An insert-based enzymatic cell culture system to rapidly and reversibly induce hypoxia: investigations of hypoxia-induced cell damage, protein expression and phosphorylation in neuronal IMR-32 cells.
An insert-based enzymatic cell culture system to rapidly and reversibly induce hypoxia: investigations of hypoxia-induced cell damage, protein expression and phosphorylation in neuronal IMR-32 cells.
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DOI：
10.1242/dmm.013078
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发表时间：
2013-11
影响因子：
4.3
通讯作者：
Albrecht M
中科院分区：
文献类型：
--
作者：
Huang Y;Zitta K;Bein B;Steinfath M;Albrecht M
缺血-再灌注损伤和组织缺氧具有高度的临床相关性，因为它们与各种病理生理状况如心肌梗死和中风相关。然而，引起细胞损伤的潜在机制仍然没有完全理解，这至少部分是由于缺乏用于诱导快速和短暂缺氧条件的细胞培养系统。本研究的目的是建立一个模型，是适合于短暂和长期缺氧相关的细胞和分子效应的调查，并获得采用神经元培养系统的缺氧介导的机制的见解。采用半透膜插入系统与缺氧诱导酶葡萄糖氧化酶和过氧化氢酶组合，以在培养基中快速且可逆地产生缺氧条件。进行过氧化氢测定、葡萄糖测量和蛋白质印迹以验证系统并评估所产生的缺氧对神经元IMR-32细胞的影响。使用基于插入的双酶模型，在培养基中迅速诱导缺氧条件。葡萄糖浓度逐渐降低，而过氧化氢水平没有改变。此外，可以通过添加和随后去除含酶插入物来进行快速和可逆（开-关）的缺氧产生。采用神经元IMR-32细胞，我们发现，3小时的缺氧导致细胞损伤的形态学迹象和乳酸脱氢酶（细胞损伤的生化标志物）的水平显着增加。低氧条件也增加了细胞的半胱氨酸天冬氨酸蛋白酶原-3和过氧化氢酶的量，以及促生存激酶Akt的磷酸化，但不是Erk 1/2或STAT 5。总之，我们提出了一个新的框架，在细胞水平上研究缺氧介导的机制。我们声称，该模型是同类模型中的第一个，使研究人员能够在体外快速可逆地诱导缺氧条件，而不会对培养细胞产生缺氧诱导剂的不必要干扰。该系统可以帮助进一步解开与各种组织和器官临床相关的缺氧相关机制。
Ischemia-reperfusion injury and tissue hypoxia are of high clinical relevance because they are associated with various pathophysiological conditions such as myocardial infarction and stroke. Nevertheless, the underlying mechanisms causing cell damage are still not fully understood, which is at least partially due to the lack of cell culture systems for the induction of rapid and transient hypoxic conditions. The aim of the study was to establish a model that is suitable for the investigation of cellular and molecular effects associated with transient and long-term hypoxia and to gain insights into hypoxia-mediated mechanisms employing a neuronal culture system. A semipermeable membrane insert system in combination with the hypoxia-inducing enzymes glucose oxidase and catalase was employed to rapidly and reversibly generate hypoxic conditions in the culture medium. Hydrogen peroxide assays, glucose measurements and western blotting were performed to validate the system and to evaluate the effects of the generated hypoxia on neuronal IMR-32 cells. Using the insert-based two-enzyme model, hypoxic conditions were rapidly induced in the culture medium. Glucose concentrations gradually decreased, whereas levels of hydrogen peroxide were not altered. Moreover, a rapid and reversible (onoff) generation of hypoxia could be performed by the addition and subsequent removal of the enzyme-containing inserts. Employing neuronal IMR-32 cells, we showed that 3 hours of hypoxia led to morphological signs of cellular damage and significantly increased levels of lactate dehydrogenase (a biochemical marker of cell damage). Hypoxic conditions also increased the amounts of cellular procaspase-3 and catalase as well as phosphorylation of the pro-survival kinase Akt, but not Erk1/2 or STAT5. In summary, we present a novel framework for investigating hypoxia-mediated mechanisms at the cellular level. We claim that the model, the first of its kind, enables researchers to rapidly and reversibly induce hypoxic conditions in vitro without unwanted interference of the hypoxia-inducing agent on the cultured cells. The system could help to further unravel hypoxia-associated mechanisms that are clinically relevant in various tissues and organs.
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影响因子：
3.4
作者：
Baumann RP;Penketh PG;Seow HA;Shyam K;Sartorelli AC
通讯作者：
Sartorelli AC
影响因子：
2.5
作者：
Delivoria-Papadopoulos, Maria;Ashraf, Qazi M.;Mishra, Om P.
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影响因子：
3.4
作者：
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作者：
Gotoh M;Sano-Maeda K;Murofushi H;Murakami-Murofushi K
通讯作者：
Murakami-Murofushi K
影响因子：
2.5
作者：
Abudara, V;Jiang, RG;Eyzaguirre, C
通讯作者：
Eyzaguirre, C