骆驼蓬脂转移蛋白基因真核表达载体的构建及其体内外抗瘤效应的研究

目的:构建骆驼蓬脂转移蛋白(lipid transfer protein from Peganum harmala,PhLTP)基因真核表达质粒,并探讨其对黑色素瘤B16细胞在体内外的抗肿瘤作用。方法:将PhLTP基因亚克隆至pcDNA3.1上,获得重组质粒pcDNA3.1-PhLTP;用脂质体转染法将重组质粒及空载体外转染B16细胞,MTT检测其对B16细胞生长的影响。建立B16荷瘤小鼠模型,设重组质粒(pcDNA3.1-PhLTP)、空载(pcDNA3.1)、生理盐水和阳性药物(CTX)组,分别处理小鼠后测量各组肿瘤体积并称瘤重,计算抑瘤率。光镜观察鼠脾、肝等组织变化;免疫组织化学法检测各瘤体中PhLTP、血管内皮生长因子(VEGF)及碱性成纤维细胞生长因子(bFGF)的表达。结果:pcDNA3.1-PhLTP转染B16细胞72 h后,细胞增殖能力明显受到抑制(P0.01)。注射pcDNA3.1-PhLTP组的小鼠肿瘤生长速度明显减慢,肿瘤体积小于空载和生理盐水组(P0.05)。显微镜下可见重组质粒组肿瘤细胞有不同程度的点、片状坏死,而肝、肺等无明显病理损伤。重组质粒组肿瘤组织中有PhLTP蛋白的表达,且VEGF和bFGF的阳性表达指数都低于空载和生理盐水组(P0.01)。结论:成功构建了重组表达质粒pcDNA3.1-PhLTP,体内外实验结果显示其能有效地抑制B16细胞的生长,预示了该重组质粒在治疗黑色素瘤中的潜在应用价值。     

小鼠白细胞介素21瘤苗的构建及其抗肿瘤效应研究

目的:建立稳定表达小鼠白细胞介素21(mIL-21)的肿瘤细胞瘤苗,观察其在小鼠体内是否能够诱导有效的抗肿瘤免疫反应及免疫记忆效应。方法:将已鉴定的重组质粒pcDNA3.1/mIL-21用脂质体法转染Sp2/0细胞制备瘤苗,RT-PCR法鉴定瘤苗中mIL-21的表达。通过流式细胞仪检测细胞周期来反映瘤苗体外增殖活性,再将其接种BALB/c小鼠,监测肿瘤生长情况,观察mIL-21瘤苗诱导的抗肿瘤效应;用ELISA法检测血清IFN-γ和IL-4含量。结果:得到稳定表达mIL-21的瘤苗Sp2/0-mIL-21。与对照组相比,体外增殖活性无差异。皮下接种BALB/c小鼠后,肿瘤生长缓慢,部分小鼠无瘤体生长并长期存活;用野生株Sp2/0瘤细胞再次攻击未长肿瘤的实验小鼠,4周后亦未见肿瘤生长。接种瘤苗小鼠血清中IFN-γ水平明显上升,IL-4无明显增高。结论:成功构建了mIL-21瘤苗Sp2/0-mIL-21,其能诱导有效的抗肿瘤免疫反应及免疫记忆效应。     

高表达Snail蛋白诱导黑色素瘤EMT细胞模型的构建及鉴定

目的:构建稳定表达Snail蛋白的可用于肿瘤上皮-间质化研究的肿瘤细胞模型.方法:采用亚克隆方法从质粒pCMV6-mSnail中PCR扩增小鼠Snail基因,连接至表达质粒pL-tdTomato-Neo,筛选重组质粒并经双酶切及测序鉴定.构建成功的重组质粒pL-tdTomato-mSnail转染小鼠黑色素瘤B16细胞,G418筛选稳定细胞株.采用荧光定量PCR和Western blot技术检测胞内Snail及上皮/间质标志物的变化.建立裸小鼠皮下移植瘤模型,活体成像系统观测移植瘤.结果:重组质粒pL-tdTomato-mSnail成功构建,其稳定转染株B16/dT-mSN胞体发出强烈红色荧光.胞内Snaill水平显著上调,E-钙粘蛋白下调,波形蛋白表达上调,呈现典型的上皮-间质化表型.结论:成功获得稳定高表达小鼠Snail蛋白的EMT细胞模型,且可用于体内外荧光成像观测,为研究Snail蛋白在介导肿瘤EMT过程中的生物作用提供了重要的实验工具.     

MIC3-EGFP融合蛋白真核表达质粒的构建及其在COS-7细胞中的表达

目的:构建以绿色荧光蛋白(greenfluoreseeneeprotein,GFP)为报告基因的重组表达质粒pEGFP-C2-MIC3并检测MIC3-EGFP融合蛋白其在COS-7细胞中的表达及定位.方法:通过基因重组的方法构建pEGFP-C2-MIC3重组真核表达质粒,并通过酶切和基因测序鉴定.脂质体法转染体外培养的COS-7细胞,转染后24h在活细胞状态下用倒置荧光显微镜直接观察MIC3-EGFP融合蛋白在COS-7细胞中的分布.结果:PCR检测,酶切鉴定及测序证实目的基因MIC3正确连接到pEGFP-C2的多克隆位点.pEGFP-C2-MIC3重组体转染COS-7后,在细胞质表达.结论:成功地构建了pEGFP-C2-MIC3融合蛋白真核表达质粒,在COS-7细胞中获得表达.     

小鼠白细胞介素33真核表达质粒的构建及表达

克隆小鼠IL-33基因构建其真核表达质粒,并转染COS-7细胞检测其表达。提取C57BL/6小鼠肺组织总RNA,经反转录聚合酶链式反应（RT-PCR）扩增小鼠IL-33基因,酶切后插入pcDNA^TM3.1/myc HisA构建其真核表达质粒pcDNA-3.1-IL-33,重组质粒转染COS-7细胞,RT-PCR和免疫印迹法（western blotting）检测目的基因表达。结果显示,pcDNA3.1-IL-33中插入的片段序列测定结果与小鼠IL-33cDNA序列一致,重组质粒转染COS-7细胞后检测到相应mRNA及蛋白表达。成功克隆了小鼠IL-33基因cDNA,并构建其真核表达质粒。     

纤黏连蛋白重组多肽CH50抑制黑色素瘤MMP-2和MMP-9表达、激活的研究

目的 研究纤黏连蛋白重组多肽CH50对黑色素瘤B16细胞侵袭能力的影响,探讨CH50多肽抑制肿瘤生长、侵袭的机制。方法 体外培养小鼠黑色素瘤B16细胞、小鼠腿部皮下注射B16细胞建立肿瘤动物模型。采用明胶电泳法检测B16细胞和黑色素瘤组织中基质金属蛋白酶MMP-2和MMP-9的表达和激活;以CH50多肽体外处理B16细胞或体内表达CH50,观察CH50下调MMPs表达以及对肿瘤细胞侵袭能力的抑制作用。结果 B16细胞在体外培养条件下主要表达MMP-2,而在肿瘤微环境中则同时表达MMP-2和MMP-9。肿瘤组织中MMPs的表达明显高于体外培养B16细胞。CH50多肽对体外培养B16细胞的MMPs表达和激活无明显抑制作用,但处理后的B16细胞进入体内后表达MMPs的能力受到明显抑制。体内转染表达的CHSO多肽亦可明显抑制肿瘤表达MMPs、并抑制肿瘤侵袭能力。结论 纤黏连蛋白重组多肽CHSO可以抑制肿瘤微环境中基质金属蛋白酶MMP-2和MMP-9的表达和激活,从而抑制黑色素瘤生长及侵袭能力。     

人可溶性VEGFR2和IL-12融合基因真核表达质粒的构建与表达

[目的]构建和表达人可溶性血管内皮生长因子受体2(VEGFR2)与白细胞介素12(IL-12)融合基因的真核表达质粒,并初步验证其抗肿瘤活性。[方法]根据Gen Bank公布的基因序列,设计并分别全基因合成全长人VEGFR2和IL-12基因,VEGFR2与IL-12通过Furin/2A(F2A)进行连接,融合基因克隆入真核表达载体p VAX1,构建p VAX1-VEGFR2-F2A-IL-12表达质粒。将p VAX1-VEGFR2-F2A-IL-12质粒瞬时转染293T细胞后,通过ELISA法测其真核表达能力。质粒注射B16荷瘤小鼠模型后,分析其抗肿瘤效果。[结果]质粒p VAX1-VEGFR2-F2A-IL-12构建成功;ELISA检测293T细胞转染上清液结果显示,VEGFR2和IL-12都能够在293T细胞中得到表达。质粒注射荷瘤小鼠模型后,显示出了较强的抑制B16肿瘤生长的能力,达到50%。[结论]p VAX1-VEGFR2-F2A-IL-12质粒成功构建和表达,该质粒具有较好的抑制B16肿瘤生长的能力,其抑制率达50%。     

siRNA干扰MMP-9和FAK双基因抑制小鼠黑色素瘤生长和在体迁移

目的:观察干扰MMP-9和FAK双基因对恶性黑色素瘤高转移细胞B16F10体内转移的影响。方法:构建PGV102-MMP9-siRNA、PGV102-FAK-siRNA重组质粒载体,脂质体^TM2000介导转染小鼠黑色素瘤B16F10细胞,RT-PCR检测基因的干扰效果;建立C57BL/6小鼠皮下移植瘤模型观察细胞在体成瘤和肿瘤的生长情况,常规组织切片,H&E染色观察肿瘤组织病理学特征;经C57BL/6小鼠尾静脉注射细胞5×10⁵个/只,24天后计数小鼠肺转移结节数评价肿瘤细胞在体迁移能力。结果:RT-PCR结果表明,重组质粒转染细胞组的MMP-9和FAK的mRNA水平显著低于正常细胞组(P<0.01),转染细胞组C57BL/6小鼠皮下成瘤的肿瘤生长速率、黑色素瘤肺转移结节数明显低于正常细胞组(P<0.01)。结论:干扰B16F10细胞MMP-9和FAK双基因可明显抑制小鼠体内恶性肿瘤的生长和迁移。     

pEGFP-HPV16E7重组融合蛋白质粒的构建及其表达

应用基因重组技术,构建增强绿色荧光蛋白(EGFP)与人乳头瘤病毒16型E7(HPV16E7)的重组融合表达质粒,经限制性内切酶酶切鉴定和PCR分析后,用基因转染技术将其导入小鼠肝癌细胞,荧光显微镜下观察融合蛋白的表达.酶切鉴定和PCR分析证实重组质粒中插入目的基因片段的大小、方向和插入位点均正确,在转染的小鼠肝癌细胞中观察到绿色荧光蛋白的表达.构建的pEGFP-HPV16E7融合表达质粒能直观地反映转染细胞中EGFP-HPV16E7融合蛋白的表达.由于转化率与表达率融为一体,故有利于对转染细胞的筛选,缩短转染细胞在体外的筛选的时间适用于对HPV16E7分子生物学特性、致瘤机理及APC提呈等的研究.为建立表达HPV16E7的实体瘤动物模型奠定了基础.     

小鼠脂联素与绿色荧光蛋白融合基因表达载体的构建及在COS-7细胞中的表达

以绿色荧光蛋白(GFP)基因(gfp)为报告基因,构建小鼠脂联素(mADPN)基因(mAd)与gfp的融合基因mAd/gfp表达载体pCI-neo-apoEHCR-hAATp-mAd-gfp,脂质体法转染体外培养的COS-7细胞,荧光显微镜观察GFP在细胞中的表达可间接反映mADPN的表达,并通过RT-PCR在核酸水平进一步确证mAd的表达.荧光显微镜观察及RT-PCR结果均证明mADPN在COS-7细胞中获得了高效表达,表明mADPN重组表达载体pCI-neo-apoEHCR-hAATp-mAd可以在真核细胞COS-7中高效表达mADPN,为进一步探讨mAd在小鼠体内的表达提供了可行性依据.     

体内连续进化——从噬菌体到真核基因组的进化故事（英文）

进化是生物多样性产生和保留的自然进程。通过对编码蛋白质的基因进行有目的地设计和改造,获得性能更优异的蛋白质用于生产生活,是蛋白质工程的目的所在。为了在实验室中通过定向进化的蛋白质工程模拟自然进化的实现过程,研究人员通过在快速增殖的原核生物和简单的真核生物中引入靶向诱变元件,建立了各种体内连续进化系统。本综述介绍了体内连续进化平台的现状,重点关注噬菌体和酵母中人工进化技术的研究进展,并对其在生物技术领域中的成功应用进行了总结,最后简要展望了体内连续进化这一新兴领域的发展方向。     

In planta transformation of Arabidopsis thaliana

Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions. After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. We have investigated the reproducibility and the overall efficiency of this simple in planta transformation procedure. In addition, we determined the T-DNA copy number and inheritance in the transformants and examined whether transformed progeny recovered from the same Agrobacterium-treated plant represent one or several independent transformation events. Our results indicate that in planta transformation is very reproducible and yields stably transformed seeds in 7–8 weeks. Since it does not employ tissue culture, the in planta procedure may be particularly valuable for transformation of A. thaliana ecotypes and mutants recalcitrant to in vitro regeneration. The transformation frequency was variable and was not affected by lower growth temperature, shorter photoperiod or transformation vector. The majority of treated plants gave rise to only one transformant, but up to nine siblings were obtained from a single parental plant. Molecular analysis suggested that some of the siblings originated from a single transformed cell, while others were descended from multiple, independently transformed germ-line cells. More than 90% of the transformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes. Of those, 60% contained one functional insert, 16% had two T-DNA inserts and 15% segregated for T-DNA inserts at more than two unlinked loci. The remaining transformants displayed non-Mendelian segregation ratios with a very high proportion of sensitive plants among the progeny. The small numbers of transformants recovered from individual T₁ plants and the fact that none of the T₂ progeny were homozygous for a specific T-DNA insert suggest that transformation occurs late in floral development.National Research Council of Canada Publication No. 38003     

光皮桦组织培养离体再生研究

采用正交试验等设计,系统开展了光皮桦组织培养高效再生体系研究。结果表明:光皮桦茎段外植体最佳诱导培养基及激素组合为MS+0.50mg.L-1 6-BA+0.10mg.L-1 TDZ+30mg.L-1蔗糖+5.50g.L-1琼脂,丛生芽最佳生根培养基为1/2MS+1mg.L-1 IBA+20g.L-1蔗糖+5.50g.L-1琼脂;丛生芽在最佳生根培养基上培养15d后获健壮生根苗,移栽成活率达90%。该实验结果为光皮桦的优良品种快繁以及遗传转化体系建立奠定了良好的基础。     

In vitro chromosome doubling of nineZantedeschia cultivars

Tetraploid plants have been produced from nineZantedeschia cultivars usingin vitro colchicine treatment. Rapidly-multiplying shoot cultures were treated on a medium containing 0.05% colchicine for 1, 2 or 4 days to induce chromosome doubling. Following the treatment, most shoots were killed but the surviving shoots were multiplied for several subcultures. These shoots were then rootedin vitro and transferred to a greenhouse. Plants were screened 2 months later by measuring stomatal length, and 110 out of 565 plants were selected as putative tetraploids with a stomatal length significantly greater than in diploid control plants. Chromosome counts were carried out on root tips from 44 plants and confirmed that 38 were tetraploids, 2 were chimeras (predominantly tetraploid with a few octoploid cells), and 4 were diploids. Stomatal length has been rechecked in mature tetraploid plants of the cultivar Black Magic, demonstrating that stomatal length is a good indicator of ploidy level inZantedeschia. This study has shown that multiplying colchicine-treated shootsin vitro for several subcultures prior to transfer to soil produced very few chimeras. The stomatal length measurements are non-destructive and allow the rapid screening of a population for tetraploids.     

以转录因子AP-1为靶的decoy核酸体内外抗肿瘤研究

合成了双链寡聚核苷酸——decoy核酸,其与靶转录因子AP-1有高亲和性,可进入细胞作为decoy顺式元件,通过抑制特异的转录因子和调控区域的结合,调控基因转录而改变基因的表达.在体内外抗肿瘤试验中, decoy核酸有显著抑制肿瘤细胞增殖的作用,可以成为潜在性的肿瘤基因治疗药物.     

In vitro organogenesis of Citrus volkameriana and Citrus aurantium

In vitro organogenesis of Citrus volkameriana and C. aurantium was studied considering three explant types: epicotyl segment, internodal segment, and hypocotyl segment with attached cotyledon fragment. The explants were cultured in medium according to Grosser and Gmitter (EME) supplemented with 0, 0.5, 1.0, 1.5, and 2.0 mg dm^{− 3} 6-benzyl-aminopurine (BAP), incubated firstly in darkness for 4 weeks, and then transferred to 16-h photoperiod for 2 weeks. Comparing epicotyl and internodal segments, a higher percentage of responsive explants and a higher number of shoots per explant were obtained with epicotyl segments, regardless of the BAP concentration. For C. volkameriana the highest percentage of responsive epicotyl segments (42 %) was obtained in EME with 1.0 mg dm⁻³ BAP, while for C. aurantium (59 %) in EME with 0.5 mg dm⁻³ BAP. The organogenesis efficiency was the best with the use of the hypocotyl segment with attached cotyledon fragment (77 % for C. volkameriana and to 75 % for C. aurantium). With this explant the morphogenesis occurred only in the hypocotyl region. The in vitro organogenesis was characterized by histological analyses showing that the morphogenic process started in the cambium region near the explant cut end.     

In vitro production of solasodine from Solanum trilobatum

Sucrose concentration in the culture medium affected chlorophyll content, trichome development and amount of solasodine in regenerated plantlets of Solanum trilobatum. High chlorophyll content and glandular trichomes were observed in the plants grown on Murashige and Skoog basal medium supplemented with 131.85 mM sucrose. The solasodine was quantified using reverse phase high performance liquid chromatography. The plantlets cultivated on this medium yielded 35.97 mg g⁻¹ (d.m.) solasodine whereas the field plants used as control yielded only 2.32 mg g⁻¹ (d.m.) of solasodine.     

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.     

体外转录的自我复制型mRNA疫苗研究进展*

自我复制型mRNA是一种灵活的疫苗平台,该平台的开发基于甲病毒表达载体,其中复制必需基因得以完整保留,而结构蛋白基因则被来自病原的抗原基因替换。由于避免了病原培养、毒力返强和现存免疫的干扰,使其成为疫苗快速设计的理想平台。大量研究数据显示,此类疫苗可应用在人、小鼠、兔、猪、禽甚至鱼类体内诱导体液免疫和细胞免疫。过去,自我复制mRNA疫苗的研究采用重组单载体的模式,基因组骨架来源于辛德毕斯病毒、塞姆利基森林病毒和委内瑞拉马脑炎病毒。现在,反式复制型RNA和核酸修饰的反式复制型RNA作为下一代技术平台被寄予厚望。对基于甲病毒表达载体的mRNA疫苗技术的研究进展进行概述,重点介绍针对以流感病毒、新型冠状病毒和寨卡病毒等为代表的自我复制型mRNA疫苗研究现状,并探讨了该技术平台的未来发展方向。