90 Hyperthermia Induced Stress Proteins In Equine Superficial Digital Flexor Tendon
90 Hyperthermia Induced Stress Proteins In Equine Superficial Digital Flexor Tendon
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马指屈肌腱浅层中的 90 种热疗诱导应激蛋白
DOI：
10.1136/bjsports-2014-094114.89
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发表时间：
2014
影响因子：
18.4
通讯作者：
Salavati M
中科院分区：
文献类型：
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作者：
Salavati M
前肢浅指屈肌腱（SDFT）的损伤是运动马中常见的损伤（Patterson-Kaneet al.，2009年）。在高速运动过程中，SDFT的核心温度已被记录上升到48 ℃，这是由于拉伸-反冲回路滞后内的能量损失（Wilson和Goodship，1994），这被假设为细胞死亡和最终肌腱损伤的一个促成因素。为了进一步了解高温诱导损伤的机制，我们研究了通过与高温相关的应激蛋白诱导细胞凋亡。仅使用无损伤史的肌腱。将拉伸区域无菌解剖，并使用多刀片切割装置（Dudhiaet al.，2007年）。在8%O2、5%CO2和87%N2（不含血清的DMEM）中过夜培养后，通过将培养基加热至45 ℃并保持10 min，然后冷却至37 ℃来诱导高热。用10 K Ω热敏电阻传感器（Omega，UK）监测培养基温度（图1）。从对照组（37 ℃）和加热组中收集样品，加热至加热后8 h。将外植体立即固定在4%PBS缓冲福尔马林中，并使用特异性抗体进行免疫组织化学处理：连接蛋白32、膜联蛋白V、热休克蛋白25（HSP 25）、基质金属蛋白酶1（MMP 1）、ADAMts 4。Annexin V和HSP 25在2 h时表达显著升高，并在8 h时仍保持升高，与0 h和对照组相同时间点相比，加热外植体（图2）。尽管膜联蛋白V的表达显著高于对照组，但其在8小时内保持相对稳定。MMP 1是短暂的，显着增加，在2和4小时，但下降了8小时，而ADAMts 4 postheating.DiscussionA短暂发作的高温诱导早期凋亡信号（膜联蛋白V和HSP 25）在SDFT外植体的表达没有显着变化。连接蛋白-32的上调表明间隙连接的形成增加，这可能促进细胞间凋亡信号转移的增加，并协调凋亡向邻近腱细胞的扩散。2007;6（4）：547-556Patterson-Kane JC，EC Firth.Vet J. 2009;181（2）：79-89Wilson AM，Goodship AE.J Biomech. 1994;27（7）：899-905
IntroductionInjuries of forelimb superficial digital flexor tendon (SDFT) are a common injury in athletic horses (Patterson-Kaneet al., 2009). During high speed locomotion the core temperature of SDFT has been recorded to rise up to 48oC, as a result of energy loss within the hysteresis of the stretch-recoil loop (Wilson and Goodship, 1994), which has been hypothesised as a contributing factor to cell death and ultimately tendon damage. To further understand the mechanism of hyperthermia induced damage we investigated the induction of apoptosis via stress proteins associated with hyperthermia.MethodsEquine SDFT (from horses aged 7 ± 1.5 years) were obtained from an abattoir. Only tendons with no history of injury were used. The tensile region was dissected aseptically and cut into 20 mm x 2 mm x 2 mm explants using a multiple blade cutting apparatus (Dudhiaet al., 2007). After overnight culture in 8% O2, 5% CO2and 87% N2(DMEM with no serum) hyperthermia was induced by heating the media to 45oC and holding for 10 min before cooling to 37oC. Media temperature was monitored with 10KΩ thermistor sensors (Omega, UK) (Figure 1). Samples were collected from both control (37oC) and the heated group up to 8 h after heating. Explants were immediately fixed in 4% PBS buffered formalin and processed for immunohistochemistry using specific antibodies: Connexin-32, Annexin V, Heat shock protein 25 (HSP25), Matrix metalloproteinase 1 (MMP1), ADAMts4.ResultsConnexin-32, Annexin V and HSP25 were expressed significantly higher by 2 h and remained elevated up to 8 h compared to 0 h and the equivalent control time point in heated explants (Figure 2). Although Annexin V expression was significantly higher than the control group it remained relatively stable up to 8 h. MMP1 was transiently and significantly increased at 2 and 4 h but declined by 8 h, whereas there was no significant change in the expression of ADAMts4 post-heating.DiscussionA transient episode of hyperthermia induced early apoptotic signals (Annexin V and HSP25) in SDFT explants. The upregulation of Connexin-32 suggests increased formation of gap junctions which may facilitate an increase in transfer of apoptotic signals between cells and orchestrate the spread of apoptosis to neighbouring tenocytes.ReferencesDudhia J,et al. Aging Cell. 2007;6(4):547–556Patterson-Kane JC, EC Firth.Vet J. 2009;181(2):79–89Wilson AM, Goodship AE.J Biomech. 1994;27(7):899–905