【作者】 周桔；

【导师】 瞿树林； 汤长发；

【作者基本信息】 湖南师范大学 ， 运动人体科学， 2007， 硕士

【摘要】 目的：通过采用不同持续时间低氧后训练的大鼠低氧训练模型，研究低氧和／或训练对大鼠心肌细胞低氧诱导因子(Hypoxia Induced Factor-1α，HIF-1α)及对凋亡相关因子的影响，比较低氧和／或训练过程中大鼠心肌细胞HIF-1α及凋亡相关因子Bcl-2、Bax蛋白表达的变化及相关性。方法：60只SD大鼠随机分为正常对照组(A)、低氧8h组(B)、低氧12h组(C)、常氧训练组(D)、低氧8h训练组(E)和低氧12h训练组(F)，(n=10)。D、E、F组大鼠每天在坡度为0的动物跑台上以25m／min的速度训练1h。训练完后，将B、E组和C、F组放入氧浓度为12.5％(相当于海拔4000m)的低氧舱内8h和12h。实验期为4wk，5d／wk。按实验设计，所有大鼠均在实施腹腔注射，麻醉理想后断头处死，取全血检测RBC、Hb、HCT值，取心肌组织制备心肌组织石蜡切片，用免疫组化法检测大鼠心肌细胞HIF-1α、心肌凋亡细胞和B细胞淋巴瘤／白血病癌基因-2(B cell lymphoma／leukemia 2，Bcl-2)、Bax(Bcl-2 associated x,Bax)因子的蛋白表达以及TUNEL标记法在光镜下观察凋亡心肌细胞核数目。结果：(1)检测RBC、Hb、HCT三个指标的结果显示：B组与E组比较均具有显著性差异(P＜0.05)；A组和D组比较亦均具有显著性差异(P＜0.05)。(2)心肌组织切片进行HE染色显示，正常对照组(A)和低氧8h组(B)大鼠心肌细胞结构完整，核大，呈圆形；低氧8h训练组(E)、低氧12h组(C)大鼠的心肌细胞结构逐渐变化；低氧12h训练组(F)、常氧训练组(D)可见大鼠心肌细胞明显肿胀，间隙明显增大，核变小，不规则。(3)TUNEL染色切片统计结果显示：E组和F组比较，具有显著性差异(P＜0.05)；A组与D组比较，具有显著性差异(P＜0.05)。(4)免疫组化染色切片观察大鼠心肌组织HIF-1α的蛋白表达显示：将实验各组心肌样本的石蜡切片进行免疫组化染色，在显微镜下观察发现：HIF-1α免疫组织化学阳性物质定位于细胞核内，呈弥散或颗粒状或二者混合。呈强染性的心肌细胞核中可见丰富的阳性反应颗粒，细胞核周围的胞浆内亦有阳性表达。统计结果显示：B组、C组分别与A组比较，均具有显著性差异(P＜0.05)；E组和F组比较具有显著性差异(P＜0.05)；A组和D组比较，具有显著性差异(P＜0.05)。(5)免疫组化染色切片观察大鼠心肌组织Bcl-2和Bax的表达显示，Bcl-2、Bax免疫组织化学阳性物质定位于胞浆内，偶见胞膜(或)核膜，细胞核被苏木素复染成蓝色。统计结果显示：A组、B组、C组之间Bcl-2、Bax的表达均具有显著性差异(P＜0.05)；E组、F组之间比较，具有显著性差异(P＜0.05)；A组和D组比较具有显著性差异(P＜0.05)。(6)大鼠心肌细胞凋亡与HIF-1α、Bax、Bax／Bcl-2比值的相关性以及HIF-1α与Bax表达的相关性显示：大鼠心肌细胞凋亡与HIF-1α、Bax、Bax／Bcl-2比值之间存在正相关(P＜0.05)；心肌细胞HIF-1α的蛋白表达与Bax之间存在正相关(P＜0.05)。结论：(1)低氧刺激SD大鼠有氧代谢能力提高，常氧训练以及低氧8小时训练模型使SD大鼠有氧代谢能力降低。(2)低氧和／或训练均可诱导大鼠心肌组织HIF-1α、Bcl-2以及Bax的蛋白表达；随着低氧时间的延长，大鼠心肌细胞凋亡加重。(3)Bcl-2与Bax参与调控心肌细胞的凋亡，二者之间的平衡关系可能是维持或导致凋亡是否发生的重要因素。HIF-1α的表达可能协同Bcl-2家族凋亡相关因子的表达，在低氧和／或训练导致的心肌细胞凋亡中发挥重要作用。更多还原

【Abstract】 Objects: This research aimed at discussing the effects of Hypoxiaof different time and training apoptosis of Rats, in order to findexpression of HIF-1α、Bcl-2 and Bax of rats and the relationshipsbetween HIF-1αand related genes of apoptosis.Methods: 60 male SD rats were randomly divided into 6 groups:normoxia group (A), hypoxia of 8h group (B), hypoxia of 12h group (C),normoxia training group(D), hypoxia of 8h training group(E), hypoxiaof 12h training group (F). The rats of group D、E and F were introducedto treadmill running on an incline of 0 at 25m/min for lb. Aftertraining, the rats of group B, E, C and F were exposed to hypoxicchamber with 12.5％concentrations of oxygen (equal to altitude 4000m)for 8h and 12h every day for 4 weeks.Results: (1) The RBC, Hb and HCT were showed: the comparisonsbetween group B and E, and group A and D were significant(P＜0.05).(2) The slice was showed in HE dyeing: In the group A and B, thestructure of rats’ myocardial cell is integrated and clear, core isbig and circular; In the group C and F, myocardial cell structurechanges gradually: In the group D and F, myocardial cell begin tobe obviously swelling and the gap is obviously enlarged between cell,sinusoid is narrowed even vanishes, the core becomes small.(3) The TUNEL dyeing slice showed that: between the groups A andD, there was a significant difference(P＜0.05); and between the groupsE and F, there was a significant difference(P＜0.05).(4)Immunohistochemical technology and computer image processingmethod were applied to exam the location and content of HIF-1αoncardiac tissue. The result showed HIF-1αof cardiac tissue in B andC were significant compared with A; HIF-1αof cardiac tissue in groupE and F、A and D were all significant.(5) The dyeing slice of Bcl-2 and Bax showed: With the extension of time, male-dyeing grains increased gradually and the grey degreewas strengthened. The statistic results showed that Bcl-2 and Bax ofcardiac tissue among A, B and C groups were significant; hypoxiainduced factor-1αof cardiac tissue in group E and F, A and D wereall significant.(6) The relations between Myocardial apoptosia and theae itemsshowed that there was a highly significant positive correlation amongAI and HIF-1α, Bcl-2 and Bax(P＜0.05).Conclusion: (1)The hypoxia could increase the level of RBC, Hb andHCT, but the hypoxia and training could decrease the level of RBCHb and HCT.(2) HIF-1α, Bcl-2 and Bax of cardiac tissue was increasedsignificantly by intervention with hypoxia, training and hypoxiatraining. With the hypoxia time longer, HIF-1αof cardiac tissue wasmuch higher.(3) Bcl-2 and Bax participated in regulating the Myocardium’sapoptosis, and the balance relationship between the two were importantfactors if they would maintain or induce apoptosis. HIF-1αmay controland regulate the mechanism of Bcl-2 protein family and other relatedgenes on apoptosis of cardiac cell.更多还原