3T3-L1脂肪细胞对MIN6胰岛素细胞中Kir6.2表达的抑制作用

为明确脂肪细胞对胰岛素细胞中KATP通道表达的直接影响,MIN6胰岛素细胞被分为两组：一组为对照组,一组与分化的3T3-L1脂肪细胞共培养1周。运用半定量RT-PCR方法测定MIN6细胞中KATP通道蛋白Kir6．2的表达变化,Fura-2荧光方法测定MIN6细胞内钙浓度的变化,放射免疫测定方法明确MIN6细胞的胰岛素分泌功能。结果显示,与3T3-L1脂肪细胞共培养1周后,MIN6细胞中Kir6．2的表达明显减少,其表达水平降低为对照组的65．3％。对照组MIN6细胞在0．1mmoi／L甲苯磺丁脲(KATP通道关闭剂)的刺激下,表现为细胞内钙水平显著性升高和胰岛素分泌显著性增加,而共培养组MIN6细胞则失去了甲苯磺丁脲刺激所引起的细胞内钙升高及胰岛素分泌反应。以上实验结果表明,3T3-L1脂肪细胞可以通过分泌一些活性因子直接降低MIN6细胞中KATP通道蛋白的表达和合成,损害MIN6细胞的胰岛素分泌功能。实验结果提示脂肪细胞直接参与2型糖尿病中胰岛β细胞功能障碍的发生。     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-Mediated genetic transformation in tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis> [L.] O. Kuntze)

We have developed a system to produce transgenic plants in tea (Camelia sinensis [L.] O. Kuntze) viaAgrobacterium tumefaciens-mediated transformation of embryogenic calli. Cotyledon-derived embryogenic callus cultures were cocultivated with anA. tumefaciens strain (AGL 1) harboring a binary vector carrying the hygromycin phosphotransferase (hpt II), glucuronidase (uid A), and green fluorescent protein (GFP) genes in the tDNA region. Following cocultivation, embryogenic calli were cultured in medium containing 500 mg/L carbenicillin for 1 wk and cultured on an antibiotic selection medium containing 75 mg/L hygromycin for 8–10 wk. Hygromycin-resistant somatic embryos were selected. The highest production efficiency of hygromycin-resistant calli occurred with cocultivation for 6–7 d in the presence of 400 μM acetosyringone (AS). Hygromycin-resistant somatic embryos developed into complete plantlets in regeneration medium containing half-strength Murashige and Skoog (MS) salts with 1 mg/L benzyl amino purine (BAP) and 9 mg/L giberellic acid (GA₃). Transformants were subjected to GFP expression analysis, β-glucuronidase (GUS) histochemical assay, PCR analysis, and Southern hybridization to confirm gene integration.     

An in vitro three-dimensional coculture model of cerebral microvascular angiogenesis and differentiation

Summary The microvasculature of the developing brain is plastic and responds differently to the many insults associated with preterm birth. We developed three-dimensional in vitro culture models for the study of the responses of the developing cerebral micro-vasculature. Beagle brain microvascular endothelial cells (BBMEC) were isolated by differential centrifugation from newborn beagle pups on postnatal Day 1 and placed in three-dimensional culture dispersed in a collagen gel. Alternatively, BBMEC were placed in a three-dimensional coculture with neonatal rat forebrain astrocytes. Cultures were analyzed for extracellular matrix components at 1 and 6 d, and total RNA was extracted for Northern analyses. Urokinase plasminogen activator activity was assayed in both mono- and cocultures of the two cell types. Studies of three-dimensional BBMEC/astrocyte cocultures demonstrated progressive tube formation with only low levels of endothelial proliferation. By 6 d in three-dimensional coculture, the BBMEC formed capillarylike tubes with a wrapping of glial processes, and basement membrane protein synthesis was noted. Urokinase plasminogen zymography suggested intercellular signaling by the two cell types. These data suggest that the three-dimensional beagle brain germinal matrix microvascular endothelial cell/neonatal rat astrocyte coculture provides a good model for the investigation of microvascular responses in the developing brain.     

Ability of various inserts to promote endothelium cell culture for the establishment of coculture models

To select an insert suitable for human umbilical vein endothelial cell (HUVEC) culture, we compared several available inserts of 0.2 to 0.45 m porosity: Cellagen (ICN), Transwell-COL (Costar), Millicell-HA and CM (Millipore), Anopore (Nunc), Cyclopore (Falcon) in comparison with a control surface (Thermanox). The requirements were: (i) to promote attachment, adhesion and proliferation of HUVEC (judged by [³H]thymidine incorporation into DNA at days 1, 3, 7); (ii) to allow HUVEC visualization by inverted, fluorescence microscopy for uptake of DiI-Ac-LDL and scanning electron microscopy, performed at day 9 after seeding.Because Transwell and Cellagen are collagen precoated and CM has to be coated for cell culture, we performed collagen coating (types I + III or IV) for non-pretreated inserts for the purpose of comparison. Our preferences comprise Transwell-COL, Cyclopore not coated or coated (whatever the collagen type), and Cellagen. However, on a quality/price ratio criterion, Cyclopore, even uncoated, is the insert of choice. The HA, CM and Anopore inserts, even coated, do not allow HUVEC growth but do not alter positive uptake of acetylated LDL.Abbreviations HUVEC human umbilical vein endothelial cells     

Two new motile phototrophic consortia: “Chlorochromatium lunatum” and “Pelochromatium selenoides”

Two new phototrophic consortia, “Chlorochromatium lunatum” and “Pelochromatium selenoides”, were observed and collected in the hypolimnion of several dimictic lakes in Wisconsin and Michigan (USA). The two consortia had the same morphology but different pigment composition. The cells of the photosynthetic components of the consortia were half-moon-shaped. This morphology was used to differentiate them from the previously described motile phototrophic consortia “Chlorochromatium aggregatum” and “Pelochromatium roseum”. These phototrophic cells did not resemble any described unicellular green sulfur bacteria. The predominant pigments detected were bacteriochlorophyll d and chlorobactene for the green-colored “Clc. lunatum”, and bacteriochlorophyll e and isorenieratene for the brown-colored “Plc. selenoides”. Their pigment compositions and the presence of chlorosomes attached to the inner face of the cytoplasmic membrane in both kinds of photosynthetic cells confirmed this new half-moon-shaped morphotype as a green sulfur bacterium. Both consortia were found thriving in lakes with low concentrations of sulfide (< 60 μM), below the layers of “Clc. aggregatum” and “Plc. roseum”. The green consortia were observed in lakes where the oxic-anoxic interface was located at shallow depths (2–7 m), while the brown consortia were found at greater depths (8–16 m). The two newly described consortia were never detected together at the same depth in any lake. Received: 30 April 1997 / Accepted: 17 January 1998     

Growth factor interactions between mouse mammary cell lines cocultured in collagen gels

Summary Three related mouse mammary cell lines were cultured in collagen gels and assayed for growth factor responsiveness and interaction via soluble factors. The CL-S1 cell line is nontumorigenic and grows poorly in collagen gel culture. The +SA and −SA cell lines exhibit different degrees of malignant behavior in vivo and have different growth properties in vitro. In collagen gel culture, +SA growth was stimulated by serum but not by epidermal growth factor (EGF), whereas both serum and EGF were required for optimal growth of −SA cells of early passage number as well as CL-S1 cells. −SA cells of later passage repeatedly exhibited a change so as to no longer require serum while retaining EGF responsiveness. [¹²⁵I]EGF binding analyses indicated that CL-S1 cells bound EGF with less affinity than did −SA cells whereas +SA cells bound almost to ligand. When cell lines were maintained in separate collagen gels but shared the same culture medium, growth of +SA or −SA cells was slightly enhanced in the presence of CL-S1 cells and −SA cell growth was enhanced by the presence of +SA cells. Using the normal rat kidney fibroblast line NRK (clone 49F) as an indicator, serum-containing conditioned media from each cell line and from each pair of cell lines cultured in collagen gels were tested for transforming growth factor (TGF) activity. Both the −SA and CL-S1 lines tested positive for TGF-α production and possibly released a TGF-β activity. These results suggest mechanisms by which cell populations in and around tumors can modify one another’s growth characteristics. The work was supported by a grant from the American Institute for Cancer Research, by American Cancer Society Institutional grant IN-119, by funds from the Poncin Trust (Seattle-First National Bank), and by grants CA-39611 and CA46885 from the National Institutes of Health, Bethesda, MD.     

放线菌与枯草芽孢杆菌的共培养及其对活性次生代谢产物的影响

为探讨共培养对放线菌产生活性次生代谢产物的影响,结合抗菌活性测定及HPLC-PDA分析,研究了22株放线菌的单培养及其与枯草芽孢杆菌的共培养发酵代谢产物的差异,并选取抗菌活性较强的链霉菌FXJ2.014进一步研究其代谢产物。发现FXJ2.014、FXJ1.296、AS4.1252三株菌与枯草芽孢杆菌共培养时产生其在相同条件下单培养时没有的物质,其中链霉菌FXJ2.014单培养时主要产生醌霉素A,共培养时产物中增加了醌霉素结构类似物FXJ2.014-HB。进一步的抗菌、抗肿瘤活性测定结果表明,两者的生物活性有较显著的差异,且FXJ2.014-HB对多种肿瘤细胞系的抑制活性普遍弱于高毒性的醌霉素A,为有潜力的细胞毒性较小的抗生素。共培养是一条很有希望的发掘放线菌活性次生代谢产物的新途径。     

Enhanced chondrogenesis in a coculture system with genetically manipulated dedifferentiated chondrocytes and ATDC5 cells

Articular cartilage repair after injury is a great challenge worldwide due to its nerveless and avascular features. Tissue engineering is proposed as a promising alternative for cartilage regeneration. In this study, an adenoviral vector carrying the transforming growth factor-β3 (TGF-β3) gene was constructed and introduced into dedifferentiated chondrocytes, which were then cocultured with ATDC5 cells in an alginate hydrogel system. The results showed that the experimental groups exhibited better cell viability and higher levels of cartilage-related genes than the control groups. In this coculture system, the chondrogenic differentiation of ATDC5 cells was effectively induced by TGF-β3 and other latent cytokines that were produced by the transfected chondrocytes. Thus, this method can avoid the degradation of exogenous TGF-β3, and it can protect ATDC5 cells during virus transfection to maintain cell viability and chondrogenic differentiation capability. Taken together, this study provides fresh insights for applying this genetically manipulated coculture system to cartilage repair in the future.     

Isolation and Identification of l-Deoxy-l-(L-asparagino)-D-fructose Formed in the Autoclaved Culture Medium

To elucidate the effect of nutrition during induction on peripheral muscle responsiveness to insulin, the incorporation of radiolabeled glucose to glycogen and the uptake of radiolabeled deoxyglucose were studied in isolated diaphragms from the fetuses of normal and diabetic pregnant rats in vitro. Basal- and insulin-stimulated incorporation of [1-¹⁴C]glucose into diaphragm glycogen were greater in the fetuses of diabetic mothers (IDM) than in normal fetuses, but there was no difference in the degree of stimulation by insulin of labeled glucose into glycogen between normal fetuses and IDM. Diaphragms from normal fetuses and IDM had the same basal uptake of 2-deoxy-[1-³H]glucose as well as insulin-stimulated uptake. Consequently the sensitivity of glucose uptake to insulin was similar both in normal fetuses and IDM. These data indicate that glucose utilization (incorporation of labeled glucose into glycogen) was increased in IDM, but that the response of glucose uptake and glycogenesis to insulin was not altered.     

A three-dimensional in vitro ovarian cancer coculture model using a high-throughput cell patterning platform

In vitro 3D cancer models that provide a more accurate representation of disease in vivo are urgently needed to improve our understanding of cancer pathology and to develop better cancer therapies. However, development of 3D models that are based on manual ejection of cells from micropipettes suffer from inherent limitations such as poor control over cell density, limited repeatability, low throughput, and, in the case of coculture models, lack of reproducible control over spatial distance between cell types (e.g., cancer and stromal cells). In this study, we build on a recently introduced 3D model in which human ovarian cancer (OVCAR-5) cells overlaid on Matrigel^™ spontaneously form multicellular acini. We introduce a high-throughput automated cell printing system to bioprint a 3D coculture model using cancer cells and normal fi broblasts micropatterned on Matrigel^™. Two cell types were patterned within a spatially controlled microenvironment (e.g., cell density, cell-cell distance) in a high-throughput and reproducible manner; both cell types remained viable during printing and continued to proliferate following patterning. This approach enables the miniaturization of an established macro-scale 3D culture model and would allow systematic investigation into the multiple unknown regulatory feedback mechanisms between tumor and stromal cells and provide a tool for high-throughput drug screening.