不同低温对大鼠脊髓缺血再灌注损伤后Bax及Bcl-2蛋白表达的影响

Differential effects of mild hypothermia and deep Hypothermia on the expression of Bax and Bcl-2 in rat spinal cord ischemia- reperfusion injury

目的：观察不同低温对大鼠脊髓缺血再灌注损伤的保护作用及其对凋亡蛋白Bax、Bcl-2表达的影响。方法将60只SD大鼠随机分为4组，低温18℃实验组（深低温组），低温28℃实验组（中低温组），常温手术对照组（常温组），常温假手术组（假手术组）。前3组采用手术夹闭大鼠左肾动脉与左右髂总动脉分叉上端腹主动脉90 min致脊髓缺血，然后松开再灌注，深低温组、中低温组在阻断前20 min分别降至18、28℃，解除阻断后30 min内将体温升至37.0℃。常温组阻断前无需降温每只大鼠再灌注72 h、1周按照TARLOV法对下肢进行评分。每组分为24 h、72 h、1周3个不同时间点取L1-L5段脊髓，每个时间点5只大鼠，采用普通病理HE染色、Nissl染色，从形态学上观察脊髓组织，TUNEL法观察细胞凋亡情况，免疫组化染色及Western blot法观察脊髓组织Bax蛋白及Bcl-2蛋白表达情况。结果 TARLOV神经评分结果显示，深低温组、中低温组下肢恢复情况明显好于常温组（P＜0.01），而中低温组评分又高于深低温组（P＜0.01）。组织形态学观察假手术组未发现明显病理学改变，深低温组、中低温组、常温组3组均出现不同程度病理改变，且中低温组轻于深低温组，深低温组组轻于常温组；TUNEL结果显示，假手术组几乎未发现凋亡细胞，常温组神经元细胞凋亡情况较深低温组及中低温组明显。免疫组化及Western blot结果显示，深低温组、中低温组各时间点Bax蛋白表达量均低于常温组，但中低温组降低比深低温组明显，组间两两比较，差异有统计学意义（ P＜0.05）。深低温组、中低温组、假手术组各时间点Bcl-2蛋白表达量均高于常温组，且中低温组较深低温组高，组间两两比较，差异有统计学意义（ P＜0.05）。结论低温保护下肢神经功能及减少神经元的死亡，对大鼠脊髓缺血再灌注损伤有保护作用，并且可能通过抑制Bax蛋白的表达及活化Bcl-2蛋白的表达，抑制神经元凋亡发挥其保护作用。过低温度则可能通过抑制Bcl-2蛋白表达而使低温保护效果降低。

Objective To evaluate the expression changes of Bax and Bcl2 after spinal cord ischemia reperfusion injury （ SCII） and exposition in the hypothermia during the operation in the rats.Methods Sixty rats were randomly di-vided into 4 groups：Group A （18℃ administration）, Group B （28℃ administration）, Group C （normal atmospheric temperature） and Group D （ sham operation controls） .Spinal cord ischemia was induced by clamping the aorta both below the left renal artery and above the aortic bifurcation for 90min.Hypothermia was induced 15 minutes before ischemia and maintained during ischemia, then rewarmed to normothermia after reperfusion in 30 min.The motor function of the lower limbs was evaluated according to Tarlov Score in at 72nd hour and 1st week.The spinal cord samples were taken at the 24th hour, 72nd hour and 1st week for evaluation of the histopathological changes, neuron apoptosis and the expression of Bax and Bcl-2 by using hematoxylin staining, Nissl staining, TNUEL, immunohistochemistry and western blot analysis. Results Hypothermia significantly improved hind limb function at Tarlov Score evaluation, and higher score was observed in Group B than Group A.No neurologic deficit was observed in Group D, while various degrees of neurologic deficit were＆amp;nbsp;observed in the other 3 groups.More apopotosis neurons, higher Bax expression, whereas lower Bcl-2 expression were observed in Group C than other 3 Groups.Compared with Group A, Bax was less expressed while Bcl-2 higher in Group B, with less apopotosis neurons.Conclusion Hypothermia preserves hind limb motor function and reduces neuronal deaths, which plays an important protective role in SCII in rat.It may protects the spinal cord from SCII through the inhi-bition of Bax expression and activation of Bcl-2.But deep hypothermia may somewhat inhibit the expression of Bcl-2, so that shows a worse result than mild hypothermia.