32 Hypoxia Protects Against Hypertermia-induced Stress In Equine Flexor Tendon Tenocytes
32 Hypoxia Protects Against Hypertermia-induced Stress In Equine Flexor Tendon Tenocytes
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32 缺氧可以防止马屈肌腱细胞的高温引起的应激
DOI：
10.1136/bjsports-2014-094114.32
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发表时间：
2014
影响因子：
18.4
通讯作者：
Dudhia J
中科院分区：
文献类型：
--
作者：
Dudhia J
趾浅屈肌腱（SDFT）损伤是马最常见的骨科损伤之一。SDFT适合作为能量储存肌腱，以帮助高速运动，然而，重复的周期性加载可导致核心肌腱温度达到48°c .2这样的高温可能通过凋亡途径诱导细胞损伤和细胞活力丧失，使肌腱易于永久性损伤。然而，生理上相关的氧条件对细胞应激途径的影响尚未被研究。我们假设与大气（20.9%）氧气水平下的培养相比，低氧培养条件会减弱马SDFT细胞的热疗诱导应激。方法将sequine SDFT细胞培养在含10%胎牛血清的DMEM中，在37°C、20%和8%的氧气条件下，在> - 75%的合流度下，然后通过将温度升高至45°C，短暂诱导热疗10分钟。热休克蛋白25 （HSP-25）、HSP-72（保护性应激途径）和Daxx（死亡结构域相关蛋白）在加热后1、2、4和8 h的表达通过免疫细胞化学检测，Annexin A1（凋亡相关蛋白）在免疫细胞化学中的表达。使用AlamarBlue®（Promega， UK）评估细胞活力。结果热休克后，shsp -25和HSP-72的表达分别增加了2倍和10倍（图1），而Daxx的表达保持不变（数据未显示）。无论温度条件如何，Annexin A1同型二聚体（核异构体）在20%氧气条件下的表达高于8%氧气条件（图1）。膜联蛋白A1的单体形式在两组中丰度都很低，并且不受加热的影响（数据未显示）。加热24 h后，细胞增殖开始下降，但72 h后细胞恢复。在20%氧气条件下，恢复速率延迟至加热后48 h。(图2)。热休克诱导热休克蛋白25和热休克蛋白75对高温的反应表明，在小细胞中存在一种减少细胞损伤的保护机制。在生理氧水平（8%）下，这种反应尤其急性，其中膜联蛋白A1同型二聚体的表达减少和Daxx诱导的缺乏进一步表明，细胞对短时热疗具有抗性。此外，在20%的氧气条件下，增殖恢复延迟。然而，反复的体内高温是否减轻了小细胞对恢复的抵抗力还需要进一步的研究。参考文献1 Patterson-Kane JC， Firth EC。兽医J. 2009；李建军，张建军，张建军，等。生物医学杂志。1994；27 (7): 899 - 905
IntroductionSuperficial digital flexor tendon (SDFT) damage is one of the most common orthopaedic injuries in horses. The SDFT is adapted as an energy storing tendon to aid high-speed locomotion, however repetitive cyclical loading can result in core tendon temperatures reaching 48°C.2Such high temperatures may induce cell damage via apoptotic pathways and loss of cell viability, predisposing the tendon to permanent damage.1However the effect of physiologically relevant oxygen conditions on tenocyte stress pathways has not been investigated. We hypothesised that hypoxic culture condition will attenuate hyperthermia induced stress in equine SDFT tenocytes compared to cultures in atmospheric (20.9%) oxygen levelsMethodsEquine SDFT cells were cultured in DMEM containing 10% foetal bovine serum at 37 °C in both 20% and 8% oxygen conditions at >75% confluency, before transiently inducing hyperthermia by elevating the temperature to 45 °C for 10 min. Expression of heat shock protein-25 (HSP-25), HSP-72 (protective stress pathways) and Daxx (Death domain-associated protein) was assayed by western blot and Annexin A1 (apoptosis associated protein) by immunocytochemistry at 1, 2, 4 and 8 h post heating. The viability of the cells was assessed using AlamarBlue®(Promega, UK).ResultsHSP-25 and HSP-72 expression increased over time post heat-shock by 2- and 10-fold respectively (Figure 1), whereas Daxx expression remained unchanged (data not shown) Annexin A1 homodimer (nuclear isoform) had constitutive higher expression in 20% oxygen compared to 8% regardless of temperature conditions (Figure 1). The monomeric form of Annexin A1 was of low abundance in both groups and was not affected by heating (data not shown). Tenocyte proliferation was initially decreased after heating at 24 h but the cells showed recovery by 72 h. Recovery rates were delayed in 20% oxygen up to 48 h post heating. (Figure 2).DiscussionThe induction of HSP-25 and HSP-75 in response to hyperthermia suggests a protective mechanism exists in tenocytes to reduce cell damage. The response is particularly acute under physiological oxygen levels (8%) where the reduced expression of Annexin A1 homodimer and the lack of Daxx induction further suggests that tenocytes are resistant to a short episode hyperthermia. In addition the recovery of proliferation was delayed under 20% oxygen. However, whether repeat bouts of hyperthermiain vivoalleviate the resistance of tenocyte to recover needs further investigation.References1 Patterson-Kane JC, Firth EC.Vet J. 2009;181(2):79–892 Wilson AM, Goodship AE.J Biomech. 1994;27(7):899–905