不同倍性泥鳅鳍细胞系的建立及生物学特性分析简

采用组织块培养法启动二倍体、三倍体和四倍体泥鳅鳍组织细胞原代培养,采用胰蛋白酶消化法传代,目前二倍体、三倍体和四倍体细胞已经分别传至59代、68代和68代。三种细胞均为成纤维细胞样细胞。鳍组织无菌处理方法是:先用质量浓度为10%的碘伏浸泡15min;后用青霉素和链霉素的混合液(500 IU/mL青霉素,500μg/mL链霉素)浸泡30min;原代培养和早期传代培养所用的培养基为DMEM/F12,添加体积分数为20%的胎牛血清、10 ng/mL人碱性成纤维细胞生长因子(bFGF)、20 ng/mLI型胰岛素样生长因子(IGF-I)以及10μg/mL硫酸软骨素。30代以后所用培养基为20%FBS-DMEM/F12。细胞培养在25℃、5%CO2培养箱中。在此条件下,二倍体、三倍体和四倍体的倍增时间分别为48.43h、36.01h和41.45h;二倍体、三倍体和四倍体的特征染色体数目分别为50条、75条和100条;测定的二倍体、三倍体和四倍体细胞及核的体积比分别为1︰1.37︰2.37和1︰1.53︰1.97;细胞经液氮冷冻保存60d后,解冻复苏后测定存活率分别为(80.88±1.38)%、(84.48±1.13)%、(81.57±1.28)%。不同倍性的泥鳅细胞系的建立丰富了鱼类细胞系的种类,为揭示多倍体鱼类生长、遗传等机制打下基础。     

不同倍性大青杨的光合特性及叶片解剖结构比较

通过对二年生二倍体、三倍体和四倍体大青杨叶片结构和光合特性的研究,探讨不同倍性大青杨生长性状差异的原因。结果表明：不同倍性大青杨的净光合速率（Pn）、光饱和点（LSP）、光补偿点（LCP）等光合指标差异显著。三倍体的最大净光合速率（Pmax）高出二倍体21%。叶片厚度、表皮细胞厚度、栅栏组织厚度、栅栏组织与海绵组织厚度的比值,都是三倍体和四倍体大青杨比二倍体高,且均呈现增加的趋势。其中,四倍体大青杨表皮细胞最厚,上下表皮分别高出二倍体64%和17%。三倍体大青杨的栅栏组织厚度及栅栏组织与海绵组织厚度的比值最大,比值高出二倍体50%。通过叶片光合特性和解剖结构比较证明,三倍体和四倍体大青杨对光的适应性和光合作用能力强于二倍体。     

不同倍性鱼垂体细胞和超微结构比较

对繁殖季节和繁殖季节后的二倍体红鲫(Carassius auratus red var.)、三倍体湘云鲫以及四倍体鲫鲤脑垂体细胞的显微和超微结构以及组织化学特性进行了比较研究. 结果表明, 3种鱼脑垂体中都存在6种不同类型分泌细胞, 但是不同倍性鱼的垂体细胞大小存在明显差异, 就同一类细胞体积而言, 四倍体鱼垂体细胞大于三倍体垂体细胞, 三倍体垂体细胞大于二倍体垂体细胞. 在繁殖季节, 四倍体鲫鲤中腺垂体的GTH细胞所占比例最大, 其次是二倍体红鲫, 最少的是三倍体湘云鲫, 该现象与四倍体鲫鲤的性腺发育提前、三倍体湘云鲫不育有关联; 另一方面, 三倍体湘云鲫中腺垂体中STH细胞所占比例最高, 其次是二倍体, 最少的是四倍体鲫鲤, 这与三倍体湘云鲫生长速度最快、四倍体鲫鲤生长速度相对缓慢有关. 另外, 三倍体湘云鲫中腺垂体GTH细胞中的分泌颗粒和分泌小球在繁殖季节没有大量排出, 而四倍体鲫鲤和二倍体红鲫明显有大量排出, 说明三倍体湘云鲫的不育性与GTH中的激素不排出有关. 以上结果说明, 不同倍性鱼类在垂体结构方面表现出的差异与它们的生长速度、性腺发育等方面具有关联性.     

二倍体鲫鲤F2产生不同倍性卵子的证据

在检测到鲫鲤F2产生3种不同大小(直径分别为0.13 cm,0.17cm和0.2 cm)类型的卵子基础上,进行了F2(♀)×红鲫(♂)及F2(♀)×四倍体鲫鲤(♂)的交配实验.通过染色体计数和流式细胞仪分析,在F2(♀)×红鲫(♂)后代中获得了四倍体、三倍体、二倍体鱼;在F2(♀)×四倍体鲫鲤(♂)后代中获得了四倍体和三倍体鱼.这两个交配组合后代中出现的不同倍性的鱼类为证明鲫鲤F2能产生三倍体、二倍体和单倍体卵子提供了进一步证据.F2(♀)×红鲫(♂)中雄性四倍体鱼的存在说明在四倍体后代中存在基因型为XXXY的个体.对上述两个交配组合后代的四倍体鱼和三倍体鱼的性腺结构观察表明四倍体鱼是可育的,而三倍体鱼是不育的.作者认为鲫鲤F2能够产生二倍体和三倍体卵子与核内复制机制和生殖细胞的融合有关.     

人工诱导白鲫(♀)×红鲤(■)异源四倍体鱼的初步研究

人工诱导鱼类多倍体是动物染色体工程的重要课题。鉴于三倍体鱼的不育性和成活率高、生长快等特点,国内外采用理化手段直接诱导三倍体鱼已在三棘刺鱼(Gasterosleus aculeatus(L.))、鲽鱼(Pleuronectes platessa)、鲤鱼(Cyprinus carpio L.)、草鱼(Ctenopharyngodonidellus)、草鱼(♀)×团头鲂(Megalobrama amblycephala)(♂)杂种,以及白鲢(Hypophthalmichthysmolitrix),等十多种鱼获得成功。有的已开始用于实践并取得较明显的经济效益。尽管如此,诱导三倍体鱼的技术毕竟不是一劳永逸的方法。理想的方法是人工诱导获得能育的异源四倍体(或同源四倍体),再与二倍体鱼杂交得三倍体鱼,从而为大规模生产三倍体鱼提供一条行之有效的捷径。陆仁后等(1982),用草鱼尾鳍细胞诱导成同源四倍体细胞并移至泥鳅去核卵得到心跳期     

橡胶树四倍体来源鉴定及不同倍性叶片解剖结构比较

利用25对SSR引物,采用基于PCR的毛细管电泳法鉴定14株人工诱导四倍体的来源,并采用2种切片方法比较橡胶树不同倍性叶片的厚度差异。结果表明,14株四倍体中9株为同一无性系,另有5株为同一无性系或全同胞子代,14株四倍体的同源二倍体的亲本之一为热研73397。橡胶树二、三、四倍体间的叶片、上表皮、栅栏组织、海绵组织的厚度差异极显著(P0.01),且随着倍性的增加而增加。二、三、四倍体鲜叶厚度分别为159.98±16.04μm、188.52±16.29μm、204.21±16.45μm,叶片石蜡切片厚度分别为139.94±13.22μm、169.53±15.07μm、180.84±26.71μm。以鲜叶厚度177.81μm或石蜡切片厚度160.70μm为临界值可以区分人工体细胞诱导四倍体橡胶树获得的二、四倍混合群体中的二倍体和四倍体。不同倍性的叶片结构紧密度和栅海比大小排序均为:三倍体四倍体二倍体,叶片结构疏松度大小排序为:二倍体四倍体三倍体。本研究结果为橡胶树多倍体资源的鉴定和多倍体育种策略选择提供了参考。     

镉胁迫对大青杨不同倍体的生长及生理生化的影响

以大青杨不同倍体当年生扦插苗为研究对象，用不同浓度CdCl₂溶液对其进行镉胁迫处理，研究不同镉浓度对大青杨3种倍体的生长和生理生化的影响。结果表明：镉胁迫对大青杨3种倍体生长有显著的抑制作用，随着镉浓度的增加，与对照苗（二倍体，CK）相比受胁迫苗的苗高和地径都显著下降；叶绿素含量先升高后下降；大青杨二倍体和四倍体的叶片含水量随镉浓度增加呈下降趋势，而三倍体叶片含水量则在低浓度时增加，高浓度时降低；三倍体和四倍体的过氧化氢酶（CAT）和超氧化物歧化酶（SOD）的酶活性在镉浓度低时增加，在镉浓度高时降低，而二倍体中酶活性则呈下降趋势。这说明镉浓度低时3种倍体都有较好的耐受性，而在高浓度胁迫下三倍体含水量、叶绿素含量、CAT的活性和丙二醛（MDA）含量比四倍体分别高5.47%、25.47%、8.59%、28.47%，比二倍体分别高23.47%、44.63%、17.23%、31.48%，说明大青杨三倍体在镉胁迫下细胞膜损伤最小，抗氧化能力最强，有较好的抗重金属镉的能力。本研究为在重金属污染地区进行育种工作提供了理论依据。     

人类染色体数目畸变类型和机理

正常人体细胞中有４６条染色体,其中２３条来自父方,另２３条来自母方,即含有两个染色体组,称为二倍体（２ｎ）。每号染色体都是成对存在的,如果某号染色体出现了单条、多条或染色体组成倍地增减,将形成染色体数目畸变。１　畸变类型１．１　整倍性变异　即染色体组成倍地增减,若整个染色体组增加可形成多倍体,整个染色体组减少则形成单倍体。在人类中,单倍体或四倍以上的多倍体尚未见报道,三倍体和四倍体多发现于自然流产的胎儿中。１）三倍体　三倍体细胞中有３个染色体组（３ｎ）,即每一号染色体都有３条（３ｎ＝６９）。人类…     

白鲫（♀）×红鲫（♂）异源四倍体鱼的倍性操作及其生殖力的研究

采用热休克调控技术诱导出103尾第一代白鲫（♀）×红鲫（）异源四倍体鱼,并对其生殖力进行了研究。1或2龄的四倍体雄鱼能产生精子,而雌鱼不能产生正常的卵。将异源四倍体雄鱼与二倍体白鲫雌鱼交配产生倍间三倍体鱼,染色体检查证明是整三倍体（3N＝150）,但其受精率很低（11．4—51．3％,平均32．4％）．网箱养殖结果表明,倍间三倍体白鲫的生长速度比白鲫快30％以上,雌雄均不育．并用冷休克处理回收异源四倍体4N（）×白鲫2N（♀）受精卵的第二极体产生了新的四倍体鱼．文中还对第一代异源四倍体鱼的批量生产、生殖能力以及异源三倍体鱼的生产应用进行了讨论．     

家蚕胚胎细胞系BmE-SWU2的建立及其生物学特性研究

家蚕反转期胚胎组织经过一年多的原代培养,建立了BmE-SWU2细胞系。该细胞系细胞呈短梭形或圆形,细胞较小,属上皮型贴壁生长细胞系;细胞生长速度快,铺平率高于80%,繁殖力强,细胞群体倍增时间为51.8 h。BmE-SWU2细胞系的二倍体细胞（2n=56）比例达到89.76％,属二倍体细胞系,其染色体具有鳞翅目昆虫染色体的典型特征,中期染色体呈短杆状或颗粒状。BmE-SWU2细胞系对BmNPV高度敏感,TCID₅₀为1.581×10^-7。     

Characteristics of sperm of polyploid Prussian carp Carassius gibelio

Wild-captured 16 diploid, five triploid and one tetraploid Prussian carp Carassius gibelio males produced motile haploid, aneuploid (1·5n) and haploid to aneuploid (> 2n) sperm, respectively, in similar concentration and with the lowest percentage of live spermatozoa in sperm for the tetraploid male (mean ± s . d . 83·03 ± 1·76%; P < 0·05) compared to diploid and triploid males (97·37 ± 1·11% and 96·70 ± 1·45%; P > 0·05).     

Pollen competition as a unilateral reproductive barrier between sympatric diploid and tetraploid Chamerion angustifolium

Speciation requires the evolution of barriers to gene exchange between descendant and progenitor populations. Cryptic reproductive barriers in plants arise after pollination but before fertilization as a result of pollen competition and interactions between male gametophytes and female reproductive tissues. We tested for such gametic isolation between the polyploid Chamerion angustifolium and its diploid progenitor by conducting single (diploid or tetraploid) and mixed ploidy (1 : 1 diploid and tetraploid) pollinations on both cytotypes and inferring siring success from paternity analysis and pollen-tube counts. In mixed pollinations, polyploids sired most (79%) of their own seeds as well as those of diploids (61%) (correcting for triploid block, siring success was 70% and 83%, respectively). In single donor pollinations, pollen tubes from tetraploids were more numerous than those from diploids at four different positions in each style and for both diploid and tetraploid pollen recipients. The lack of a pollen donor x recipient interaction indicates that the tetraploid siring advantage is a result of pollen competition rather than pollen-pistil interactions. Such unilateral pollen precedence results in an asymmetrical pattern of isolation, with tetraploids experiencing less gene flow than diploids. It also enhances tetraploid establishment in sympatric populations, by maximizing tetraploid success and simultaneously diminishing that of diploids through the production of inviable triploid offspring.     

Chromosomal changes in eight cell strains of the tasmanian rat-kangaroo,Potorous tridactylis (Marsupialia). Male and female heart fibroblasts cultured in vitro

Eight cell strains, derived from the hearts of a male and a female Motorous, were followed during their in vitro cultivation.All three male cell strains started as normal diploid cell strains. One of them, 2Hpo stayed diploid until passage 59, when the cells were frozen and stored at –96°C. After a period of growth retardation, that lasted two months, 1Hpo showed aneuploidy, the cells having 22–24 chromosomes. The cells with 23 chromosomes formed about 30% of the population. These cells predominantly missed the chromosomes 2, 3 and Y₁, from the tetraploid set. In the other cells no consistent pattern was observed. The cell strain 4Hpo did not show aneuploidy after three months of growth retardation. At the last passage (nr. 24) before death, it showed 25% diploid cells and 40% tetraploid cells.Three female strains were initiated on fibrin clot, two on plasm clot. No differences in growth and chromosomal changes, due to the different embedding media, were observed. All the strains started as diploid (2n=12) cell strains. The chromosomal changes that occurred showed many differences. Three cell strains (5Hf, 7Hp, 52Hf) died without showing any pattern in the aneuploid cells. One cell strain (53Hf) showed an aneuploid cell population with a stemline of 14 chromosomes. The cell strain (8Hp) showed different changes in ploidy. After 50 passages, it changed from diploid to aneuploid (19 chromosomes per cell in the stemline). Twenty passages later diploid cells started to dominate the population again (80% at passage 85). Then a new aneuploid population with a stemline of 18 chromosomes (30% triploid cells) arose, and the strain survived as a permanent line.The work was carried out, in part, under the association between Euratom and the University of Leiden, contract Nr. 052-64-I BIAN, and it also received support form the Foundation for Basic Medical Research (FUNGO).     

Arrangement of spindle apparatus in mitoses of different ploidy

In non-hypotonically treated mitoses from tissue cultures of Microtus agrestis, both the constitutive heterochromatin of the sex chromosomes and the spindle apparatus were stained by the Giemsa C-banding technique. By means of counting the heterochromatic chromosomes, we determined the cell ploidy and studied the number of centrioles and the spindle arrangement of diploid, triploid, tetraploid and octoploid mitoses. Diploid and triploid prophases contained 2 centrioles in most cases, tetraploid prophases 4, binucleate cells with 2 diploid nuclei likewise 4 and binucleate cells with 2 tetraploid nuclei 8 centrioles. Nearly 99% of diploid and triploid metaphases were bipolar. Of the tetraploid metaphases only 45% were bipolar, 29.5% tripolar, 7.5% quadripolar and 18% formed as a parallel mitosis. In all examined binucleate cells that had had an asynchronous DNA synthesis, a multipolar mitosis was found.     

Effect of antibiotics on development in vitro of hamster pronucleate ova

Antibiotics are commonly added to embryo culture media, but effects on embryo development have not been examined thoroughly. Hamster ova were used to investigate whether penicillin, streptomycin or gentamicin affect embryo development in vitro. Ova were collected 10 h post activation by spermatozoa in vivo and cultured in five treatments: 1) Control: chemically-defined medium HECM-9 with no antibiotics; 2) HECM-9 with 100 IU/mL penicillin; 3) HECM-9 with 50 microg/mL streptomycin; 4) HECM-9 with 10 microg/mL gentamicin and 5) HECM-9 with both 100 IU/mL penicillin and 50 microg/mL streptomycin. Individually, penicillin, streptomycin and gentamicin did not affect embryo development to the 8-cell stage at 58 h post oocyte activation, or morula/blastocyst stages, or blastocysts alone at 82 h post activation. However, when penicillin and streptomycin were both present in the culture medium the percentages of 8-cell embryos at 58 h and blastocysts at 82 h were significantly lower than the control. No antibiotic treatment improved hamster embryo development in vitro. We caution against the use of penicillin and streptomycin together for hamster embryo culture, and show that it is not necessary to include any antibiotics in embryo culture media for up to 72 h if proper sterile technique is used with an oil overlay.     

Commercial-scale sperm cryopreservation of diploid and tetraploid Pacific oysters, Crassostrea gigas

Cryopreservation of sperm from tetraploid organisms (the possession of four chromosome sets) is essentially unexplored. This is the first cryopreservation study to address sperm from tetraploid Pacific oysters, Crassostrea gigas, and addresses the commercial production of triploid oysters (three chromosome sets). Initial motility, refrigerated storage of undiluted sperm, osmolality of extender solutions, sperm concentrations, equilibration time, and cryoprotectants of propylene glycol and dimethyl sulfoxide were evaluated with sperm from diploid and tetraploid oysters. Unlike most teleost fishes, in which the duration of active motility is typically brief, the motility of sperm from oysters lasts for hours. The present study showed that responses to treatment effects by sperm from tetraploids were different from diploids. The majority of tetraploid experiments resulted in less than 10% motility after thawing and less than 5% fertilization. The highest fertilization obtained for thawed sperm was 96% for sperm from diploid oysters and 28% for sperm from tetraploid oysters. Differential responses to treatments by sperm from tetraploid and diploid oysters may be due to differences in gonadal development. However, the use of cryopreserved sperm from tetraploid Pacific oysters produced 100% triploid offspring by fertilization of eggs from diploid females as determined by flow cytometry of larvae. This study demonstrates that sperm from tetraploid oysters can be collected, frozen, and stored for production of triploid offspring.     

Haematological characterization of loach Misgurnus anguillicaudatus: comparison among diploid, triploid and tetraploid specimens

The purpose of this study was to determine whether diploid, triploid and tetraploid loach (Misgurnus anguillicaudatus) differed in terms of their main haematological and physiological characteristics. Diploid and tetraploid fish were produced by crossing of natural diploids (2n x 2n) and natural tetraploids (4n x 4n), respectively. Triploid fish were produced by hybridization between diploid males and tetraploid females. The blood cells were significantly larger in polyploids, and the volumetric ratios of erythrocytes and leucocytes (thrombocyte and neutrophil) in tetraploids, triploids and diploids were consistent with the ploidy level ratio of 4:3:2. No significant differences were observed in haematocrit among polyploids. The erythrocyte count decreased with increased ploidy level, while total haemoglobin, mean cell volume, mean cellular haemoglobin content, and mean cell haemoglobin concentration all increased with increase in ploidy level. Erythrocyte osmotic brittleness declined in polyploids so that polyploid erythrocytes were more resistant to osmotic stress than diploid ones. Overall, loach with higher ploidy levels showed evidence of some advantages in haematological characteristics.     

三倍体白鲫的生物学特性

由人工诱导的异源四倍体白鲫雄鱼与正常的二倍体白鲫雌鱼交配获得的异源三倍体白鲫,其主要生物学特性同二倍体白鲫进行比较研究表明,三倍体白鲫具有体形较大、相对体高和相对尾柄高较高、侧线上下鳞数目较多的形态特征,仍保留着二倍体白鲫以浮游植物为主的植食性营养方式,雌雄均不育,比二倍体生长快,网箱养殖１１２天后,体重比二倍体平均增加３２．３８％。试验证实三倍体白鲫是一个具有养殖生产前景的新对象。     

Establishment of a triploid V79 cell line from tetraploid cells obtained through polyploidization using K-252a

Abstract. Triploid V79 cells were established from tetraploid cells. Diploid V79 cells were polyploidized by K-252a, an inhibitor of protein kinases, and then released from the drug for 10 days. At that time, the cell population was a mixture of diploid and tetraploid cells. Triploid cells were obtained through the cloning of tetraploid cells. They had 33 chromosomes (1.5 times the diploid number) and showed a karyotype of three homologueous chromosomes. The duration of the G₁, S and G₂/M phases was almost the same as for diploid cells. The cell volume of triploid V79 cells was about two times that of the diploid cells. An explanation for the diploid-tetraploid-triploid transition is proposed.