细胞工厂中F基因型腮腺炎病毒SP-A株在人二倍体细胞上的增殖研究

目的利用细胞工厂,探讨F基因型腮腺炎病毒SP-A株在KMB_(17)细胞上的增殖特性,为腮腺炎疫苗的研制提供基础数据。方法分别以体积分数为10%、8%和5%的血清浓度将细胞培养至单层后,观察病毒接种后细胞病变情况,初步确定最低血清含量后;在不含血清培养基条件下,按照不同接种感染复数(multiplicity of infection, MOI)接种细胞,确定病毒增殖峰值后,进一步观察不同吸附条件对病毒增殖的影响;同时进行荧光实时定量PCR,验证病毒增殖变化。结果在体积分数为5%的血清条件下,细胞可生长成致密单层;高MOI(0.500)感染细胞后,在4～5 d内进入增殖高峰,中MOI(0.050)、低MOI(0.005)感染细胞后,在5～6 d进入增殖高峰,维持48 h后,呈现缓慢下降趋势;在比较37℃吸附试验中,非吸附组病毒增殖高峰明显迟于吸附组,荧光实时定量PCR检测结果显示在病毒感染细胞6 d时病毒载量达到峰值。结论 5%新生牛血清即可获得生长状态良好的细胞,MOI对腮腺炎病毒在KMB_(17)细胞株中的增殖有较大的影响,当使用适宜范围的MOI(0.01～0.02),在非吸附的条件下,腮腺炎病毒SP-A株在KMB_(17)株中能够良好地增殖,在病毒培养至7～9 d可获得高滴度的病毒收获液。     

肠道病毒71型在RD细胞和Vero细胞中的增殖动力学特征

目的:利用噬斑法比较肠道病毒71型(EV71)在RD细胞和Vero细胞中的增殖动力学特征。方法:首先探讨培养基类型、羟乙基哌嗪乙磺酸(HEPES)、胎牛血清(FBS)、牛血清白蛋白(BSA)及甲基纤维素(MC)含量对EV71噬斑形成的影响,得到最适营养覆盖物配比;进一步,EV71以感染复数(MOI)为0.1分别接种RD细胞和Vero细胞,收集接种后不同时间点的细胞培养液,噬斑法测定各时间点培养液上清中的病毒滴度,并绘制log2(病毒滴度)-时间图,对比分析EV71在2种细胞中的增殖动力学特征。结果:终浓度含1%MC和2%FBS的MEM(1×)或DMEM(1×)为EV71噬斑形成的最适营养覆盖物;EV71在RD细胞和Vero细胞中的增殖周期均约为12 h,MOI=0.1时,EV71在RD细胞中的增殖活动较Vero细胞中活跃,增殖效率比Vero细胞中高2个数量级。结论:用RD细胞扩增EV71比Vero细胞更具优势。     

狂犬病病毒CVS-11株培养工艺的优化

目的通过优化培养工艺提高狂犬病病毒CVS-11株的滴度。方法 BSR细胞(1.5×106个细胞/孔)以不同感染复数(MOI 0.100、0.050、0.010、0.005和0.001)接种CVS-11,培养72 h收获,测定病毒滴度;以0.005和0.001 MOI,分别接种不同密度的BSR细胞(1×105、2×105、3×105、4×105、5×105、6×105、8×105和10×105个细胞/孔)培养CVS-11,于72 h收获,测定病毒滴度;以5×105个细胞/9.6 cm2和0.001 MOI,分别在48、72、96、120和144 h收获病毒,测定病毒滴度;并通过正交实验获得最佳培养条件;由6孔板(9.6 cm2/孔)放大到T75 cm2瓶培养,验证正交实验结果。结果细胞密度对CVS-11病毒滴度有一定的影响,以4×105细胞/9.6 cm2获得的CVS-11病毒滴度最高。以相同的细胞数量和收获时间,CVS-11病毒的滴度随着MOI的减小而增大。固定细胞数和MOI,病毒滴度在培养早期随时间的延长而增加,直至96 h后,随着时间的延长滴度下降。正交实验的结果表明3个因素对滴度的影响顺序是MOI细胞数量收获时间,放大培养验证了正交试验的结果。结论以4×105个细胞/9.6 cm2,0.001 MOI,培养96 h获得的CVS-11滴度最高。     

通过基因组定量研究猪瘟病毒在细胞中的增殖特性

应用间接免疫荧光、Real-time PCR和病毒感染滴度(TCID50)测定技术,分别从病毒抗原、病毒基因组RNA复制水平和病毒感染滴度变化3个方面,研究了猪瘟病毒(CSFV)在PK-15细胞中增殖的特点,用猪瘟病毒石门株感染96孔板培养的细胞,1×102个TCID50/孔,间接免疫荧光检测结果显示感染后8h能检测到被荧光抗体染色的感染细胞,随感染时间的延长,出现荧光的细胞数量逐渐增多,在感染后72h,几乎所有细胞均能出现荧光。Real-time PCR结果显示在细胞感染初期的8~24h,病毒的基因组RNA复制呈加速趋势,其拷贝数在感染后72h达到高峰。此外,在感染后8h能检测到病毒基因组负链RNA转录,不过负链RNA在病毒增殖过程中维持在较低的水平。TCID50测定结果表明CSFV的感染滴度增加趋势与基因组类似,在病毒感染8h后能检测到具有感染性的子代病毒,感染滴度在8~20h之间逐渐增长,24~48h之间增长速度稍减慢,在感染后48~52h达到高峰,能在72h之内维持较高的感染滴度。     

MOI对PK-15细胞感染PPV后细胞因子mRNA转录时相的影响

【目的】更好地了解感染复数(MOI)对猪细小病毒(PPV)感染PK-15细胞后引起细胞因子的反应,探讨宿主-病毒之间的作用关系。【方法】运用荧光定量PCR技术,测定和分析3种感染复数对PPV感染PK-15细胞引起的病毒DNA量的变化和细胞因子IFN-、IRF-3、TNF-a和IL-18分泌水平。【结果】PPV感染PK-15细胞12 h病毒开始大量迅速增殖,感染48 h且MOI为1.0时达到最高峰;PPV感染后可引起PK-15细胞中IFN-、IRF-3、TNF-a和IL-18的表达量显著增加,其中IRF-3基因在感染后1 h且MOI为10.0时表达量达到967倍。【结论】细胞因子的分泌水平显著依赖感染复数和感染时间的交互作用。     

四株草鱼呼肠孤病毒毒株的细胞感染特性比较研究

本文首次对低温保存的三株草鱼呼肠孤病毒GCRV873 、GCRV875、GCRV876与新分离的GCRV991毒株进行了细胞培养与病毒感染特性等比较研究。结果表明 ,GCRV873 、GCRV875、GCRV876在 - 30℃保存 10年后仍然具有一定的感染性 ,其滴度均在 10 2 TCID50 /mL以上 ,略低于从病鱼组织分离的GCRV991毒株的滴价。经传代培养后 ,四株GCRV的毒力逐渐升高 ,并趋于稳定 ;当感染复数 (MOI)为 0 .0 5PFU/cell时 ,测定四株GCRV的滴度均高于 10 8TCID50 /mL ,但略有差异。GCRV873 的滴度最高 ,可达到 6 .4× 10 11TCID50 /mL。连续传代的GCRV毒株在不同温度 (2 8℃、31℃、34℃、37℃、41℃ )条件下 ,均可感染CIK细胞 ;在 2 8℃时 ,感染效价最高 ,随着温度的升高 ,其感染效价逐渐降低     

猪瘟病毒与宿主细胞间的相互关系

猪瘟病毒(CSFV)是一种正链RNA病毒,在培养细胞中增殖力弱,滴度低,通常不使培养细胞产生病理变化;而感染猪导致的猪瘟发病迅速,死亡率高。本文综述了近年来在CSFV增殖过程、与宿主细胞间的相互作用方面的研究进展,并对CSFV与细胞凋亡间的关系进行了探讨。     

Ⅰ型禽腺病毒AV208株在鸡肝癌细胞中增殖规律的研究

【目的】摸索鸡肝癌细胞系(LMH)培养条件,并采用LMH细胞系对Ⅰ型禽腺病毒AV208株(FAVⅠ-AV208)进行增殖规律的研究。【方法】细胞以不同血清浓度培养并以不同接种比例传代。观察最佳感染复数下的细胞病变和病毒增殖情况,并研究病毒接种物浓度与蚀斑形成数量之间的关系。【结果】LMH细胞系的最适培养条件为,以10%FBS的培养基以1:5比例传代培养。FAVⅠ-AV208毒株可以在LMH细胞中良好增殖并达到较高的滴度(107.5TCID50/0.1 mL)。在最佳感染复数(MOI)为0.01时,72 h细胞即出现明显的细胞病变(CPE),特征为细胞变大变圆,集聚成不规则的葡萄串状。病毒接种物浓度与蚀斑形成数量间呈线性相关。【结论】LMH细胞是FAVⅠ较合适的病毒培养系统,为研制禽腺病毒重组疫苗提供了有利工具。     

病毒感染复数对狂犬病毒PM株增殖的影响

目的探讨不同感染复数(Multiplicity of infection,MOI)的狂犬病毒PM株在人二倍体细胞(2BS株)中增殖的影响,确定感染复数。方法将狂犬病毒PM株按照MOI 0.01、0.02、0.05、0.10、0.15、0.20接种至2BS细胞中,用显微镜观察接种病毒后细胞的形态变化;培养3~5 d后,第一次收获病毒液;更换培养液继续培养3~4 d后,进行第二次收获。采用小鼠脑内滴定法测定狂犬病毒滴度,通过NIH法测定灭活病毒液的效价。结果当MOI为0.05~0.10时,细胞圆缩30%~40%,细胞能维持较好的生长形态,可收获病毒,病毒收获液的滴度较高(5.0lg LD50/m L),且灭活后病毒液的效价较高(4.0IU/m L)。结论确定狂犬病毒PM株在人二倍体2BS细胞增殖的适宜MOI,为人用狂犬病疫苗生产工艺的优化提供了依据。     

细胞工厂在轮状病毒基因重配株LD9培养中的应用初探

为了探索用细胞工厂代替转瓶培养轮状病毒基因重配株LD9及收获高滴度的LD9病毒原液和提高产量的可行性,分别在2层4、层细胞工厂和3L1、5L转瓶培养Vero细胞,比较两种容器内细胞的生长状态。结果显示,以相同活细胞数2.5×104/ml同时接种两种不同培养容器时,细胞工厂培养3d已长成单层,而转瓶培养需5d;对两种容器长满单层时的细胞经胰酶消化后通过细胞仪计数、分析,结果显示,两种容器培养细胞长成单层时的单位面积细胞密度相当;对长成致密单层细胞的两种容器以相同的MOI(MOI=0.1)接种LD9病毒,转瓶培养的病毒于第8d病毒滴度达到高峰,为6.0～6.5 lgCCID50/ml;细胞工厂第5d病毒滴度达高峰,为6.5 lgCCID50/ml,并于第9d病毒滴度再次达到峰值,为6.0～6.5 lgCCID50/ml,实现二次收获病毒。     

六种藓类植物茎的比较解剖学研究

通过对6种藓类植物,即褶叶青藓(Brachythecium salebrosum(Web.et Mohr.)B.S.G.)、湿地匐灯藓(Plagiomnium acutum(Lindb.)Kop.)、侧枝匐灯藓(Plagiomnium maximoviczii(Lindb.)Kop.)、大凤尾藓(Fissidensnobilis Griff.)、大羽藓(Thuidium cymbifolium(Doz.et Molk.)B.S.G.)和大灰藓(Hypnum plumaeforme Wils.)嫩茎和老茎的石蜡切片和显微观察发现,同一藓类植株的嫩茎和老茎,茎结构稳定,不同种藓类植物茎横切面具有不同特征.植物体茎横切面形状、表层细胞的层数、细胞大小和细胞壁厚薄、皮层细胞大小和形状、中轴的有无以及比例等特征可以作为藓类植物的分科分类依据之一.     

真菌类遗传学分析的知识结构教学

本文以认知结构理论为指导,讨论了真菌类遗传分析与高等动植物遗传分析的内在联系,认为利用这种内在联系进行教学可收到好的效果并说明了作者的具体教学过程。 Abstract:In the paper, the relationship between genetic analysis of Fungi and genetic analysis of high animal and plant was discussed.A good results were obtained when we adopted this method in the teaching.     

Cause of Senescence of Nine Sorts of Flowers

The levels of endogenous phytohormones and respiratory rate in nine sorts of flowers such as Cymbidium faberi Rolfe, Nopalxochia ackermannii Kunth and others were investigated both at full bloom and senescence and meanwhile the effect of exogenous phytohormones on prolonging the blossoms and promoting ethylene production were tested. There is a high content of endogenous ethylene in all the long-lived flowere, about 3–16 folds higer than the short-lived ones. There is a high level of ABA at full blooming flowers of short-lived flowers, in which there is no or only some cytokinins in it, but the ratio of CTK (6BA+zeatin)/ABA is smaller(l.7). The endogenous ABA reached a much higher level at senescence in all nine sorts of flowers, so it is reasonable to consider that it is ABA which plays an important role of regulation in controlling flower's senescence. There is a much higher level of GA3 and zeatin in the long-lived flowers which is not demonstrated in the shortlived ones. The respiratory rate is one of the factors controtling the longevity of flowers, but it does not play a decided role. Application of 6BA and zeatin prolongs distinctly orchid’s longevity, however exogenous IAA through the promotive action on ethylene production, evidently extends the longevity of the flowers of the Nopalxochia ackermannii Kunth.     

Time of detection of recessive genes: effects of system of mating and number of examined individuals

Summary Anthers were cultured from two sets of seven lines of hexaploid wheat (Triticum aestivum L.) with different cytoplasms, the euplasmic nucleus donors, Siete Cerros 66 and Penjamo 62, as well as their six alloplasmic lines derived from wild relative species of the genera Triticum and Aegilops. Significant cytoplasmic and nuclear effects but no cytoplasmic-nuclear interaction were found for embryogenic anther response, with the best performance of Penjamo 62 in Ae. kotschyi cytoplasm. Plant regeneration was not affected significantly by the cytoplasmic background of the lines cultured. The possible genetic implications of the observed cytoplasmic and nuclear influences on the in vitro haploid induction of wheat are discussed.     

龙胆科药用植物化学成分的研究现状

龙胆科植物在我国的分布范围很广,且多数为药用植物,其多数种属的药用植物,至今其化学成分尚未被系统研究。综述了目前龙胆科药用植物的化学成分的研究现状及一般提取方法,对近年来发现的环烯醚萜及裂环烯醚萜类化合物进行了总结,为本科药用植物的更深入研究提供了参考。     

Photoregulation of seed germination of wild-type and of an aurea-mutant of tomato

Seed germination of an aurea mutant of tomato ( Lycopersicon esculentum Mill.) is promoted by continuous irradiation with red, far-red or long-wavelength far-red (758 nm) light as well as by cyclic irradiations (5 min red or 5 min far-red/25 min darkness). Far-red light applied immediately after each red does not change the germination behaviour. Seed germination of the isogenic wild-type, cv. UC-105, is promoted by continuous and cyclic red light while it is inhibited by continuous and cyclic far-red light and by continious 758 nm irradiation. Far-red irradiation reverses almost completely the promoting effect of red light. The promoting effect (in the aurea mutant) and the inhibitory effect (in the wild-type) of continuous far-red light do not show photon fluence rate dependency above 20 nmol m⁻² s⁻¹. It is concluded that phytochrome controls tomato seed germination throgh low energy responses in both the wild type and the au mutant. The promoting effect of continuous and cyclic far-red light in the au mutant can be attributed to a greater sensitivity to P_fr.     

Scales of spatial patterns of distribution of intertidal invertebrates

Few comparative studies of spatial patterns at different scales have examined several species in the same habitat or the same species over a range of habitats. Therefore, variability in patterns among species or among habitats has seldom been documented. This study quantifies spatial patterns of a suite of intertidal snails and a species of barnacle using a range of statistical techniques. Variability in densities was quantified from the scale of adjacent quadrats (over a distance of centimeters) to tens of kilometers. Significant differences in abundances occurred primarily at two spatial scales. Small-scale differences were found at the scales of centimeters or 1–2 m and, for many species on many shores, these accounted for most of the variability in abundances from place to place. These are likely to be determined by behavioural responses to small-scale patches of microhabitat. Large-scale differences in abundance were also found in most species at the scale of hundreds of meters alongshore. These are likely to be due to variation in recruitment (and/or mortality) because of limited dispersal by adults of these species. There was little or no additional variation among shores, separated by tens of kilometers, than was shown among patches of shore separated by hundreds of meters. Identification of the scale(s) at which significant differences in abundance are found focus attention on the processes (and the scales at which these processes operate) that influence patterns of distribution and abundance. Some of the advantages and disadvantages of various procedures are discussed.