【作者】 林喜秀；

【导师】 瞿树林； 陈嘉勤；

【作者基本信息】 湖南师范大学 ， 运动人体科学， 2008， 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、训练以及低氧训练对大鼠肾组织细胞凋亡相关因子的影响。方法:60只SD大鼠按体重随机分为6组,每组10只,即: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)。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组依次放入氧浓度为12.5%(相当于海拔4000m)的低氧舱内8h和12h。实验期为4wk,5d/wk。最后一次实验结束后24小时,大鼠均实施速眠新Ⅱ腹腔麻醉,其理想后断尾取全血(检测RBC、HB、HCT值)后断头处死,取肾髓质外皮质部组织制备肾组织石蜡切片,HE染色检测肾组织细胞的形态学变化;免疫组化法检测大鼠肾组织细胞HIF-1a、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、EPO的蛋白表达以及TUNEL标记法在光镜下观察肾组织凋亡细胞核数目。结果:1.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.肾组织切片进行HE染色显示:A组和B组大鼠肾组织细胞结构完整,肾小管上皮细胞未见特殊改变;C组肾小管轻度肿胀,部分刷状缘脱落,管腔内偶可见脱落的细胞;D肾小管扩张,细胞扁平,轻度浊肿,刷状缘脱落,肾小球轻度淤血;E组较D组病变明显减轻,肾小管变性范围和程度均明显小于和轻于D组,胞核基本正常,肾小管轻度肿胀;F组肾小管上皮细胞水肿样变性,以近曲小管为重,表现为肾小管管腔扩张,管内可见管形和脱落细胞;上皮细胞核染色质边集,胞浆空泡样变性。3.TUNEL染色切片统计学分析结果显示:E组和F组、C组与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肾组织HIF-1a的蛋白表达显示:将实验各组肾组织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-1a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。呈强染性的肾组织细胞核中可见丰富阳性反应颗粒,细胞核周围的胞浆内亦有阳性表达。统计学分析结果显示: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肾组织Bcl-2、Bax和EPO的表达显示,Bcl-2、Bax和EPO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组与F组、C组与F组比较之间比较,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)。EPO的表达结果显示:EPO结果显示:B组和C组分别与A组比较,B组与A组有显著性差异(P＜0.05)。6.大鼠肾组织细胞凋亡与Bax、Bax/Bcl-2比值的相关性以及HIF-1a与Bax表达的相关性显示:大鼠肾组织细胞凋亡与Bax、Bax/Bcl-2比值之间存在正相关(P＜0.05);肾组织细胞HIF-1a的蛋白表达与Bax之间存在正相关(P＜0.05)。结论:1.合适的低氧刺激使SD大鼠有氧代谢能力提高。2.低氧训练可诱导大鼠肾组织HIF-1a、EPO、Bcl-2以及Bax蛋白表达;细胞凋亡率与凋亡指数及病理损伤与运动时低氧刺激有关,以低氧12小时训练组最明显。3.Bcl-2与Bax参与调控肾组织细胞的凋亡;HIF-1a的表达可能协同Bcl-2家族凋亡相关因子的表达,在低氧训练导致的肾组织细胞凋亡中发挥双重效应。更多还原

【Abstract】 Objects:This research aimed at discussing the effects of Hypoxia of different time and training apoptosis of the Renal of Rats,in order to find expression of HIF-1a、EPO、Bcl-2 and Bax of the Renal of rats and the relationships between HIF-1aand 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), hypoxia of 12h training group(F).The rats of group D、E and F were introduced to treadmill running on an incline of 0 at 25m/min for 1h.After training,the rats of group B and E,C and F were exposed to hypoxic chamber with 12.5%concentrations of oxygen(equal to altitude 4000m) for 8h and 12h every day,five days per week,for 4 weeks.Results:1.The RBC,Hb and HCT were showed:the comparisons between 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,the structure of rats’ renal cell is integrated and clear,core is big and circular; In the group C and F,renal cell structure changes gradually;In the group D and F,renal cell begin to be 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 and D,there was a significant difference(P＜0.05);and between the groups E and F,there was a significant difference(P＜0.05);and between the groups C and F,there was a significant difference(P＜0.05).4.Immunohistochemical technology and computer image processing method were applied to exam the location and content of HIF-1aon renal tissue.The result showed HIF-1aof renal tissue in B and C were significant compared with A;HIF-1a of rnnal tissue in group E and F、A and D were all significant.5.The dyeing slice of Bcl-2、Bax and EPO showed:With the extension of time,male-dyeing grains increased gradually and the grey degree was strengthened.The statistic results showed that Bcl-2 and Bax of renal tissue among A、B and C groups were significant;Bcl-2 and Bax of renal tissue in group E and F、C and F、A and D were all significant.The statistic results showed that EPO of renal tissue among A、B and C groups were not significant;EPO of renal tissue in group E and F、C and F、A and D were not all significant.6.The relations between Renal apoptosis and these items showed that there was a highly significant positive correlation among AI and HIF-1a、Bcl-2 and Bax(P＜0.05).Conclusion:1.The hypoxia could increase the level of RBC、Hb and HCT,the proper hypoxia and training could increase the capability of exercise.2.HIF-1a of renal tissue was increased significantly by intervention with hypoxia,training and hypoxia training.HIF-1a of renal tissue in F was much higher.3.Bcl-2 and Bax of renal tissue was increased significantly by intervention with hypoxia、training and hypoxia training,and Bcl-2 and Bax of renal tissue in F was much higher.But EPO of renal tissue was not increased significantly by intervention with hypoxia、training and hypoxia training.4.Bcl-2 and Bax participated in regulating the renal apoptosis,and the balance relationship between the two were important factors if they would maintain or induce apoptosis.HIF-1a may control and regulate the mechanism of Bcl-2 protein family and other related genes on apoptosis of renal cell.更多还原