腺病毒与癌症的研究

有些病毒可以致癌,如HPV致富颈癌,“乙肝”病毒、“丙肝”病毒致肝癌等,而有些病毒如腺病毒（Adenovirus）却可以作为基因工程的载体得到实际应用。我国深圳一家基因技术公司研究人员将人P53基因通过腺病毒重组,其重组体制成注射液（商品名叫“今又生”gendicine）,用于治疗头颈部鳞病如鼻咽癌等试验取得成功,已有数百至数千人接受治疗,其产品已商品化生产。     

Ad-hBNP重组腺病毒的构建

目的:构建携带人BNP cDNA片段高效重组腺病毒Ad-hBNP,为实验提供研究工具.方法:从人心肌组织提取的RNA,用RT-PCR方法中获得hBNP扩增片段,与pUCm-T载体连接构成pUC-hBNP重组质粒;用KpnI、SalI分别双酶切pUC-hBNP和pAd-Track-CMV,将目的片段插入pAd-Track-CMV构建重组质粒pAdTrack-CMV-hBNP;pAdTrack-CMV-hBNP重组质粒经Pmcl酶切线性化后,电转法转入含有腺病毒骨架质粒的E.coli BJ5183感受态细胞中,同源重组获得重组质粒pAdEasy-hBNP;经BamHI、PacI酶切鉴定及基因序列检测后,重组成功的pAdEasy-hBNP经阳离子脂质体法转染HEK293T细胞,经过包装、扩增和纯化后,测定病毒滴度,电镜检测病毒形态.结果:转染HEK293T细胞5-6天GFP呈"彗星"状;重组腺病毒滴度为1.1×1012V.P/ml;电镜检测重组腺病毒为多面体结构.结论:应用Ad-Easy缺陷性腺病毒载体系统成功构建重组腺病毒Ad-hBNP,为进一步基因治疗研究提供分子生物学工具.     

犬瘟热病毒H蛋白基因重组腺病毒的构建及免疫效果评估

研究选取犬瘟热病毒H蛋白基因为目的基因,利用反转录聚合酶链式反应(RT-PCR)、酶切、连接等方法构建出重组人5型腺病毒穿梭质粒和骨架质粒,两者共转染293AD细胞并包装出重组腺病毒。通过电镜负染、酶切鉴定、Western blot及一步生长曲线测定等方法进行病毒的生物学特性鉴定,证明犬瘟热病毒H蛋白基因重组腺病毒构建正确。犬分别肌肉接种CDV疫苗毒和重组腺病毒,并检测免疫后第14,28,42,56,70,84d时犬血清中抗CDV中和抗体的变化情况。结果显示,犬免疫重组腺病毒后体内产生的抗CDV平均中和抗体水平高于对照组,滴度最高达2-8。研究表明,正确构建的重组腺病毒具有良好的免疫原性,诱导产生的抗犬瘟热病毒中和抗体达到犬最低免疫保护水平,为进一步研发犬瘟热病毒活载体疫苗提供理论依据。     

共表达甲型流感病毒M1和HA双基因的重组腺病毒的构建及鉴定

以我国分离的首株人H5N1亚型禽流感病毒(A/Anhui/1/2005)作为研究对象,通过引入内部核糖体进入位点序列(IRES),构建共表达H5N1亚型禽流感病毒膜蛋白基因M1和HA的重组腺病毒。PCR扩增禽流感病毒H5N1亚型M1和HA基因的全长可读框片段,先后亚克隆入pStar载体,然后扩增M1-IRES-HA片段并将其插入穿梭载体pShuttle-CMV,再与pAd-Easy载体在BJ5183菌中通过同源重组产生重组腺病毒载体,转化293细胞,包装出重组腺病毒Ad-M1/HA。将Ad-M1/HA感染293细胞,可观察到明显细胞病变效应,用免疫荧光及Western-blot方法均检测到M1和HA基因的表达。共表达M1和HA双基因的重组腺病毒的成功构建为开发新型重组腺病毒流感疫苗奠定了基础。     

质粒纳米乳剂的制备与阴离子交换色谱测定方法

以纳米乳剂为载体包裹草原兔尾鼠卵透明带3DNA疫苗,制备获得了纳米乳剂DNA疫苗,并对其进行了质量评价。采用界面乳化法制备质粒纳米乳剂,用电子显微镜测定了其粒径及其分布,根据质粒的带电特性,利用强阴离子Q SepharoseTMXL色谱柱分离纳米乳剂和游离质粒,建立了强阴离子交换柱质粒纳米乳剂包封率的快速测定方法。结果表明:制备的纳米乳剂DNA疫苗的平均粒径为(23±10)nm,包封率为80.5%。选择0.05mol/L的Tris-HCl为平衡液,流速为0.7mL/min,紫外检测波长260nm,柱温30℃,进样量为2mL的实验条件,质粒的含量和峰面积的线性关系良好(r=0.9983),加样回收率在95%以上,该方法简便快速、灵敏度高,重复性好,可用于纳米乳剂DNA疫苗包封率的快速测定。     

人肝癌靶向性CD80基因表达重组腺病毒载体的构建及鉴定

目的:构建人肝癌靶向性CD80基因重组腺病毒载体。方法:首先,从人基因组DNA中克隆AFP增强子、启动子,利用腺病毒穿梭质粒pShuttle和pShutte-CMV,采用AFP增强子替换CMV启动子,构建含有AFP启动子和增强子的穿梭质粒,命名为pShuttle-AFP。将人CD80基因克隆到pShuttle-AFP的AFP增强子和启动子之后,构建pShuttle-AFP-CD80质粒。再将其与腺病毒骨架质粒pAdEasy-1共转化E.coliBJ5183,利用同源重组构建腺病毒质粒pAd-AFP-CD80。以获得的重组子转染AD293细胞后制备重组腺病毒,并对重组的病毒进行鉴定和病毒滴度测定。结果:测序结果与Genebank中公布的AFP启动子、核心增强子和CD80基因序列相符。双酶切结果与预期结果相符。PacI酶切结果与目的结果一致。穿梭质粒pShutte-AFP-CD80和腺病毒质粒pAd-AFP-CD80经酶切和PCR鉴定均获得期望大小的目的片段。结论:成功的构建了人肝癌靶向性pAd-AFP-CD80腺病毒载体,为CD80基因在肝癌靶向性治疗的研究奠定了基础。     

长春新碱阳离子纳米结构脂质载体及其小肠吸收的研究

目的:硫酸长春新碱作为一种细胞毒型抗肿瘤药物,临床上多用其注射剂,虽应用广泛,但存在较多缺点,如药物半衰期短,代谢速率快以及毒副作用明显。本文目的是制备包载长春新碱和十二烷基磺酸钠的阳离子纳米结构脂质载体,并对其进行评价。方法:用复乳挥发法制备出目标脂质纳米粒;利用激光粒度仪对其粒径及zeta电位进行检测;利用高效液相色谱法对其包封率和载药量进行测定;透析法检测纳米粒的体外释放行为;用小肠吸收法评价纳米粒的促进吸收作用。结果:制得的纳米粒的平均粒径为(192.4±4.14)nm,多分散系数(PDI)为0.184±0.015,包封率为32.28%,Zeta电位为(30.6±4.09)m V,载药量为(1.56±0.10)%;体外释放实验显示在pH=7.4的中性释放介质中,硫酸长春新碱脂质纳米粒表现出缓释特性;小肠吸收实验表明十二烷基磺酸钠的加入和阳离子纳米粒的修饰可提高小肠对药物的吸收。结论:阳离子硫酸长春新碱纳米结构脂质载体具有缓释效果,并可以促进小肠对药物的吸收。     

一种改进的重组腺病毒载体的包装和克隆纯化方法

目的:重组腺病毒载体是目前使用最多的病毒载体之一。常用的AdEasyTM包装系统在制备条件复制型腺病毒时易混有野生型腺病毒或可复制型腺病毒(RCA),在HEK293细胞中包装出的重组腺病毒必须经过2～3轮噬斑的筛选,费时费力。本实验拟对常规包装方法进行改进。方法:将腺病毒载体质粒转染至HEK293细胞过程中的液体培养基更换为琼脂糖凝胶的混合培养液,因为凝胶形成的阻碍,包装出的病毒不能通过细胞-培养液-细胞的方式进行扩散,只能依靠细胞-细胞的方式来扩展而形成病灶(噬斑);然后随机挑选单个噬斑进行PCR鉴定,筛选出特异的目标病毒。结果:采用常规方法在HEK293细胞中初包装出的腺病毒,同源重组产生的RCA已被检测到。采用改进方法制备的重组腺病毒,能排除背景病毒的干扰,使重组腺病毒载体的包装和克隆纯化一步完成;利用此病毒初步在体外实现了对人肝癌细胞的特异性杀伤。结论:改进的方法是一种高效、简便、快捷地包装并纯化重组溶瘤腺病毒的方法,采用该方法包装的重组溶瘤腺病毒能满足实验的需要。     

应用Ad Easy腺病毒载体系统构建人肌浆网钙离子ATP酶2a(SERCA2a)

目的:利用Ad easy腺病毒表达系统构建含人肌浆网钙离子ATP酶2a(SERCA2a)基因重组腺病毒,并在HEK293细胞中扩增制备重组腺病毒.方法:将人SERCA2a基因全长c DNA(3700bp)插入到腺病毒穿梭载体pAdTrack-CMV,成功构建pAd-TrackCMV SERCA2a重组质粒,经Pme I酶切线性化,采用电击法转入到已含Ad easy质粒的电感受态菌BJ5183进行重组.挑选同源重组质粒,Pac I酶切线性化转染HEK293细胞包装成重组腺病毒颗粒,荧光检测有绿色荧光蛋白表达.将重组病毒和SD大鼠心肌细胞共培养,western-blot检测SERCA2a可以在大鼠心肌细胞过表达且影响了胞内SERCA2a的活性.结果:成功包装含人SERCA2a基因的重组腺病毒,并可以有效感染SD大鼠心肌细胞.结论:利用新型腺病毒载体在短时间内成功构建了携带有人SERCA2a基因的腺病毒,为以后进一步研究人SERCA2a基因治疗提供了新途径.     

重组腺病毒Ad-GFP的纯化

目的：用色谱法纯化制备携带绿色荧光蛋白基因的重组腺病毒。方法：采用5L生物反应器悬浮培养HEK-293N3S细胞进行病毒的扩增,冻融法裂解细胞,通过超滤、Source15Q离子交换层析和Sepharose4Fast Flow凝胶过滤层析进行分离纯化,采用分光光度法和高效液相色谱法分析重组腺病毒产品的纯度,采用组织培养半数感染剂量方法测定病毒的感染滴度。结果：通过两步色谱层析得到产品,经分光光度法分析,其D260nm/D280nm比值为1.21,高效液相色谱法分析其纯度达96.5%,产品滴度为1.0×1011U/mL,病毒颗粒数为1.76×1012/mL,比活性为5.68%。结论：确定了稳定可行的重组腺病毒色谱分离工艺,得到的产品在纯度、滴度和比活性等方面均符合国家食品药品监督管理局的标准。     

A new proposal for measurement of the adjusted mean crowding through consideration of size variability in habitat units

The mean crowding has previously been measured under the assumption that all quadrats or habitat units have the same size, even though the actual habitat units such as seeds or leaves are generally variable in size. A new index, ‘adjusted mean crowding’, which is adjusted for this variability can be given as where Q is the total number of habitat units in the whole area, x_j the number of individuals in the jth habitat unit, and a_j is defined as the ‘relative size’ of the jth habitat unit, i.e. a_y=y_y/(∑y_j/Q) where y_j is the actually measured size of the jth habitat unit. It is expected that and for the uniform distribution and the random distribution ‘per unit size’, respectively. The comparison between and regressions ( analysis) for the egg distribution pattern of Callosobruchus chinensis or C. maculatus proved that the regression is biased by a positive correlation between the egg number per seed and seed size rather than by a density-dependent change in the ovipositional behavior.     

Relationships of latitude,altitude, and body size to litter size and mean annual production of offspring inPeromyscus

Litter size was positively correlated with latitude and altitude but not with production of offspring or with body size in Peromyscus. Increased litter size in northern populations probably reflects shortening the breeding season by climate and not a greater mortality rate at northern latitudes compared to southern latitudes. Production of offspring was negatively correlated with body size but not with latitude, altitude, or litter size. This is probably due to larger species living longer and taking longer to mature.     

Of mice and wrens: the relation between abundance and geographic range size in British mammals and birds

We examine the relation between population size and geographic range size for British breeding birds and mammals. As for most other assemblages studied, a strong positive interspecific correlation is found in both taxa. The relation is also recovered once the phylogenetic relatedness of species has been controlled for using an evolutionary comparative method. The slope of the relation is steeper for birds than for mammals, but this is due in large part to two species of mammals that have much higher population sizes than expected from their small geographic ranges. These outlying mammal species are the only ones in Britain to be found only on small offshore islands, and so may be exhibiting density compensation effects. With them excluded, the slope of the abundance–range size relation for mammals is not significantly different to that for birds. However, the elevation of the relation is higher for mammals than for birds, indicating that mammals are approximately 30 times more abundant than birds of equivalent geographic range size. An earlier study of these assemblages showed that, for a given body mass, bats had abundances more similar to birds than to non-volant mammals, suggesting that the difference in abundance between mammals and birds might be due to constraints of flight. Our analyses show that the abundance–range size relation for bats is not different for that from other mammals, and that the anomalously low abundance of bats for their body mass may result because they have smaller than expected geographic extents for their size. Other reasons why birds and mammals might have different elevations for the relation between population size and geographic range size are discussed, together with possible reasons for why the slopes of these relations might be similar.     

An evolutionary correlate of genome size change in plethodontid salamanders

Variation in the amount of nuclear DNA, the C-value, does not correlate with differences in morphological complexity. There are two classes of explanations for this observation, which is known as the ''C-value paradox''. The quantity of DNA may serve a ''nucleotypic'' function that is positively selected. Alternatively, large genomes may consist of junk DNA, which increases until it negatively affects fitness. Attempts to resolve the C-value paradox focus on the link between genome size and fitness. This link is usually sought in life history traits, particularly developmental rates. I examined the relationship among two life history traits, egg size and embryonic developmental time and genome size, in 15 species of plethodontid salamanders. Surprisingly, there is no correlation between egg size and developmental time, a relationship included in models of life history evolution. However, genome size is positively correlated with embryonic developmental time, a result that is robust with respect to many sources of variation in the data. Without information on the targets of natural selection it is not possible with these data to distinguish between nucleotypic and junk DNA explanations for the C-value paradox.     

A critique of the «increasing population density effect»

The «increasing population density effect» (IPDE) proposed by Macchiarelli and Bondioli (1986) represents one of the most recent attempts to account for dental reduction in modern human populations. Under the IPDE, the marked reduction in tooth size observed in post-Pleistocene human groups is seen as merely a side-effect of a more general reduction in body size, which resulted from an increase in population density. The model is dependent upon a strong correlation between tooth size and body size, which after numerous attempts has yet to be convincingly demonstrated in humans. This paper argues that the IPDE neglects the negative consequences on individual fitness of teeth which are too large to fit into diminishing jaws or are more susceptible to pathology, and that the worldwide reduction of tooth size is the result of selection for smaller teeth due to shifts to a softer and/or more cariogenic diet. Although increased population density may have intensified this selective effect by decreasing general fitness by lowering resistance to oral infections, it was not the primary cause of dental reduction. All proposed mechanisms of dental reduction, however, are in need of additional testing, and possible directions are offered for future studies of the phenomenon.     

Population distributions of plant size and light environment of giant ragweed (Ambrosia trifida L.) at three densities

Summary Plots in a naturally occurring population of giant ragweed (Ambrosia trifida L.) near Ames, Iowa, USA were left unthinned (high density,=693 plants/m²) or were thinned in early June 1989 to create low and medium densities of 10 and 50 plants/m². Size and light environment of individual plants were measured at monthly intervals from June to September. By September, low density plants had 15 times greater biomass/plant and 30 times greater leaf area/plant than high density plants, although biomass and leaf area per unit land area decreased with decreasing density. Plants at high density allocated more biomass to stem growth, but plants at medium and low density had successively higher leaf area ratios, higher potential photosynthetic rates, higher allocation to leaves, and higher growth rates. Average light on leaves decreased with increasing density and also decreased over the growing season in the low and medium densities. The distribution of light environments of individual plants was non-normal and skewed to the left in most months, in contrast to the rightwards skew of distributions of plant size parameters. Inequality in the distributions, as measured by coefficient of variation and Gini coefficients, increased over most of the growing season. There was little effect of density on inequality of stem diameter, height, or estimated dry weight, but inequality in reproductive output greatly increased with density. There was greater inequality in number of staminate flowers produced than in number of pistillate flowers and seeds produced. Path analysis indicated that early plant size was the most important predictor of final plant size and reproductive output; photosynthesis, conductance, and light environment were also significantly correlated with size and reproduction but usually were of minor importance. Variation in growth rate apparently increased inequality in plant size at low density, whereas belowground competition and death of smaller plants may have limited increases in inequality at high density.     

Comparative analysis of breeding rates of rural and urban grey starling colonies in Tokyo area: The second report (Part 2)

1. The rates of clutch size, brood size and brood-size fledged recorded during the period 1956≈63 (2 years interrupted) in the rural and the urban colony of the grey starling were analysed comparatively.
2. The clutch size was significantly larger in the urban than in the rural one and the brood size was also larger significantly in the urban population. But, the difference in brood-size fledged was insignificant.
3. This reflected that the hatching rate was similar in both colonies (the rates in total differed but possible unusual rate was included in the clutch size of 5 eggs) and this may be determined physiologically but not depending upon food supply since smaller clutches showed higher hatching rate.
4. However, the fledging rate was higher in the rural and lower and more variable in the urban colony. This is apparently due to food which was nutritive animal food in the rural but largely mixed with fruits in the urban.
5. The fledging rate was rather irregularly variable with brood size suggesting that this is dependent upon parents' adaptability in feeding. However, the rate of 100% fledging becomes higher from brood sizes of 6 and less chicks.
6. Thus, in general larger clutch and brood sizes produced larger absolute numbers of chicks and chicks fledged respectively. But, 5 was the most frequent size in the clutch and brood sizes and 4 in the brood-size fledged.
7. From the above, the ecological evolution of urban population was discussed.

     

Factors affecting body size variation within a population of an herbivorous lady beetle,Henosepilachna niponica (Lewis)

Size variation in newly-emerged adults was examined in two different local populations of an herbivorous lady beetle, Henosepilachna niponica, for 1976–80. Mean adult size of both sexes changed rather synchronously in the two populations over 5 years. Body size of adult beetles apparently decreased with increasing leaf damage of the plants on which they developed. Adult beetles which emerged late in the season, associated with increasing food deterioration, were smaller than those which emerged early. Ecological consequences of adult size variation is discussed in terms of oviposition site selection.