MG1-ht35.These cell lines have been subcultured over 70 passages.Adaption to high temperatures was confirmed by a constant population doubling time with individual cell lines.The population doubling time of heat adapted cell lines were 1-4 h less than these of parental cell lines.Cell shapes did not show obvious change,however,the cell size of sf9-ht cells was enlarged and those of High5 and MG1 ht cells were reduced after heat adaption.When the cell lines were infected with Autographa californica nuclear polyhedrosis virus(AcMNPV)at 28℃,33℃,35℃ and 37℃,production of budded virus and occlusion bodies in each cell line was optimum at its own adapted temperature.     

重组人纤维蛋白片段诱导CIK的增殖及对肺癌耐顺铂细胞的杀伤作用

为探讨重组人纤维蛋白片段(RetroNectin)对CIK(Cytokine-induced killer cells)细胞活化、增殖的影响及CIK细胞的免疫学特性和对肺癌耐药细胞DDP-A549 (DDP: Cisplatin)的杀伤作用。提取健康人外周血中单个核细胞, Ⅰ组细胞接种于前一天用RetroNectin和CD3MAb包被好的培养瓶中, Ⅱ组接种于单独用CD3MAb包被好的培养瓶中, 接种当天加入IFN-γ, 第二天加入IL-2诱导CIK细胞。细胞计数法测定CIK细胞的增殖,并绘制细胞生长曲线, MTT法测定细胞杀伤活性,流式细胞术分析细胞表型。扫描电镜和透射电镜下观察CIK细胞对DDP-A549的杀伤和DDP-A549细胞的改变。RetroNectin对CIK细胞有很强的激活作用, 可使其细胞增殖总数达524.77倍, 其主要效应细胞CD3+CD56+可达(31.40±1.91)%; 对肺癌耐顺铂细胞DDP-A549的杀伤作用明显高于其亲代药物敏感株A549(P<0.01)。但两组CIK细胞对靶细胞的杀伤率在相同效靶比时无明显差异(P>0.05)。RetroNectin能显著增强CIK增殖活性, 但对CIK细胞的杀伤活性并无明显影响。CIK能够显著抑制DDP-A549的生长, 可用于耐顺铂肺腺癌的免疫治疗。     

Proteogenomic Analysis to Identify Missing Proteins from Haploid Cell Lines

Chromosome‐centric Human Proteome Project aims at identifying and characterizing protein products encoded from all human protein‐coding genes. As of early 2017, 19 837 protein‐coding genes have been annotated in the neXtProt database including 2691 missing proteins that have never been identified by mass spectrometry. Missing proteins may be low abundant in many cell types or expressed only in a few cell types in human body such as sperms in testis. In this study, we performed expression proteomics of two near‐haploid cell types such as HAP1 and KBM‐7 to hunt for missing proteins. Proteomes from the two haploid cell lines were analyzed on an LTQ Orbitrap Velos, producing a total of 200 raw mass spectrometry files. After applying 1% false discovery rates at both levels of peptide‐spectrum matches and proteins, more than 10 000 proteins were identified from HAP1 and KBM‐7, resulting in the identification of nine missing proteins. Next, unmatched spectra were searched against protein databases translated in three frames from noncoding RNAs derived from RNA‐Seq data, resulting in six novel protein‐coding regions after careful manual inspection. This study demonstrates that expression proteomics coupled to proteogenomic analysis can be employed to identify many annotated and unannotated missing proteins.     

Replication of Nosema Algerae In Three Insect Cell Lines

SYNOPSIS Nosema algerae , a microsporidan parasite of anopheline mosquitoes, was successfully replicated in 3 insect cell culture lines: Trichoplusia ni (TN-368); Heliothis zea (IPLB-1075); and Mamestra brassicae (IZD-Mb-0503). Infectious spores were produced in vitro. Spores were observed at 48 h postinfection, and some cells were filled with sproes by 72 h.
The number of parasites per cell increased with time. At 72 h postinfection, the infection rates for the 3 cell lines ranged from 23 to 32%. Infected cell lines were subcultured, and by the 6th passage spore production had ceased.     

Cell Culture and Selection of High Flavonoids- producing Cell Lines in Saussurea medusa

Explants of stems and leaves of Saussurea medusa Maxim. were cultured on MS medium supplemented with 0.5 mg/L BA, 2 mg/L NAA, and from which factors, such as the media, plant hormones and culture temperature, as well as the addition of phenylalanine to the medium, that affect the callus growth, were investigated. The results showed that cells grew appropriately in MS medium supplemented with 2 mg/L NAA at 25 ℃. Phenylalanine was not suitable for cell growth in solid culture but it increased flavonoid production. The calli could be distinguished by colour with naked eyes into two cell lines, a faint yellow (A) and a red coloured (B), representing respectively the different colour of metabolite accumulations. A sensitive and rapid high-performance liquid chromatographic as well as a UV spectrophotometric method have been developed for detecting the flavonoids in cultured cells. It revealed that the A line contained 1.9% flavonoids and 0.42% jaceosidin, which was 2.5 times and 3.9 times more than B line; 2.6 and 4.2 times more than the initial callus cells respectively.     

鱼类细胞系在病毒学研究中的应用进展

鱼类细胞培养尽管起步较晚,但截至目前已有近280余株不同的鱼类细胞系相继建立起来,在生理学、病毒学、毒理学、肿瘤及基因工程等多个领域发挥重要作用。主要对鱼类细胞系在病毒学研究中的最新应用,并结合作者自身的研究,尤其侧重于鱼类病毒分离、重要功能基因鉴定、抗病毒免疫和病毒致病机理研究等方面的进展作一概述。     

Synthesis and Cytotoxicity Assessment against Human Colorectal Cancer Cell Lines of Quaternary 8-Dichloromethyl Protoberberine Alkaloids

Twenty-two quaternary 8-dichloromethylprotoberberine alkaloids were synthesized from unmodified quaternary protoberberine alkaloids (QPAs) to improve their physical and chemical properties and to obtain selectively anticancer derivatives. The synthesized derivatives showed more appropriate octanol/water partition coefficients by up to values 3–4 compared to unmodified QPA substrates. In addition, these compounds exhibited significant antiproliferative activity against colorectal cancer cells and lower toxicity on normal cells, resulting in more significant selectivity indices than unmodified QPA compounds in vitro. The IC₅₀ values of antiproliferative activity of quaternary 8-dichloromethyl-pseudoberberine 4-chlorobenzenesulfonate and quaternary 8-dichloromethyl-pseudopalmatine methanesulfonate against colorectal cancer cells are 0.31 μM and 0.41 μM, respectively, significantly stronger than those of other compounds and positive control 5-fluorouracil. These findings suggest that 8-dichloromethylation can be used as one of the modification strategies to guide the structural modification and subsequent investigation of anticancer drugs for CRC based on QPAs.     

稳定表达人Fhit突变体细胞系的建立

目的:构建人脆性组氨酸三联体(Fhit)突变体真核表达载体并建立稳定表达人Fhit突变体的细胞株,以便进一步研究Fhit与复制蛋白A(RPA)在体内的相互作用。方法:将3种人Fhit突变体cDNA克隆至带有HA标签的真核表达载体pREP10上,构建人Fhit突变体真核表达载体,转染HeLa细胞,经潮霉素B加压筛选阳性克隆,用Western印迹鉴定稳定表达Fhit突变体蛋白FhitA、FhitD和FhitF的阳性细胞株。结果:经PCR鉴定及序列分析,Fhit突变体基因真核表达载体pREP10/FhitA/D/F-HA构建正确,转染人HeLa细胞,筛选出Fhit突变体表达较高的细胞株。结论:建立了3株稳定表达Fhit突变体的细胞株HeLa-FhitA/D/F,为研究Fhit与RPA的相互作用在DNA损伤应答中发挥的作用奠定了基础。     

Synthesis of Lanostane‐Type Triterpenoid N‐Glycosides and Their Cytotoxicity against Human Cancer Cell Lines

Seventeen lanostane‐type triterpenoid derivatives ( 2 – 18 ), including 11N‐glycosides ( 8 – 18 ), were synthesized from the natural triterpenoid, lanosterol ( 1 ), and were evaluated for their cytotoxicity against the human cancer cell lines, HL‐60, A549, and MKN45, as well as the normal human lung cells, WI‐38. Among them, N‐β‐d ‐2‐acetamido‐2‐deoxyglucoside ( 10 ) showed cytotoxicity against HL‐60, A549, MKN45, and WI‐38 cells (IC₅₀ 0.0078 – 2.8 μm ). However, N‐β‐d ‐galactoside ( 12 ) showed cytotoxicity against HL‐60 and MKN45 cells (IC₅₀ 0.0021 – 4.0 μm ), but not the normal WI‐38 cells. Furthermore, Western blot analysis suggested that 12 induces apoptosis by activation of caspases‐3, 8, and 9. These results will be useful for the synthesis of other tetracyclic triterpenoids or steroid N‐glycosides to increase their cytotoxicity and apoptosis‐inducing activities.     

羊草与灰色赖草F1代细胞悬浮系的建立及植株再生

本研究以羊草(L eym us ch inensis)与灰色赖草(L eym us cinereus)杂种F1代幼穗为外植体诱导愈伤组织,在3.0 m g/L 2,4-D M S培养基上继代1次后,转入不同浓度激素(2,4-D、IAA、KT)配比和不同浓度蔗糖的M S液体培养基进行振荡培养,建立杂种F1代细胞悬浮系和植株再生体系.结果表明,细胞悬浮培养时,M S 1.0 m g/L2,4-D 0.1 m g/L KT 4%蔗糖的液体培养基最佳;悬浮细胞分化时,1.0 m g/L 2,4-D 0.1 m g/L KT 4%蔗糖 M S和1.0 m g/L 2,4-D 4%蔗糖 M S培养的悬浮细胞在1.0 m g/L NAA 0.5 m g/L KT M S分化培养基上的绿苗分化率分别达到83%和80%.细胞悬浮系及再生体系的建立为杂种F1代育性恢复的研究奠定了基础.     

大鼠促甲状腺激素释放激素受体1基因的克隆及其在COS-7细胞系中的表达

目的:克隆大鼠促甲状腺激素释放激素受体1(TRH-R1)基因,构建其真核表达载体,并检测该基因在非洲绿猴肾细胞系COS-7中的表达。方法:应用RT-PCR方法,以大鼠脑源RNA为模板,扩增获得TRH-R1基因,定向克隆到pDsRed2-N1中,以LipofectAMINE 2000试剂转染pDsRed2-N1-TRH-R1表达载体至COS-7细胞系中进行瞬时表达。结果:测序结果表明,从大鼠脑源总RNA中克隆到正确的TRH-R1基因全长编码序列;显微照相观察到所构建的TRH-R1表达载体质粒在COS-7细胞系中获得有效表达。结论:大鼠TRH-R1基因的克隆、真核表达载体的构建及在COS-7细胞系中表达获得成功,为进一步研究其功能奠定了基础。     

Use of a renal tubule cell line (LLC-PK1) to study the nephrotoxic potential of a kappa-type bence-jones protein

Summary The cytotoxicity of a Bence-Jones protein was assessed using a porcine renal tubule cell line (LLC-PK1), with the aim of developing a model for studying the potential nephrotoxicity of these proteins. The effects of a kappa Bence-Jones protein on cell viability were studied by means of biochemical methods (supravital dye uptake and measurement of cellular enzyme activities) and morphological electron microscopy. After a 24-h-treatment with Bence-Jones protein, a moderate cytotoxicity (about 15%) was noted but only a minor difference compared to treatment with bovine albumin in the same conditions. The morphological study showed a few cells in the process of lysis, but their numbers were insufficient for the demonstration of a clear cytotoxic effect. Immunocytochemical studies showed Bence-Jones protein fixation on some cells, especially on the outer membrane. Labeling of the hyaloplasm and basal pole of a few cells pointed to internalization of protein by LLC-PK1 cells. Although the cytotoxicity of the Bence-Jones protein tested here was only moderate, the use of this model enabled its cytotoxic effect to be distinguished from that ofβ-lactoglobulin. This isolate could serve as a “moderate control” for a later study with a BJP having caused acute renal failure.     

Identifying Monoaminergic,GABAergic, and Cholinergic Characteristics in Immortalized Neuronal Cell Lines

We measured the concentration of neurotransmitters in immortalized neural cell lines of hippo-campal, septal, brainstem and cerebellar origin. While in most of the cell lines, concentrations of monoamines, -aminobutyric acid (GABA) and acetylcholine were low, in some they were markedly higher. This made it quite easy to identify possible monoaminergic, GABAergic or cholinergic cell lines. However all the cell lines contained glutamate and aspartate and there were no outstanding differences in levels of these amino acids differences between the cell lines. Deprivation of serum, which made the cells acquire a more differentiated morphology, caused an increase in the intracellular concentrations of some compounds and a switch from multiple to a single transmitter in the case of some cell lines. It suggested that measurement of transmitter concentrations combined with serum deprivation studies, may provide an indication of the neurochemical characteristics of immortalised neuronal cell lines.     

Combinatory effects of citrinin and ochratoxin A in immortalized human proximal tubule cells

Ochratoxin A (OTA) and citrinin (CIT) are two mycotoxins often occurring together in grains and cereals. Although both are nephrotoxic and can induce apoptosis, combination effects have not been examined up to now. Therefore, the aim of this study was to take a close look at the interactions of citrinin and OTA in cultured human proximal tubule-derived cells (IHKE cells). The cytotoxicity of both mycotoxins was studied, measuring the metabolic activity and the cell number. Furthermore, caspase 3-activation as a marker for apoptosis was examined for both mycotoxin alone and in combination. The results show that citrinin had an antagonistic effect on ochratoxin A induced caspase 3-activation in concentrations of 2.5 and 5 μmol/l. Higher concentrations (7.5 and 15 μmol/l) lead to additive effects, lower citrinin concentrations (0.25 and 1 μmol/l) did not show any effect at all. The observed decrease in caspase 3-activity was specific for the combination with OTA, since the combination of citrinin with cisplatin did not show any effect. Citrinin did not influence of the OTA-induced apoptosis when added two hours after applying ochratoxin A. Also the combination of both toxins decreased the uptake of OTA into the cells which might be an explanation for the antagonistic effect of citrinin in certain concentrations. However, the transport into cells can not be the only explanation. so further examinations are necessary. Presented at the 27^th Mykotoxin-Workshop. Dortmund, Germany, June 13–15, 2005.     

亚麻胚性细胞悬浮培养体系的建立和影响因素

亚麻品种‘双亚五号’的胚性愈伤组织诱导最佳培养基为MB（MS无机盐加B5维生素）＋1.0mg·L^-12,4-D;细胞初始悬浮培养的最佳培养基为MB＋0.2mg·L-16-BA;细胞继代悬浮培养的最佳培养基为改良的MB（NH4NO3减半,KNO3加倍）＋0.02mg·L^-12,4-D＋0.2mg·L^-16-BA;适宜的蔗糖量为50g·L^-1;适宜的继代接种量为1.0～1.5g·（25mL）^-1;继代间隔为5～7d。     

水翁悬浮细胞系的建立及其悬浮培养的生长特性

建立了水翁悬浮细胞系,并对其悬浮培养的生长特性作了初步探讨。以水翁新生芽尖作为外植体,接种于添加有不同浓度和配比的生长调节物质及各种附加物的MS固体培养基中,诱导培养产生初代愈伤组织;挑选Ⅰ和Ⅱ型的愈伤组织进行继代培养改良,考察愈伤组织的生长状况和统计生长量来决定最佳继代培养基的配方和得到适合悬浮培养的愈伤组织;将以上得到的愈伤组织转接于最佳继代液体培养基中,于24±1℃,120r/min条件下振荡培养,筛选分散度好、较均匀、生长快、色浅透明的细胞作为种子传代,数次传代后得到性能良好的悬浮细胞系;以细胞生长量(鲜重)为指标,绘制了水翁悬浮细胞的生长曲线。研究表明:2.0mg/L的2,4-D的诱导率最高(92%,初代愈伤组织为Ⅰ型),Ⅱ型愈伤组织的最高诱导率为75%;最佳的继代培养基配方为MS 0.5mg/L 2,4-D 0.5mg/L 6-BA 1.0mg/L IAA 0.5mg/L IBA 0.5mg/L NAA 0.1mg/L KT 700mg/L LH,形成Ⅱ型愈伤组织的生长量可达3.28g/瓶(鲜重);液体继代培养3代后,可得到性能良好的悬浮细胞系;水翁悬浮细胞的生长曲线表明,最佳接种期为培养后的16~18d。