24 research outputs found
Validation of biological recognition elements for signal transduction as first step in the development of whole cell biosensors
Choosing the proper combination of receptor element, cell type and measurable signal requires major consideration for developing cell-based biosensors. In order to use physiologically relevant cellular responses towards (geno)toxic conditions, information on the mechanism of action and of the expected outcome of exposure needs to be considered
Radiation Response of Murine Embryonic Stem Cells
To understand the mechanisms of disturbed differentiation and development by
radiation, murine CGR8 embryonic stem cells (mESCs) were exposed to ionizing radiation and
differentiated by forming embryoid bodies (EBs). The colony forming ability test was applied for
survival and the MTT test for viability determination after X-irradiation. Cell cycle progression was
determined by flow cytometry of propidium iodide-stained cells, and DNA double strand break
(DSB) induction and repair by ÎłH2AX immunofluorescence. The radiosensitivity of mESCs was
slightly higher compared to the murine osteoblast cell line OCT-1. The viability 72 h after
X-irradiation decreased dose-dependently and was higher in the presence of leukemia inhibitory
factor (LIF). Cells exposed to 2 or 7 Gy underwent a transient G2 arrest. X-irradiation induced
ÎłH2AX foci and they disappeared within 72 h. After 72 h of X-ray exposure, RNA was isolated and
analyzed using genome-wide microarrays. The gene expression analysis revealed amongst others a
regulation of developmental genes (Ada, Baz1a, Calcoco2, Htra1, Nefh, S100a6 and Rassf6),
downregulation of genes involved in glycolysis and pyruvate metabolism whereas upregulation of
genes related to the p53 signaling pathway. X-irradiated mESCs formed EBs and differentiated
toward cardiomyocytes but their beating frequencies were lower compared to EBs from
unirradiated cells. These results suggest that X-irradiation of mESCs deregulate genes related to the
developmental process. The most significant biological processes found to be altered by
X-irradiation in mESCs were the development of cardiovascular, nervous, circulatory and renal
system. These results may explain the X-irradiation induced-embryonic lethality and
malformations observed in animal studies
Hypergravity attenuates Reactivity in Primary Murine Astrocytes
Neuronal activity is the key modulator of nearly every aspect of behavior, affecting cognition, learning, and memory as well as motion. Hence, disturbances of the transmission of synaptic signals are the main cause of many neurological disorders. Lesions to nervous tissues are associated with phenotypic changes mediated by astrocytes becoming reactive. Reactive astrocytes form the basis of astrogliosis and glial scar formation. Astrocyte reactivity is often targeted to inhibit axon dystrophy and thus promote neuronal regeneration. Here, we aim to understand the impact of gravitational loading induced by hypergravity to potentially modify key features of astrocyte reactivity. We exposed primary murine astrocytes as a model system closely resembling the in vivo reactivity phenotype on custom-built centrifuges for cultivation as well as for live-cell imaging under hypergravity conditions in a physiological range (2g and 10g). We revealed spreading rates, migration velocities, and stellation to be diminished under 2g hypergravity. In contrast, proliferation and apoptosis rates were not affected. In particular, hypergravity attenuated reactivity induction. We observed cytoskeletal remodeling of actin filaments and microtubules under hypergravity. Hence, the reorganization of these key elements of cell structure demonstrates that fundamental mechanisms on shape and mobility of astrocytes are affected due to altered gravity conditions. In future experiments, potential target molecules for pharmacological interventions that attenuate astrocytic reactivity will be investigated. The ultimate goal is to enhance neuronal regeneration for novel therapeutic approache
Streamlining Culture Conditions for the Neuroblastoma Cell Line SH-SY5Y: A Prerequisite for Functional Studies
The neuroblastoma cell line SH-SY5Y has been a well-established and very popular in vitro model in neuroscience for decades, especially focusing on neurodevelopmental disorders, such as Parkinsonâs disease. The ability of this cell type to differentiate compared with other models in
neurobiology makes it one of the few suitable models without having to rely on a primary culture of neuronal cells. Over the years, various, partly contradictory, methods of cultivation have been reported. This study is intended to provide a comprehensive guide to the in vitro cultivation of undifferentiated SH-SY5Y cells. For this purpose, the morphology of the cell line and the differentiation of the individual subtypes are described, and instructions for cell culture practice and long-term cryoconservation are provided. We describe the key growth characteristics of this cell line, including proliferation and confluency data, optimal initial seeding cell numbers, and a comparison of different
culture media and cell viability during cultivation. Furthermore, applying an optimized protocol in a long-term cultivation over 60 days, we show that cumulative population doubling (CPD) is constant over time and does not decrease with incremental passage, enabling stable cultivation, for example, for recurrent differentiation to achieve the highest possible reproducibility in subsequent analyses. Therefore, we provide a solid guidance for future research that employs the neuroblastoma cell line
SH-SY5
Hypergravity attenuates Reactivity in Primary Murine Astrocytes
Neuronal activity is the key modulator of nearly every aspect of behavior, affecting cognition, learning and memory as well as motion. Alterations or even disruptions of the transmission of synaptic signals are the main cause of many neurological disorders. Lesions to nervous tissues are associated with phenotypic changes mediated by astrocytes becoming reactive. Reactive astrocytes form the basis of astrogliosis and glial scar formation. Astrocyte reactivity is often targeted to inhibit axon dystrophy and thus promote neuronal regeneration. Here, we use increased gravitational (mechanical) loading induced by hypergravity to identify a potential method to modify key features of astrocyte reactivity. We exposed primary murine astrocytes as a model system closely resembling the reactivity phenotype in vivo on custom-built centrifuges for cultivation as well as for livecell imaging under hypergravity conditions in a physiological range (2g and 10g). This resulted in significant changes to astrocyte morphology, behavior and reactivity phenotypes, with the ultimate goal being to enhance neuronal regeneration for novel therapeutic approaches
The Use of ProteoTuner Technology to Study Nuclear Factor ÎșB Activation by Heavy Ions
Nuclear factor ÎșB (NF-ÎșB) activation might be central to heavy ion-induced detrimental processes such as cancer promotion and progression and sustained inflammatory responses. A sensitive detection system is crucial to better understand its involvement in these processes. Therefore, a DD-tdTomato fluorescent protein-based reporter system was previously constructed with human embryonic kidney (HEK) cells expressing DD-tdTomato as a reporter under the control of a promoter containing NF-ÎșB binding sites (HEK-pNFÎșB-DD-tdTomato-C8). Using this reporter cell line, NF-ÎșB activation after exposure to different energetic heavy ions (Âčâ¶O, 95 MeV/n, linear energy transferâLET 51 keV/”m; ÂčÂČC, 95 MeV/n, LET 73 keV/ÎŒm; Âłâ¶Ar, 95 MeV/n, LET 272 keV/”m) was quantified considering the dose and number of heavy ions hits per cell nucleus that double NF-ÎșB-dependent DD-tdTomato expression. Approximately 44 hits of Âčâ¶O ions and â45 hits of ÂčÂČC ions per cell nucleus were required to double the NF-ÎșB-dependent DD-tdTomato expression, whereas only â3 hits of Âłâ¶Ar ions were sufficient. In the presence of Shield-1, a synthetic molecule that stabilizes DD-tdTomato, even a single particle hit of Âłâ¶Ar ions doubled NF-ÎșB-dependent DD-tdTomato expression. In conclusion, stabilization of the reporter protein can increase the sensitivity for NF-ÎșB activation detection by a factor of three, allowing the detection of single particle hitsâ effects
Streamlining Culture Conditions for the Neuroblastoma Cell Line SH-SY5Y: A Prerequisite for Functional Studies
The neuroblastoma cell line SH-SY5Y has been a well-established and very popular in vitro model in neuroscience for decades, especially focusing on neurodevelopmental disorders, such as Parkinson’s disease. The ability of this cell type to differentiate compared with other models in neurobiology makes it one of the few suitable models without having to rely on a primary culture of neuronal cells. Over the years, various, partly contradictory, methods of cultivation have been reported. This study is intended to provide a comprehensive guide to the in vitro cultivation of undifferentiated SH-SY5Y cells. For this purpose, the morphology of the cell line and the differentiation of the individual subtypes are described, and instructions for cell culture practice and long-term cryoconservation are provided. We describe the key growth characteristics of this cell line, including proliferation and confluency data, optimal initial seeding cell numbers, and a comparison of different culture media and cell viability during cultivation. Furthermore, applying an optimized protocol in a long-term cultivation over 60 days, we show that cumulative population doubling (CPD) is constant over time and does not decrease with incremental passage, enabling stable cultivation, for example, for recurrent differentiation to achieve the highest possible reproducibility in subsequent analyses. Therefore, we provide a solid guidance for future research that employs the neuroblastoma cell line SH-SY5Y
Die Etablierung der Neuroblastomzelllinie SY5Y als Modellsystem zur AbschÀtzung strahleninduzierter SchÀden neuronaler Zellen bei einer bemannten Marsmission
Die kosmische Strahlung ist der gröĂte Risikofaktor fĂŒr die bemannte Raumfahrt, insbesondere
fĂŒr Langzeitmissionen wie die Reise zum Mars. Die kosmische Strahlung ist in sich inhomogen
und setzt sich aus verschiedensten Komponenten und StrahlenqualitÀten zusammen. Eine
Vorhersage von Hoch-Dosis-Ereignissen wie SonnenstĂŒrmen ist schwierig bis unmöglich. Eine
ausreichende Abschirmung ist derzeit technisch nicht umsetzbar und beim heutigen Stand der
Entwicklung sogar kontraproduktiv. Daher muss das VerstÀndnis der Wirkung ionisierender
Strahlung auf den Menschen und insbesondere auf das nur wenig erforschte Gehirn vertieft
werden.
In dieser Arbeit wird die Neuroblastomzelllinie SY5Y als mögliches Modellsystem fĂŒr die
Auswirkungen ionisierender Strahlung auf das menschliche Gehirn untersucht sowie zell- und
strahlenbiologisch etabliert
Induktion von neuronaler Regeneration durch Hypergravitation
Einleitung:
Eine uneingeschrĂ€nkte Funktion von neuronalen Zellen ist unerlĂ€sslich fĂŒr jeden Aspekt des menschlichen Verhaltens. Wahrnehmung, Erinnerung, Lernen sowie jegliche Art von Bewegung sind grundsĂ€tzlich von der AktivitĂ€t unseres Nervensystems abhĂ€ngig. Eine Ursache oder Konsequenz der meisten neurologischen Erkrankungen sind daher VerĂ€nderungen oder Störungen der synaptischen AktivitĂ€t.
Um wirksame Behandlungen von neurologischen Störungen zu entwickeln, mĂŒssen synaptische Signale wieder in das zuvor verletzte neuronale Netzwerk integriert werden. Besonders nach schweren LĂ€sionen, wie z.B. nach Traumata und RĂŒckenmarksverletzungen, sowie durch Beeinflussung von Tumorgewebe oder epileptischen AnfĂ€llen wird vermehrt Narbengewebe gebildet, welches die Regeneration von neuronalen AuslĂ€ufern und damit synaptischen Signalen in das verletzte Gewebe nahezu vollstĂ€ndig inhibiert. Diese Art von Verletzung kann daher zu irreparablen SchĂ€den der mentalen oder physischen LeistungsfĂ€higkeit von Patienten fĂŒhren.
Fragestellung:
Wir streben an, den sehr ineffizienten Vorgang der neuronalen Regeneration nach Verletzungen von Nervengewebe durch die Exposition in Hypergravitation zu induzieren.
Unsere Hypothese ist, dass artifiziell erhöhte Schwerkraft eine potentiell sehr innovative und wirkungsvolle Methode darstellen könnte, um Komponenten des neuronalen Zytoskelettes zu stabilisieren, was weiterhin dazu fĂŒhren sollte, dass die Projektionen der Nervenzellen der Inhibition des neuronalen Narbengewebes entgegenwirken und in gesteigertem MaĂe wachsen (regenerieren) können. Daher sollten die AuslĂ€ufer unter Einfluss von Hypergravitation viel ausgeprĂ€gter in der Lage sein auszuwachsen und sich anschlieĂend neu in geschĂ€digtes Gewebe zu integrieren.
Weiterhin soll die neuronale Entwicklung und AktivitĂ€t bei Exposition mit ionisierender Strahlung untersucht werden, um Strahlung als Risikofaktor fĂŒr neurodegenerative Erkrankungen z.B. in der bemannten Raumfahrt zu bewerten.
Methodik:
In der vorliegenden Studie werden primĂ€re murine hippokampale Neuronen eingesetzt, welche ein nah-verwandtes Modell-System fĂŒr humane Nervenzellen darstellen. Die neuronale Entwicklung unter dem Einfluss von Hypergravitation (2g) und Strahlenexposition wird bei allen Entwicklungsstadien im Vergleich zu Kontrollen (1g, unbestrahlt) untersucht, wie z.B. das Neuritenwachstum, die Polarisation, die Synaptogenese, sowie abschlieĂend die Integration in ein maturiertes, funktionelles neuronales Netzwerk.
Ergebnisse:
Die Exposition von primĂ€ren Neuronen an erhöhte Gravitation (2g) induzierte ein gesteigertes Auswachsen initialer Neuriten (ca. 30%), sowie ein erhöhtes Neuriten-Wachstum (Elongation) (ca. 20%) im Vergleich zu Kontrollen bei 1g. In spĂ€teren Entwicklungsstadien wurden trotz potentiellen VerĂ€nderungen des neuronalen Zytokslettes maturierte synaptische Kontakte ausgebildet. Weiterhin wurden primĂ€re Astrozyten (Glia-Zellen, die neuronales Narbengewebe nach Verletzungen bilden) durch Hypergravitation in ihrem Wachstum und ihrer Ausbreitung (Narbenbildung) gehemmt. Diese Beobachtungen sind in groĂer Ăbereinstimmung mit einem stabilisierten Tubulin- und einem destabilisierten Aktin-Zytoskelett.
Schlussfolgerungen:
Unsere Ergebnisse belegen, dass neuronale Regeneration durch den Einfluss von Hypergravitation in primĂ€ren Neuronen induziert und das Wachstum von primĂ€ren Astrozyten (Glia-Zellen) durch die Kultivierung unter Hypergravitationsbedingungen gehemmt wird. Dieser Ansatz kann fĂŒr weitere Studien angewandt werden, um die grundlegenden Mechanismen aufzuklĂ€ren und die Effizienz neuronaler Regenerationsprozesse steigern zu können
Cell death bypass mechanisms in DNA damage response of mammalian cells after exposure with heavy ions relevant for Space radiation environment
For humans in space, there are two limiting factors namely the microgravity and the galactic cosmic radiation. For sake of better risk assessment for the astronaut, the cellular response to such ionizing radiation qualities, as predominate in Space, needs to be better understood. One of the key elements to be investigated is the cell cycle control as a central element in DNA damage response being a central switch for cellular decision making between life and death. Thus cell death bypass mechanisms in DNA damage response of mammalian cells will be investigated after heavy ion exposure relevant for the Space radiation environment. The RBE-LET dependency will be determined for different biological endpoints. Of special interest are cellular survival, the activation of transcription factors and the identification of genes expressed in DNA damage response after high LET exposure.ćčłæ26ćčŽćșŠæŸć°ç·ć»ćŠç·ćç 究æéçČćç·ăăæČ»çèŁ
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