湖北双蝴蝶的组织培养研究

以湖北双蝴蝶带芽茎段、不带芽茎段及叶片为外植体,以MS为基本培养基,通过添加不同的植物生长物质种类和浓度配比,建立湖北双蝴蝶组培快繁体系。结果如下:在所有实验方案中,带芽茎段的出愈率最高,是理想的离体快繁材料。较适宜的初代培养基为MS+BA2.0mg/L+蔗糖3.0%,增殖培养基为MS+BA2.0mg/L+NAA0.1mg/L+蔗糖3.0%,而根的诱导则在1/2MS+NAA0.5mg/L+蔗糖1.5%的培养基上进行较为适宜。同时对组织培养过程中湖北双蝴蝶植株再生的方式进行了讨论。     

药用植物灯盏花的组织培养

以灯盏花花葶、花盘及叶柄为外植体,MS为基本培养基,通过不同的激素种类和浓度配比,建立灯盏花组培快繁体系。结果如下:在所有实验方案中,花葶的出愈率最高,是理想的快速繁殖材料。较适宜的诱导愈伤组织的培养基为MS+BA1.0mg/L+IBA0.05mg/L+蔗糖3.0%,诱导不定芽的培养基为MS+BA2.0mg/L+IAA1.0mg/L+蔗糖3.0%或MS+Kt3.0+IAA0.5mg/L+蔗糖3.0%,而根的诱导则是在1/2MS+NAA1.0Mg/L+蔗糖3.0%的培养基上进行。同时对组织培养过程中灯盏花植株再生的方式进行了讨论。     

Maintenance of the Adult Rat Superior Cervical Ganglion In Vitro: Comparison of Organ and Explant Culture Systems

Abstract: Concentrations of selected intermediates of energy metabolism whole rat superior cervical ganglia maintained in vitro by an organ culture technique were compared with values measured in small slices of this maintained under essentially the same conditions. Rates of incorporation [³H]leucine into trichloroacetic acid-precipitable material in whole ganglia mained constant for at least 48 h: however, the oxidation-reduction state tissue as indexed by (NAD):(NADH) ratios calculated from measured amounts of lactate and pyruvate decreased more than 50% within ³h in vitro . Ganglion explants prepared by cutting the tissue into ³⁰⁰-pm transverse sections played (NAD):(NADH) ratios that were about three times greater than noted in whole ganglia maintained in vitro for the same period of time. explants contained significantly higher concentrations of pyruvate and α-ketoglutarate than whole ganglia maintained in culture. Maintenance of vorable metabolic state may support the extensive growth of neurites seen explant cultures of superior cervical ganglia. Outgrowth of processes containing catecholamines could be detected readily in explant cultures of ganglia adult rats; however, this was somewhat slower and less consistent than growth observed in explants from neonatal rats. Outgrowth of neurites adult ganglia was minimal without the addition of Nerve Growth Factor.     

Effect of conditions of cultivation on quantitative assessment of the viability of an organism's tissues]

Viability of mouse kidney tissue explants as estimated quantitatively according to their growth in plasmafree culture depends on some factors connected with cultivation conditions. Methods of serum preparation, pH of the medium, as well as peculiarities of tissue explants distribution in cultivation, their size and fastness of attachment to the substrate proved to influence the quantitative estimation of the tissue explants viability.     

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.     

Association in organ culture of the hypophysis and glandular tissue of the testis of Gobius niger L.]

Pituitaries of Gobium niger are previously cultured separately for periods of time varying from 6 to 20 days. Therafter, their gonadotropic potency is tested by associating them during three days with explants of glandular tissue from testis as effectors: in each case, a new fish gives a controll pituitary and three testicular explants. The latter are distributed into (1) controll explant, (2) explant associated with controll pituitary, (3) explant associated with a previously cultured pituitary. The results indicate that a gonadotropic potency subsists in the pituitary even after 20 days of isolated culture.     

Infectivity of monomorphic and pleomorphic trypanosoma brucei stocks cultivated at 28 C with various tsetse fly tissues

Noninfective procyclic forms of Trypanosoma brucei stocks derived from the pleomorphic EVE 10 were cultivated at 28 C in Cunningham's liquid medium in the presence of head-salivary gland, alimentary tract, and abdominal body wall explants of Glossina morsitans morsitans. After 8 to 10 days of cultivation some of the procyclic forms transformed into metacyclic stages infective for mice. Infectivity persisted for varying periods up to 66 days, when the experiments were terminated. Only 10 explants of alimentary tract or abdominal body wall tissues were required in the flasks to render the culture infective for most of the mice inoculated. Similar trypanosome suspensions grown with 10 head-salivary gland explants produced an infection on only one occasion. Cultures of procyclic organisms derived from the monomorphic stock 427 grown inthe presence of all three types of tsetse fly explants produced only sporadic infections in mice. Metacyclic forms failed to develop in trypanosome populations of stock EVE 10 cultivated at 28 C in the liquid medium alone or supplemented with mouse embryo tissues.     

Immunohistochemical identification of proliferating cells in organ culture using bromodeoxyuridine and a monoclonal antibody

The ability to measure cell proliferation is important in the study of cancer biology. The usual technique for quantitating proliferating cells in tissue explant and organ culture by detection of [3H]-thymidine incorporation into DNA by autoradiography is tedious and time-consuming. We have developed a technique for identification and quantitation of bromodeoxyuridine (an analogue of thymidine) in cultured tissue explants. Fetal mouse colon explants were exposed in vitro to bromodeoxyuridine (BUdR) or [3H]-thymidine for 3 to 72 hr and then for various periods to unlabeled thymidine. The tissues were stained with a monoclonal anti-bromodeoxyuridine antibody and in parallel [3H]-thymidine incorporation was detected by autoradiography. Incorporation of BUdR was measured by quantitating the amount of pigment deposited over nuclei after immunohistochemical staining, using an optical data digitizer. It was found that both techniques identified proliferating cells. Dividing cells were present both in crypts and in the surrounding stroma in Day 14 fetal mouse colon cultures. The immunohistochemical technique was more rapid and less cumbersome than autoradiography.     

In vitro explant culture of mantle epithelium of freshwater pearl mussel

The in vitro culture of nacre secreting pallial mantle explants of freshwater pearl producing mussel, Lamellidens marginalis (Lamarck) included depuration of pearl mussels with different physical and chemical agents to eradicate various commensals, removal of pallial mantle ribbon, aseptic preparation of explants from the ribbon and transfer of those explants into tissue culture petri dishes. Special synthetic tissue culture media enriched with additives viz., inactivated calf fetal serum and antibiotics were poured into plates with explants. The culture plates were incubated at 30 degrees C in a CO2 incubator at 5%, CO2. The cultures could be maintained for 42-45 days without any contamination. After 12 hr epithelial like cells began to migrate out and formed a complete cell sheet surrounding the explant within 12-15 days. The epithelial cells in the culture indicated functional viability as subsequently after 38-40 days of culture, typical aragonitic 'nacre' crystals of CaCO3 could be observed throughout the culture plates.     

Proteoglycan synthesis in organ cultures from regions of bovine tendon subjected to different mechanical forces.

Synthesis of proteoglycans by morphologically and chemically distinct regions of bovine flexor tendon was investigated in explant cultures. Proximal regions of the flexor tendon which experience only tensile forces and have low contents of proteoglycans initially exhibited relatively low rates of proteoglycan synthesis but high rates of collagen synthesis. The predominant proteoglycan produced by all proximal explants was of small hydrodynamic size and appeared similar to that extracted from proximal tissue. In contrast, explants derived from the distal tendon region, which experiences frictional and compressive forces in addition to tensile forces, and has a high content of proteoglycans, showed relatively high initial rates of proteoglycan synthesis and lower rates of collagen synthesis. These distal explants produced primarily large proteoglycans on the first day in culture. Turnover of newly synthesized proteoglycans was not detectable in proximal tissue, and was low in distal tissue. Loss of unlabelled proteoglycan from proximal and distal explants was not detected during the 12 days of culture. These observations suggest that the increase in specific types of proteoglycans in regions of tendon subjected to frictional and compressive forces is the result of elevated synthesis rates in this tissue. Two alterations in proteoglycan synthesis occurred during the 12-day culture period. (1) The rate of proteoglycan synthesis by all explants increased with time in culture. (2) The proportion of small proteoglycans synthesized by distal explants increased from 32% of the total proteoglycan produced on day 1, to 80% of that produced on day 12. Explants from proximal tendon continued to produce only small proteoglycans throughout the 12 days in culture. This switch in proteoglycan phenotype, resulting in decreased synthesis of large proteoglycans by the distal tissue, may be due to a lack of compressive forces on the cultured explants.     

Functional differentiation in mouse mammary gland epithelium is attained through DNA synthesis,inconsequent of mitosis

Virgin mouse mammary gland in explant culture will differentiate and synthesize casein and α-lactalbumin when insulin, hydrocortisone, and prolactin (IFPRL) are present in the culture medium. Explants whose DNA synthesis has been blocked differentiate cytologically, mobilize lipid, synthesize RNA, and incorporate ³H-amino acids into proteins to the same extent as unblocked tissue. Nevertheless, casein synthesis as measured by immunoprecipitation with casein-specific antiserum remains at the zero-time level in blocked explants while unblocked explants produce casein at five- to eightfold greater levels. Electrophoretic analysis of immunoprecipitated radioactive proteins showed that the IFPRL-treated virgin tissue made all four size classes of mouse casein. Immunoperoxidase studies of explants revealed that the number of mammary epithelial cells positive for casein was 2–8% in blocked and 24–31% in unblocked, in good agreement with the radioimmunoprecipitation results. Immunoelectron microscopy demonstrated the accumulation of casein within the cisternae of the granular endoplasmic reticulum and in Golgi vacuoles in the unblocked epithelial cells. Similar accumulation did not occur in blocked cultures despite the secretory appearance of the cells. Autoradiographic analysis of blocked and unblocked explants, incubated in the presence of IFPRL and [³H]thymidine for 72 hr, showed that 53–57% of the epithelial cells synthesized DNA in unblocked explants, whereas only 2% incorporated the label in the presence of cytosine arabinoside. When explants were incubated with IFPRL and various concentrations of colchicine, only 5–6% of the epithelial cells were found to enter mitosis. Since cell duplication cannot account for the severalfold increase in casein-producing cells in the unblocked explants, the results suggest that the requirement for DNA synthesis in this system may involve either polyploid cells or the augmentation of specific sequences necessary for the facilitation of terminal differentiation. Similar requirements for DNA synthesis were not observed in mammary explants from pregnant and primiparous (but nonpregnant) mice.     

A method for the isolation and serial propagation of keratinocytes,endothelial cells,and fibroblasts from a single punch biopsy of human skin

Summary When multiple types of cells from normal and diseased human skin are required, techniques to isolate cells from small skin biopsies would facilitate experimental studies. The purpose of this investigation was to develop a method for the isolation and propagation of three major cell types (keratinocytes, microvascular endothelial cells, and fibroblasts) from a 4-mm punch biopsy of human skin. To isolate and propagate keratinocytes from a punch biopsy, the epidermis was separated from the dermis by treatment with dispase. Keratinocytes were dissociated from the epidermis by trypsin and plated on a collagen-coated tissue culture petri dish. A combination of two commercial media (Serum-Free Medium and Medium 154) provided optimal growth conditions. To isolate and propagate microvascular endothelial cells from the dermis, cells were released following dispase incubation and plated on a gelatin-coated tissue culture dish. Supplementation of a standard growth medium with a medium conditioned by mouse 3T3 cells was required for the establishment and growth of these cells. Epithelioid endothelial cells were separated from spindle-shaped endothelial cells and from dendritic cells by selective attachment toUlex europeus agglutinin I-coated paramagnetic beads. To establish fibroblasts, dermal explants depleted of keratinocytes and endothelial cells were attached to plastic by centrifugation, and fibroblasts were obtained by explant culture and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing fetal bovine serum (FBS). Using these isolation methods and growth conditions, two confluent T-75 flasks of keratinocytes, one confluent T-25 flask of purified endothelial cells, and one confluent T-25 flask of fibroblasts could be routinely obtained from a 4-mm punch biopsy of human skin. This method should prove useful in studies of human skin where three cell types must be grown in sufficient quantities for molecular and biochemical analysis.     

Glycoprotein expression in foetal and adult mouse cerebellum

1. Explants of cerebellum from foetal mouse were cultured in vitro for up to 12 days. Some glycoprotein components displayed time-dependent changes in concentration in the cultured explants. 2. The specific activity of several enzymes involved in the biosynthesis or degradation of N-linked glycoproteins, increased markedly in the cerebellar explants as a function of time in culture. 3. Glycoprotein expression in foetal mouse cerebellum is compared with that in the adult tissue.     

Evidence that somatomedin is synthesized by multiple tissues in the fetus

Production of somatomedin-C, a growth hormone-dependent peptide believed to mediate the growth-promoting actions of growth hormone, has been assessed using explants of fetal mouse tissues. Quantitation of this peptide in media of explants cultured for 3 days has been accomplished with a membrane receptor assay for somatomedin and a specific radioimmunoassay for somatomedin-C. Somatomedin-C is produced by the 11-day-gestation fetal mouse liver, increases exponentially in parallel with liver growth until the 16th day of gestation, and falls postnatally. Media somatomedin is believed to be derived by de novo synthesis since saline extracts of liver and most other fetal tissues contain only a small fraction of the activity in culture media. The immunoreactive material secreted into media appears to be closely related to human somatomedin-C since it produces dilution curves which are parallel to those of pure hormone, migrates on Sephacryl 200 at a size similar to that of one of the components of human serum somatomedin-C, dissociates into small molecular weight material with acid treatment, and isofocuses in a range comparable with that of somatomedin-C purified from human serum. Eleven-day limb bud mesenchymal micromass cultures and 17-day-gestation intestine, heart, brain, kidney, and lung also synthesize immunoreactive somatomedin-C in serum-free medium. For these tissues, the media activity was far in excess of the tissue extractable activity. Somatomedin activity in excess of the tissue extractable activity, however, was not found in media from 17-day-gestation placenta. The finding that multiple tissues synthesize somatomedin-C raises the possibility that the primary biological actions of this hormone are exerted locally at its sites of origin. Although a function of this type by a peptide has not been widely suspected, it seems plausible that the cells of fetal tissues are capable of producing local mitogens in much the same manner as the postulated inducers of tissue differentiation.     

Human bronchial explants in long-term culture: Establishing a baseline for secretion

Summary A serum-free culture medium was employed to evaluate both qualitative and quantitative aspects of secreted proteins derived from human bronchial explants over a period of 26 to 50 d. It was found that protein and hexose were secreted at a reasonably constant rate during this period. Viability of explants was assessed by histological examination, attachment to scored grids of the petri dish, incorporation of labeled precursors into tissue proteins, and by fluorographic analysis of the sodium dodecyl sulfate-polyacrylamide gel pattern of the secreted material. This culture system is useful as a model for the study of secretory products and processes and how they are affected by various stimuli.     

酸性培养基对喜树叶片细胞壁降解的影响

在组织培养过程中对喜树叶片外植体进行解剖学观察。发现在组织培养条件下,喜树叶片在培养基的酸性环境中细胞壁呈现模糊微弱降解、明显降解和完全降解直至消失的解剖学特征。在同样的组培条件和相同的时间内,同一喜树叶片不同部位出现细胞壁程度不同的降解和消失现象,可能是喜树叶片因上表皮凸凹不平进而导致其不同部位与酸性培养基接触的程度不同,因而使培养基中的酸性物质对喜树叶片上表皮的不同部位影响出现异质化。本文对培养基中的酸性成分对喜树叶片细胞壁降解的影响有了进一步的认识和理解。     

Synthesis of two lactogenic proteins by the mouse placenta in vitro.

The culture medium of mouse placental tissues was analyzed on acrylamide gel electrophoresis to localize lactogenic substances. Placental explants from 12- or 14-day-pregnant BALB/cHe mice were organ-cultured for 6 days in leucine-free Way-mouth's medium supplemented with 3H-leucine (10 mu/Ci/ml) and insulin (0.12I.U./ml). The medium was collected every other day and subjected to acrylamide gel electrophoresis. The electrophoretic pattern of radioactive leucine incorporated into proteins was examined on stained 7% gels. Five protein bands were associated with high radioactivity. The location of lactogenic activity on acrylamide gel was when investigated by the technique of organ culture of mouse mammary tissues. Placental explants from 12- to 14-day-pregnant BALB/cHe mice were organ-cultured in Waymouth's medium supplemented with insulin for 2 days. After electrophoresis, proteins were eluted by keeping the segment of acrylamide gel in phosphate buffer, dialyzed and dissolved in tissue culture medium 199 supplemented with insulin and cortisol. Mammary tissues from 8-day-pregnant KA2 mice were cultured for 3 days in the medium containing each eluate. Mammary glands always responded to eluted proteins from two positions of 7% gel, as judged in histological sections. The data suggest the presence of two lactogenic substances in the mouse placenta.     

Desmin expression during early mouse tongue morphogenesis.

Occipital somites provide progenitor cells for craniofacial muscle development including the tongue musculature. Serum-derived factors are assumed to be pre-requisite for myogenesis in vitro. To test these assertions, we designed experiments to determine whether early mouse tongue development in general, and desmin localization in particular, were expressed during the development of embryonic mouse first branchial arch explants cultured in serumless, chemically-defined medium. Immunohistochemical techniques determined the chronology and positions of desmin expression during early craniofacial development. Occipital somites expressed desmin at E9 (9 days +/- 2 h post-fertilization, 18-20 somites). A discrete cell migration pathway initiating in the somites and terminating in the lateral lingual processes of the tongue primordium was defined based upon desmin expression patterns in E9-E11 embryos and computer-assisted three dimensional reconstructions. The in vitro model system was permissive for tongue morphogenesis, allowing development and fusion of the lateral lingual processes with the tuberculum impar. During culture myoblasts were not observed to fuse into myotubes with sarcomeric assembly, even though explant myoblasts produced muscle-specific protein. E10 explants cultured for 9 days demonstrated a five-fold increase in cell number that expressed desmin (P less than 0.05) when compared to the E10 starting material. We interpret these results to indicate that the tongue myogenic cell lineage was determined between E8 and E11, and that this resident population expanded within explants cultured in serumless medium by several explanations: (i) cells other than progenitor myoblasts (e.g., satellite cells) were induced to become myoblasts, and/or (ii) progenitor myoblasts within the original explants expanded by cell division in the absence of serum factors.(ABSTRACT TRUNCATED AT 250 WORDS)