MTT法检测石刁柏悬浮细胞技术的研究

本研究确定了MTT法测定悬浮培养的石刁柏细胞活力所需的最适条件为:比色波长560nm,最适缓冲液为柠檬酸钠-Vc缓冲液,渗透促进剂为二甲基亚砜,洗脱剂为酸化异丙醇,0.133%浓度的MTT溶液适于对未知培养时期的细胞计数,0.267‰浓度的MTT溶液适于鉴定细胞活力;同时,本研究通过MTT对培养细胞的生长过程进行了测定,确定细胞活性最强时期为指数期;并研究了细胞数量与吸光值之间的关系以及培养时间对MTT染色的影响.     

MTT法和CCK-8法检测细胞活性之测试条件比较

利用HL60作为研究对象,对比了MTT法和CCK-8法在最佳检测时刻和检测波长等参数的差异。对于CCK-8法,最佳测试时刻在细胞液培养4 h后,最佳测试波长为490 nm。实验结果表明用CCK-8法检测的OD值与活细胞数的线性相关度要优于MTT法,是一种灵敏度较高、重复性好的的细胞活性检测方法。     

MTT法检测细菌细胞数的主要影响因素分析

MTT法是生物活细胞计数的灵敏、快速和便捷方法。报告对MTT法应用于细菌细胞数定量分析时的分析前培养时间、MTT反应时间、MTT剂量和检测波长等主要影响因素的分析结果,同时报告MTT反应产物溶解检测与直接检测,以及MTT法与平板菌落计数法定量检测的结果比较。结果如下：Formazan生成量与细菌活细胞数呈正相关,对数生长期变化最明显;MTT为0.25 mg/mL,反应2 h时吸光值与菌落数具有良好的线性关系（r=0.999 8）;Formazan的最大吸收波长为570～580 nm;MTT反应产物溶解检测和直接检测结果经统计学分析无显著性差异（P〉0.05）;细菌细胞数在107～109 cfu/mL范围内,MTT法与菌落计数法定量结果呈现良好的线性关系（r≥0.991 7）。     

昆虫体内多胺的高效液相色谱(HPLC)测定

建立丹磺酰氯柱前衍生HPLC快速测定昆虫体内多胺含量的方法。以C18(250mm×4.6mm,5μm)为固定相,甲醇和水为流动相,梯度洗脱,柱温40℃,流速1mL/min,荧光检测波长激发波长(Ex)280nm,发射波长(Em)515nm,测得腐胺(put)、亚精胺(spd)和精胺(spm)三者回收率分别为98.7%,99.2%和97.8%,回归方程线性良好(r值均大于0.99),分析时间为16min。该法简洁、快速、灵敏度高、重现性好,可有效分析昆虫及其他生物样品中微量多胺的含量。     

不同光环境对粘虫生长发育的影响

【目的】研究不同光环境条件对粘虫Mythimna separata生长发育存活的影响,探讨影响粘虫生存的关键光环境因子,揭示昆虫对光环境的适应能力,为粘虫发生分布情况进行准确预测提供实验依据。【方法】选取特定波长和光强的LED灯作为供试光源,以不同发育阶段粘虫为供试昆虫,测定10种供试光源条件下粘虫各发育阶段的存活与发育情况。【结果】365nm波长光源照射能显著降低粘虫卵孵化率,比对照组卵孵化率降低了50.33%;420 nm波长光源照射能显著降低粘虫幼虫存活率、幼虫化蛹率和蛹羽化率,且能延长幼虫期和蛹期,比对照组幼虫存活率、化蛹率和蛹羽化率分别降低了72.00%、65.67%和72.87%,比对照组幼虫期和蛹期分别延长了12 d和11.4 d;420 nm和460 nm波长光源照射能显著降低粘虫成虫存活率,比对照组成虫存活率分别降低了44.44%%和40.00%。【结论】光强1000lx,波长365、420、460 nm的光环境能够抑制粘虫的生长发育,不利于粘虫生存。     

绿色荧光蛋白基因在昆虫细胞中的克隆与表达

将绿色荧光蛋白(GFP)基因亚克隆到转移载体pVLneo的多角体蛋白基因(ocu)启动子下游,与杆状病素AcNPV DNA共转染昆虫细胞,通过同源重组和G418筛选,构建了整合有GFP基因的重组病毒。在昆虫细胞中表达的GFP,MW为30kDa,在荧光显微镜下呈现美丽的绿色,荧光光谱表明其激发波长395nm,发射波长509nm。Southern blot杂交证明,重组病毒的1kb EcoRI片段与GFP cDNA探针有很强的杂交信号,这是GFP基因在杆状病毒基因组中整合的直接证据。     

两种新型培养细胞数量检测技术的探索

将SRB比色法和结晶紫比色法与MTT比色法与MTT比色法在96孔板培养HeLa细胞的数量及活力测量精确度和操作方法上进行了对比。试验结果显示：SRB比色法和结晶紫比色法的最佳检测波长均为490nm。最佳染液浓度分别为0.4%和0.25%。SRB比色法的检测精确度略逊于MTT法，结晶紫比色法与MTT法无显著差异。另外，此两种检测方法的操作过程更为简便，所需时间也较短，可在一定范围内取代MTT比色法。     

雨生红球藻(Haematococcus pluvialis)的光谱特性研究

实验测定了雨生红球藻不同生长阶段的色素组成,吸收光谱,荧光光谱,并对其进行了分析。结果表明,用490nm波长激发时,雨生红球藻在绿色细胞阶段存在710nm和730nm附近的长波长荧光发射峰,而在红色细胞阶段仅存在730nm的长波长荧光发射峰,预示着雨生红球藻不同生长阶段在PSⅠ结构,组成,及其色素蛋白的排布等方面有很大差异。     

桃蚜对不同单色光趋性反应的测定

为了探讨蚜虫对不同色光选择反应的定量指标, 采用滤光片技术测定了有翅和无翅桃蚜Myzus persicae对不同波长单色光的趋性反应。结果表明: 有翅蚜对490~550 nm范围内的单色光表现出明显趋性, 其中对538.9和549.9 nm的绿偏黄色光趋性最强, 平均位移分别达25.29和22.97 cm, 其次为491.5 nm的蓝绿色光, 而对于波长576.0 nm的黄色光并没有表现出明显趋性。无翅蚜对不同单色光的趋性反应则没有明显的峰值, 最高相对平均位移仅1.41。行为测定结果与前人电生理测定的结果基本一致, 说明以位移作为小体昆虫趋光性强弱的指标是可靠的。     

新型长波长光敏剂对HL-60细胞光毒作用的参数研究

细菌叶绿素锌作为一类潜在的长波长光敏剂,具有重要生物医学研究价值。本文采用MTT法研究Zn-BCA-PDT灭杀HL-60细胞的浓度、暗孵育时间、照光时间和光波长等参数,总结对HL-60白血病肿瘤细胞进行Zn-BCA-PDT的较佳处理方案:细胞密度1×105个/mL,Zn-BCA浓度10μg/mL,暗室孵育3h,照光时间50min,光源波长为800nm。     

A study of communication specificity between cells in culture

We have examined the specificity of communication between cells in culture by co-culturing cells derived from mammalian, avian, and arthropod organisms. Both mammalian and avian culture cells have similar gap junctional phenotypes, while the insect (arthropod) cell lines have a significantly different gap junctional structure. Electrophysiological and ultrastructural methods were used to examine ionic coupling and junctional interactions between homologous and heterologous cell types. In homologous cell systems, gap junctions and ionic coupling are present at a high incidence. Also, heterologous vertebrate cells in co-culture can communicate readily. By contrast, practically no coupling (0-8%) is detectable between heterologous insect cell lines (Homopteran or Lepidopteran) and vertebrate cells (mammalian myocardial or 3T3 cells). No gap junctions have been observed between arthropod and vertebrate cell types, even though the heterologous cells may be separated by less than 10 nm. In additional studies, a low incidence of coupling was found between heterologous insect cell lines derived from different arthropod orders. However, extensive coupling was detected between insect cell lines that are derived from the same order (Homoptera). These observations suggest that there is little or no apparent specificity for communication between vertebrate cells in culture that express the same gap junctional phenotype, while there is a definite communication specificity that exists between arthropod cells in culture.     

HCV结构蛋白在昆虫细胞中的表达及病毒样颗粒的组装

本文利用Bac-to-Bac杆状病毒表达系统构建了含有丙型肝炎病毒(Hepatitis C Virus,HCV)结构蛋白编码基因的重组杆状病毒vAcHCVspl,并获得了HCV结构蛋白在昆虫细胞Sf21中的表达。HCV mRNA转录和蛋白质表达时相分析表明,感染后16h HCV结构蛋白编码基因开始转录,72h达最高峰;蛋白质表达则是在感染后48h开始,72h达到高峰。电镜观察表明vAcHCVspl感染的Sf21细胞96h时在细胞质中可见很多空泡,空泡中可见50nm的球形颗粒,为HCV结构蛋白组装的病毒样颗粒。     

Chromatic exclusivity hypothesis and the physical basis of floral color

This paper presents the results of floral spectral studies on 1275 flowers from India, Brazil, Israel, Germany, and Norway. Floral spectral reflectance from 400 to 700 nm (nm) was used to quantitatively represent ‘human-perceived’ color of flowers in Red, Green, Blue color space. Floral spectral reflectance from 350 to 600 nm was used to discern and objectively represent ‘insect pollinator-perceived’ flower colors in color hexagon. We leverage the advantage offered by ‘quantified human perception’ provided by ‘human-perceived’ floral colors to represent the distribution of floral hues and uncover the relationship between the composition of incoming solar radiation and predominant ‘human-perceived’ floral colors at the tropics and the higher latitudes. Further, the observed species-level mutual exclusivity of ‘insect pollinator-perceived’ floral colors is stated as chromatic exclusivity hypothesis. We compare ‘human-perceived’ and ‘insect pollinator-perceived’ floral colors at Trivandrum (India) and provide a physical explanation for short and long ‘wavelength triads’ of insect pollinator and human visual sensitivity respectively.     

光谱和光强度对棕榈蓟马雌成虫行为反应的影响

室内研究了光谱、光强度对棕榈蓟马雌成虫的趋、避光行为的影响。结果显示:在340—605 nm波谱内棕榈蓟马雌成虫对14个单色光刺激的趋光行为反应为多峰型。其中蓝光483 nm处峰最高,趋光反应率达34.96%,其次为绿光498—524 nm、562—582 nm、紫外光340 nm处;其避光行为反应共有3个峰,其中紫外光380 nm处最高,避光率18.08%,另外2个峰分别在橙光605 nm、紫光420 nm处。在趋光率较高的单色光(340、483、524、582 nm)和避光率较高的单色光(380、605 nm)以及白光刺激下,棕榈蓟马雌成虫的趋光率随光强增强的增强而提高,而避光率则随着光强的增强而降低;光强最弱时仍均有一定趋光率,最强时均未出现高端平台。因此:棕榈蓟马雌成虫对不同单色光具有明显的选择性,光谱和光强度对其趋光行为和避光行为有较大影响,光强度的影响作用与波长因素有关。     

The beads in the Golgi complex-endoplasmic reticulum region

The region between the rough endoplasmic reticulum (ER) and the Golgi complex has been studied in a variety of insect cell types in an attempt to find a marker for the exit gate or gates from the ER. We have found that the smooth surface of the rough endoplasmic reticulum near Golgi complex transitional elements has beadlike structures arranged in rings at the base of transition vesicles. They occur in all insect cell types and a variety of other organisms. The beads can be seen only after staining in bismuth salts. They are 10-12 nm in diameter and are separated from the membrane and one another by a clear halo giving them a center to center spacing of about 27 nm. The beads are not sensitive to nucleases under conditions which disrupt ribosomes or remove all Feulgen staining material from the nucleus. Under conditions similar to those used to stain tissue, bismuth does not react in vitro with nucleic acids. The component of the beads that stains preferentially with bismuth is therefore probably not nucleic acid.     

A C-terminal aldehyde insect kinin analog enhances inhibition of weight gain and induces significant mortality in Helicoverpa zea larvae

The first reported examples of C-terminal aldehyde analogs of an insect neuropeptide are described. They are hexapeptide insect kinin analogs Boc-VFFPWG-H and Fmoc-RFFPWG-H. Activity observed for these modified analogs in an in vitro insect diuretic assay confirms that the C-terminal aldehyde group is tolerated by an insect kinin receptor. The two analogs demonstrate greatly enhanced activity over standard C-terminal amide insect kinins in a larval weight gain inhibition assay in the corn earworm Helicoverpa zea. Treatment with Boc-VFFPWG-H led to significant increases in larval mortality at doses of 500pm (45%) and 5nm (67%). Boc-VFFPWG-H represents a lead analog in the development of novel, environmentally friendly pest insect management agents based on the insect kinin class of neuropeptides.     

单管黑白双光灯的诱虫效应

在棉田单管黑白双光灯比黑光灯诱虫量有较显著的提高,对害虫天敌昆虫的诱获量却明显比黑光灯少。本文并分析了单管黑白双光灯的诱虫原理及诱虫超过黑光灯的原因。     

Field trials measuring the effects of ultraviolet-absorbing greenhouse plastic films on insect populations

Field studies were conducted to compare insect population levels in greenhouse crops covered with plastics that block the transmission of UV light in two wavelength ranges. Crops grown in greenhouses under a plastic that blocked UV light at wavelengths of 380 nm and below had lower numbers of aphids and thrips compared with a plastic that blocked UV light at wavelengths of 360 nm and below. This is consistent with the results found for thrips in previous studies using small, completely enclosed tunnels with no plant material. The effects were not as dramatic in the commercial greenhouses, perhaps due to unfiltered light entering through the open sides of those greenhouses. There was no reduction in greenhouse whitefly, Trialeurodes vaporariorum Westwood, populations under the <380 UV-absorbing plastic compared to <360 nm UV-absorbing plastic in these field trials. This is inconsistent with results found in small, completely enclosed tunnels, where sticky traps caught a significantly higher proportion (95 +/- 2%) of released greenhouse whiteflies inside tunnels covered with <360 nm absorbing plastic compared with the <380 nm absorbing plastic. The results of these studies suggest that the type of greenhouse plastic used in a structure can affect population levels of some insect species, and may be useful tools in developing integrated pest management programs for insect management. The design of the greenhouse and amount of unfiltered light that enters the system appear to be important factors in determining the level of effect.     

太阳能灭虫器对吐鲁番葡萄产区害虫的诱捕效果及对天敌安全性评价

本文研究了FWS-DBL-1新型太阳能灭虫器在320～580、360、400、460和520 nm 5种不同波长下对吐鲁番葡萄产区害虫的诱捕效果及对天敌安全性。结果表明:在5种波长下,灭虫器诱捕昆虫的种类相似,以鞘翅目、鳞翅目、半翅目、脉翅目、双翅目、直翅目和膜翅目等为主。诱捕的主要昆虫是蜉金龟Aphodius sp.、白云斑鳃金龟Polyphylla alba vicaria Semenov、额喙丽金龟Adoretus nigriforns Steven、毛喙丽金龟Adoretus hirsutus Ohaus、绣罗夜蛾Leucanitis picta Christoph和淘赏夜蛾Catocala puerpera Giorna等。其中:灭虫器在400 nm波长下的诱捕量最多,其次是320～580 nm和360 nm波长。综合分析比较:400 nm波长对鞘翅目害虫的诱捕效果最好,360 nm波长对鳞翅目和双翅目害虫的诱捕能力最强,520 nm波长对半翅目害虫的诱捕效果最好。对天敌的安全性研究表明:FWS-DBL-1太阳能灭虫器对天敌有一定的诱捕作用,益害比为1∶4～1∶5。诱捕量较多的是步甲科和草蛉科的天敌,占天敌总诱捕量的85.82%。在这5个波长中,360 nm波长下,灭虫器对天敌的诱捕作用最小,对天敌的安全性相对较高。     

昆虫细胞程序性死亡的研究进展

在昆虫发育和抵抗病原微生物的入侵过程中,细胞凋亡与自噬性死亡现象十分常见。昆虫细胞凋亡的研究已经取得了许多的成果,但是有关细胞自噬程序性死亡的研究还正在深入。昆虫细胞凋亡的信号通路至少有3条：一条类似于线虫细胞的凋亡信号通路,另一条类似于哺乳动物细胞的凋亡信号通路, 还有一条不依赖于胱天蛋白酶的凋亡信号通路。在昆虫的多种组织细胞中,细胞凋亡与自噬程序性死亡在信号通路上存在互串(cross talking),可以相互促进、抑制或替代。了解昆虫细胞程序性死亡对防治害虫具有一定的意义。