花烛突变体叶色嵌合性状的分化特征与保持方法

以花烛品种‘Sonate’(Anthurium andraeanum‘Sonate’)的叶色嵌合型无菌苗为材料,对顶芽失去嵌合性状的16个单株分别进行单芽培养,观察侧芽及茎基部再生过程中叶色嵌合性状的分化特征;并据此归纳花烛突变体叶色嵌合性状的保持方法。结果表明:处于增殖和生根阶段的花烛突变单株侧芽再生植株的嵌合率分别为25.0%～75.0%和25.0%～66.7%,总的嵌合率分别为48.4%和47.8%,具有嵌合性状的侧芽再生植株均萌发于嵌合叶片的叶腋处;处于生根阶段的突变单株的茎基部也能再生出嵌合植株,嵌合率为33.3%～80.0%,总的嵌合率达到64.7%。研究结果显示:通过单芽离体培养的方法可以解决花烛突变体顶芽叶色嵌合性状消失的问题,且应用侧芽再生(增殖与生根阶段)或基部再生(生根阶段)的方法可以保持突变植株的嵌合性状。     

芦笋丛生芽增殖培养技术

本试验以芦笋新品种"津宁"为材料,探讨植物生长调节剂对不同外植体产生丛生芽的效应。试验结果表明,NAA、6-BA和KT三者配合使用有利于丛生芽的增殖:以茎尖为增殖外植体时,培养基用MS+NAA0.1 mg/L+6-BA0.2~0.3 mg/L+KT 0.1 mg/L,30 d时平均每瓶可以产生20.5个丛生芽;以茎中段为外植体时,培养基用MS+NAA 0.1 mg/L+6-BA 0.3~0.4 mg/L+KT 0.1 mg/L,30 d时平均每瓶可产生33.5个丛生芽;以茎基为外植体时,培养基用MS+NAA 0.1 mg/L+6-BA 0.3~0.5 mg/L+KT 0.1 mg/L,30 d时平均每瓶可以产生43.1个丛生芽。进行试管苗工厂化生产时,增殖外植体以茎基为最佳。     

栀子带芽茎段愈伤组织诱导分化及丛生芽增殖和生根研究

以栀子带芽茎段为试材,对其愈伤组织诱导分化及丛生芽增殖与生根进行初步研究。结果表明,栀子带芽茎段愈伤组织快速诱导和分化最佳培养基是MS+KT 1 mg/L+NAA 0.05 mg/L;栀子带芽茎段丛生芽增殖和生根最佳培养基是MS+KT 2.0 mg/L+NAA 0.05 mg/L。     

油茶组培苗器官发生的形态学与解剖学研究

以油茶岑软3号组培苗为研究材料,对其器官发生过程进行形态学和解剖学观察。结果表明:在选定的培养基上,外植体不经过愈伤组织,直接由器官诱导形成增殖芽,从而有效地保持了其遗传的稳定性。油茶增殖芽不定根由诱导的根原基发生,它起源于形成层薄壁细胞定向分化,不断向外进行伸长生长,并穿透愈伤组织而形成。这种不定根发生形式属皮部生根类型。油茶增殖芽单芽生根培养25 d后在不定根内形成次生维管形成层和木栓形成层,培养40 d以上根内的输导分子与茎内的输导分子相连。     

一个芸香科属间嫁接嵌合体的拉丁名称:+Citroponcirus‘Hormish’

柑桔属(Citrus L.)和枳属(Poncirus Raf.)属间嫁接嵌合体是1986年从福建省连城县莒溪镇一个专业户果园的芦柑(Citrus reticulataBlanco)嫁接于枳(Poncirus trifoliata(L.)Raf.)的2 a生植株发生冻害后由嫁接口蘖芽产生变异获得的.枝条有单身复叶、二出复叶和三出复叶,一些枝条出现三出复叶枳枝,表现出芦柑和枳的嵌合;幼果密被茸毛,为枳的性状,成熟果实为芦柑形态并具有芦柑风味,产生一部分为枳一部分为芦柑的嵌合果;果实汁胞以橙红色为主,兼有少量淡黄色汁胞和橙黄嵌合汁胞.依据<国际栽培植物命名法规>,芸香科柑桔属和枳属属间嫁接嵌合体拉丁名称的属名是柑桔属和枳属的属名合并建立的新"属名",定名为+枳合芦属(+Citroponcirus),栽培品种加词采用这个嫁接嵌合体发现者的人名表示,这个属间嫁接嵌合体的拉丁名称是+Citroponcirus'Hormish';以公式表示为Citrus reticulata+Poncirus trifoliata.在施文格(Swingle)柑桔分类系统中,+Citroponcirus'Hormish'是目前世界上芸香科嫁接嵌合体中唯一的属间嫁接嵌合体.     

绿色荧光蛋白嵌合体小鼠的建立和鉴定

为研究嵌合体动物中供体胚胎干细胞 (ES)在宿主胚胎发育中的走向和定位 ,同时探讨绿色荧光蛋白(GFP)基因作为报告基因在转基因动物制作中的应用价值 ,本研究将pEGFP N1基因导入小鼠ES D3 细胞系 ,得到稳定表达GFP的胚胎干细胞亚系ES D3 GFP ,通过对昆明小鼠的囊胚腔注射 ,获得了 4只表达绿色荧光蛋白的嵌合体小鼠。其中 1只存活至成年 ,3只出生时死亡。荧光显像及组织PCR检测显示了绿色荧光蛋白在小鼠体内的嵌合情况。以绿色荧光为指标可实现活体水平的动态观察 ,本实验首次观察到以GFP为指标所示的机体嵌合情况与根据毛色嵌合推测的机体嵌合情况存在很大差异 ,以GFP为嵌合指标更加全面而准确 ;但不排除GFP对小鼠发育存在一定毒性的可能 ;另外 ,有结果显示供体ES细胞在宿主体内除了大片补丁状嵌合外 ,还存在细胞散在嵌合的情况 ,后者提示了在组织中利用GFP对ES细胞实施单细胞追踪和实时观察的可行性 ,为胚胎发育和疾病发生的相关研究提供了新的观察方法     

蟹爪兰茎段消毒及其丛生芽增殖和生根初步研究

以蟹爪兰茎段为材料,采用组织培养和单因子实验方法,研究蟹爪兰无菌体系的建立及植物生长调节剂对茎段丛生芽增殖和生根的影响。结果表明:蟹爪兰茎段最佳消毒方式是75%酒精30 s+0.1%HgCl₂ 10 min;茎段丛生芽增殖最佳培养基是MS+KT 4 mg/L+NAA 0.1 mg/L;生根最佳培养基是1/2 MS+NAA 0.5 mg/L。     

毛茛组织培养与植株再生(简报)

以毛茛（Ranunculus japonicus）茎尖、带芽茎段为外植体进行组织培养。试验结果表明,毛莨茎尖、带芽茎段萌发快,茎段丛生芽增殖数多于茎尖;壮苗和生根可在MS0培养基上一次完成,生根率达85％。     

亳芍茎尖组织培养的研究

选取亳芍茎尖为试验材料,探究不同培养条件对亳芍组织培养的影响,结果表明:亳芍茎尖在1/2MS+6-BA 1.0 mg/L培养基上培养39 d后,茎尖分化出芽的同时也形成较多的丛生芽;丛生芽在1/2MS+6-BA1.0mg/L培养基上增殖速度最快;来自不同启动培养基上的丛生芽在相同培养基上,接种15 d后观察,不同来源的丛生芽长势不同,30 d后仍存在一定的差异;幼苗在1/2MS+IBA 0.1生根效果最好。     

家兔早期胚胎细胞发育能力的研究

本文利用胚泡注射法制作嵌合体对家兔交配后96,120和144小时的ICM细胞的发育能力进行了研究。供体胚胎取自青紫兰灰免,受体胚胎取自新西兰白兔,结果表明96和120小时供胚的ICM细胞与96小时受胚胚泡组合后均能参与发育,形成嵌合兔,144小时者未获得嵌合体。由于120小时的ICM细胞发育的2只表型为雄性的嵌合兔,其中1只不育,其性腺和外周血核型表明不育兔为xx/xy性嵌合,性腺中有处于不同发育程度的卵巢和精细管,外周血含xx和xy两种核型。本实验结果首次证明家兔交配后120小时胚泡的ICM细胞仍具有参与嵌合体发育的能力。它不仅能参与体细胞的分化,并具有形成生殖细胞的能力。交配后144小时胚泡的ICM细胞其发育能力似乎已发生了局限。     

Comparison of the effects of serpin A1, a recombinant serpin A1-IGF chimera and serpin A1 C-terminal peptide on wound healing

Serpin A1 (alpha1-antitrypsin, alpha1-proteinase inhibitor), a potent neutrophil elastase inhibitor, has therapeutic potential as a wound-healing agent. We compared the in vitro wound-healing action of serpin A1-IGF, a recombinant fusion protein of serpin A1(M351E-M358L) and insulin-like growth factor I with that observed in the presence of natural serpin A1 or A1-C26, the synthetic C-terminal 26 residue peptide of serpin A1, previously shown to have mitogenic and antiviral activities. All agents reduced wound sizes in monolayers of the kidney epithelial cell line LLC-PK1 and in primary cultures of human skin fibroblasts. Wound reduction in primary human keratinocytes was only observed with the serpin A1-IGF chimera. None of the factors stimulated cell proliferation using a colorimetric assay, with the exception of the serpin A1-IGF chimera, which caused a significant increase of cell proliferation and thymidine incorporation in human skin fibroblasts. However, wound healing by the A1-IGF chimera was reduced in keratinocytes in the presence of mitomycin C, suggesting a role of cell proliferation in wound reduction. The hydrophobic A1-C26 peptide significantly increased the production of collagen I in skin fibroblasts, an appealing asset for skin care applications.     

Mer receptor tyrosine kinase signaling: prevention of apoptosis and alteration of cytoskeletal architecture without stimulation or proliferation

Mer is a member of the Axl/Mer/Tyro3 receptor tyrosine kinase family, a family whose physiological function is not well defined. We constructed a Mer chimera using the epidermal growth factor receptor (EGFR) extracellular and transmembrane domains and the Mer cytoplasmic domain. Stable transfection of the Mer chimera into interleukin 3 (IL-3)-dependent murine 32D cells resulted in ligand-activable surface receptor that tyrosine autophosphorylated, stimulated intracellular signaling, and dramatically reduced apoptosis initiated by IL-3 withdrawal. However, unlike multiple other ectopically expressed receptor tyrosine kinases including full-length EGFR or an EGFR/Axl chimera, the Mer chimera did not stimulate proliferation. Moreover, and in contrast to EGFR, Mer chimera activation induced adherence and cell flattening in the normally suspension-growing 32D cells. The Mer chimera signal also blocked IL-3-dependent proliferation leading to G(1)/S arrest, dephosphorylation of retinoblastoma protein, and elongation of cellular processes. Unlike other agonists that lead to a slow (4-8 days) ligand-dependent differentiation of 32D cells, the combined Mer and IL-3 signal resulted in differentiated morphology and growth cessation in the first 24 h. Thus the Mer chimera blocks apoptosis without stimulating growth and produces cytoskeletal alterations; this outcome is clearly separable from the proliferative signal produced by most receptor tyrosine kinases.     

Paternally inherited effects of gamma radiation on mouse preimplantation development detected by the chimera assay

It has previously been shown that type B spermatogonia in male mice treated with 0.05 Gy of X rays undergo an alteration expressed by progeny embryos as a cellular proliferation disadvantage in a chimera assay. We wished to obtain information on the assay's detection limit to ionizing radiation and on the radiosensitive target in male germ cells. Male mice were briefly irradiated with 137Cs gamma rays at nominal absorbed doses of 0.0, 0.0015, 0.005, 0.010, or 0.05 Gy and then mated for the next 8 weeks to untreated females. Four-cell embryos from treated males (experimental embryos) were paired with FITC-labeled embryos from untreated males (control embryos) to form aggregation chimeras. The chimeras were cultured for 30-40 h and examined under phase-contrast and UV illumination for the number of unlabeled cells (from the experimental embryo) and total chimera cell number, which were then expressed as "proliferation ratios" (No. unlabeled cells/total chimera cell No.). Significant decreases in proliferation ratios were observed at postirradiation weeks 4, 6, and 7 for the 0.01-Gy dose group and at weeks 5-6 for the 0.05-Gy dose group. In addition, significantly lower ratios were observed with early and mid four-cell embryos, but not with late four-cell embryos. These results suggest that mouse male germ cells express a radiosensitive target(s) whose detection limit by the assay lies at an absorbed dose between 0.005 and 0.010 Gy for brief gamma irradiation and whose effect on embryonic cell proliferation might decay by the second cleavage.     

Growth promotion of genetically modified hematopoietic progenitors using an antibody/c-Mpl chimera

Thrombopoietin is a potent cytokine that exerts proliferation of hematopoietic stem cells (HSCs) through its cognate receptor, c-Mpl. Therefore, mimicry of c-Mpl signaling by a receptor recognizing an artificial ligand would be attractive to attain specific expansion of genetically modified HSCs. Here we propose a system enabling selective expansion of genetically modified cells using an antibody/receptor chimera that can be activated by a specific antigen. We constructed an antibody/c-Mpl chimera, in which single-chain Fv (ScFv) of an anti-fluorescein antibody was tethered to the extracellular D2 domain of the erythropoietin receptor and transmembrane/cytoplasmic domains of c-Mpl. When the chimera was expressed in interleukin (IL)-3-dependent pro-B cell line Ba/F3, genetically modified cells were selectively expanded in the presence of fluorescein-conjugated BSA (BSA-FL) as a specific antigen. Furthermore, highly purified mouse HSCs transduced with the retrovirus carrying antibody/c-Mpl chimera gene proliferated in vitro in response to BSA-FL, and the cells retained in vivo long-term repopulating abilities. These results demonstrate that the antibody/c-Mpl chimera is capable of signal transduction that mimics wild-type c-Mpl signaling.     

Functional and structural characterization of chimeras of a bacterial genotoxin and human type I DNAse

Chimeras composed of the cdtB gene of a novel bacterial genotoxin and the human type I DNAse I gene were constructed and their products characterized relative to the biochemical and enzymatic properties of the native proteins. The product of a cdtB/DNAse I chimera formed a heterotrimer with the CdtA and CdtC subunits of the genotoxin, and targeted mutations increased the specific activity of the hybrid protein. Expression of active chimeric gene products established that the CdtB protein is an atypical divalent cation-dependent endonuclease and demonstrated the potential for genetically engineering a new class of therapeutic agent for inhibiting the proliferation of cancer cells.     

Nonspecific suppressor elements in murine allogeneic radiation chimeras.

Spleen cells from long-term mouse allogeneic radiation chimeras were tested for their ability to modulate the graft-versus-host (GVH) or plaque-forming cell (PFC) response of normal lymphocytes transplanted in lethally X-irradiated recipients. In vivo GVH proliferation of normal lymphocytes (syngeneic to donor cells of the chimera) against antigens of host-type in which the chimeric state had been established was reduced by chimera cells. Inhibition varied, some chimeras suppressing GVH more than others and a few not suppressing at all. The suppressive effect was abrogated if the chimera cells were treated with anti-θ; treatment with anti-IgM did not eliminate this activity. When mixtures of normal donor lymphocytes and chimera cells were given to irradiated recipients genetically different from host or donor, reduction of donor cell GVH also occurred. Further, chimera cells reduced the GVH activity of normal host cells in irradiated recipients differing from the host at one H-2 locus and from the donor at minor histocompatibility loci. The modulating effect of spleen cells from chimeras on the PFC response by normal lymphocytes also varied. Six chimeras induced a 25 to 90% suppression, two enhanced the response, and one showed no effect. Where suppression occurred, treatment of chimera cells with anti-θ most often, but not always, restored PFC production. Our results show that the suppressive action of splenic lymphoid cells by chimeras is highly nonspecific and variable in expression. We suggest that tolerance in chimeras may be mediated by nonspecific suppressor elements leading to unresponsiveness to a variety of antigens including SRBC.     

Activation of EphB2 and its ligands promotes vascular smooth muscle cell proliferation.

EphB2 and its ligands regulate interactions between endothelial and mesenchymal cells in developing arteries. In adult arteries, the relationship between smooth muscle cells and overlying intact endothelium is responsible for maintaining the health of the vessel. Heparin inhibits vascular smooth muscle cell growth in culture and intimal hyperplasia following endothelial denudation. Using gene microarrays, we identified the tyrosine kinase receptor EphB2 as being differentially expressed in response to continuous intravenous heparin administration in the rabbit model of arterial injury. EphB2 protein levels increased in cultured bovine vascular smooth muscle cells following serum stimulation and were decreased in a dose-dependent fashion by heparin. Fc chimeras of the binding domain of the EphB2 ligands blocked the formation of the EphB2 ligand-receptor complex and reduced growth of serum-stimulated vascular smooth muscle cells in a dose-dependent fashion. Activation of the ligand by an Fc chimera to EphB2 followed a parabolic dose-response growth curve, indicating growth stimulation until the chimera begins to compete with native receptors. Co-administration of EphB2/Fc chimera with heparin shifted the dose-response curve to the right. These data indicate a possible new route of Heparin's antiproliferative effect and a role of EphB2 and its ligands in vascular smooth muscle cell proliferation.     

Oral vaccination against classical swine fever with a chimeric <Emphasis Type="Italic">Pestivirus</Emphasis>: comparative investigations of liquid and lyophilized virus

The goal of this study was to evaluate the efficacy of the chimeric marker vaccine candidate CP7_E2alf in different formulations for oral immunization against classical swine fever (CSF). In the first experiment, three wild boars were vaccinated orally with the liquid chimeric virus CP7_E2alf, whereas in the second experiment, four wild boars and four domestic pigs were immunized with the lyophilized chimera administered in gelatine capsules and new baits. The chimera was safe for wild boars and domestic pigs. All animals vaccinated orally with the liquid chimera were seropositive 21 days after vaccination, and neither viraemia nor virus shedding were observed after challenge with a highly virulent CSF virus. At necropsy, viral genome of the challenge virus was detected in some organs of all surviving wild boars. Lyophilized chimera administered orally did not protect the animals. The reasons for the inefficacy of the lyophilized vaccine virus are discussed. Irrespective of the negative result with lyophilized virus, the study and previous experiences (Reimann et al., Virology, 307:213–227, 2004) suggest that the chimera CP7_E2alf is a potential CSF marker vaccine candidate.     

Insect cell production of a secreted form of human alpha(1)-proteinase inhibitor as a bifunctional protein which inhibits neutrophil elastase and has growth factor-like activities.

alpha(1)-proteinase inhibitor (API) is a potential therapeutic agent in all diseases in which elastase released by neutrophils has to be effectively neutralized. We ligated the cDNA of human API to the C-terminal section of an insulin-like growth factor II analogue (BOMIGF), known to be properly folded and secreted in insect cells using the baculovirus expression system. The BOMIGF-API chimera was recovered from the incubation medium of the infected cells. It shared the properties of both IGFs and API. It inhibited neutrophil elastase and formed SDS-stable complexes with the enzyme. The attachment of the large API protein to the C-terminal end of the 10 kDa IGF analogue did not destroy the IGF-mediated stimulation of thymidine incorporation into bovine fetal erythroid cells. We tested the capacity of the chimera to affect fibronectin-dependent TF-1 cell migration. BOMIGF-API significantly restored TF-1 cell migration in the presence of elastase, which is the enzyme of burn wound fluid most probably involved in fibronectin degradation. Some of the beneficial uses for this chimera may include all instances for which inhibition of elastase-mediated extracellular matrix destruction as well as stimulation of cell migration and proliferation are required for tissue repair.     

An assay using embryo aggregation chimeras for the detection of nonlethal changes in X-irradiated mouse preimplantation embryos

We have developed a short-term in vitro assay for the detection of sublethal effects produced by very low levels of ionizing radiation. The assay utilizes mouse embryo aggregation chimeras consisting of one irradiated embryo paired with an unirradiated embryo whose blastomeres have been labeled with fluorescein isothiocyanate (FITC). X irradiation (from 0.05 to 2 Gy) and chimera construction were performed with four-cell stage embryos, and the chimeras were cultured for 40 h to the morula stage. The morulae were partially dissociated with calcium-free culture medium and viewed under phase contrast and epifluorescence microscopy to obtain total embryo cell number and the cellular contribution of irradiated (unlabeled) and control (FITC labeled) embryos per chimera. In chimeras where neither embryo was irradiated, the ratio of the unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.50 (17.8 +/- 5.6 cells per unlabeled embryo and 17.4 +/- 5.5 cells per FITC-labeled partner embryo). However, in chimeras formed after the unlabeled embryos were irradiated with as little as 0.05 Gy, the ratio of unlabeled blastomeres to the total number of blastomeres per chimera embryo was 0.43 (P less than 0.01). The apparent decreases in cell proliferation were not observed in irradiated embryos that were merely cocultured with control embryos, regardless of whether the embryos were zona enclosed or zona free. We conclude that very low levels of radiation induce sublethal changes in cleaving embryos that are expressed as a proliferative disadvantage within two cell cycles when irradiated embryos are in direct cell-to-cell contact with unirradiated embryos.