低溫療法保護心肌細胞對抗缺血缺氧再灌流之機轉

Hypothermia Therapy Protect against Ischemia Hypoxia Reperfusion Injury in Cardiomyocyte

動物實驗及醫學研究利用低溫療法範圍在31°C至34°C可以有效降低心肌損傷及頭部外傷，但低溫療法對於減少心肌細胞凋亡壓力之詳細機轉目前尚未闡明。因此本實驗目的在於闡明低溫療法是否可以透過降低細胞凋亡改善心肌細胞缺血再灌流引發之細胞死亡，進而保護心臟的功效。心肌細胞取自胎鼠心肌組織分離出之細胞株(H9c2)。本實驗分為三組:控制組、缺血再灌流組、低溫治療組。藉由心肌細胞缺血缺氧再灌流模式，透過膠體電泳分析去氧核糖核苷酸(Deoxyribonucleicl acid, DNA)片段化、利用鈣依賴型磷脂結合蛋白(Annexin V)及碘化丙啶(Propidium Iodide, PI)雙染法並使用流式細胞儀偵測細胞凋亡、利用西方墨點法分析半胱胺酸天門冬胺酸蛋白酶(Cysteine asparate protease-3, Caspase-3)蛋白質的表現量、利用酵素結合免疫吸附法(Enzyme-linked immunosorbent assay, ELISA) 分析Caspase-3蛋白質的活性。實驗結果顯示缺血再灌流組與控制組比較，細胞密度減少、細胞存活率明顯減少。造成細胞死亡的原因是透過增加細胞凋亡、DNA產生片段化及caspase-3d 蛋白質表現量增加並且活化。低溫治療組可有效降低心肌缺血再灌流所誘導之Caspase-3活化作用，並降低細胞凋亡及DNA片斷化。以上結果顯示，低溫療法確實可以有效降低缺血缺氧再灌流損傷所導致的細胞死亡，達到心肌細胞保護作用。

Animal models and clinical studies have shown that hypothermia in the range of 31 to 34°C significantly limits myocardial and traumatic brain injury. The effect of hypothermia on cardiomyocyte injury induced by apoptotic stress remains unclear. We attempted to ascertain whether hypothermia can protect cardiomyocytes against ischemia hypoxia–induced cell apoptosis. This was an experimental study utilizing cardiomyocytes (H9c2) from an embryonic rat heart–derived cell line. Cell lines were dived into three groups: Normal control group (cell lines under normal ambient temperature and normoxia), ischemia and reperfusion group (I/R, cells were incubated in glucose-serum free medium under 37℃-0.2% O2 condition for 24 hours, and then reoxygenation to 21% O2), hypothermia therapy group (H, cell lines were incubated under 32℃at the start of reperfusion). DNA fragmentation was assayed by gel electrophoresis. Apoptotic cells were detected by both Annexin V and propidium iodide (PI) double labeling, and analyzed with a flow cytometer. Cysteine asparate protease-3 (Caspase-3) expression was detected by Western blot and caspase-3 activation as determined by enzyme-linked immunosorbent assay (ELISA). It was found that, compared with normal control, I/R group showed significantly decreased cell density and cell viability via enhanced cell apoptosis, DNA fragmentation and caspase-3 overexpression and activation. Hypothermia therapy adopted at the start of reperfusion significantly reduced the I/R-induced apoptosis and caspase-3 activation in H9c2 cells. These results suggest that ischemia and reperfusion injury in H9c2 can be attenuated by hypothermia in vitro.