兔睾丸缺血再灌注损伤及其保护措施的机理研究

发布时间：2018-05-02 02:14

本文选题：睾丸 + 缺血再灌注　；参考：《中国人民解放军医学院》2015年博士论文

【摘要】：目的基于睾丸扭转建立兔子睾丸缺血再灌注损伤模型,结合氧化应激指标及细胞凋亡因子检测探索睾丸缺血预处理、后处理及联合处理对睾丸缺血再灌注损伤的保护作用及潜在调控机理。方法选择雄性新西兰大白兔30只,随机分为5个组：A组：对照组,暴露右侧精索,不作缺血处理并关闭；B组：缺血再灌注组(I/R),使用无创血管夹闭右侧精索致睾丸缺-60min,再灌注3d；C组：预处理组,缺血再灌注前夹闭精索3次(缺血5min/次+灌注5min/次),夹闭右侧精索致缺血60min,再灌注3d；D组：后处理组,夹闭右侧精索致缺血60min后,进行3次缺血再灌注(再灌注5s/次+再缺血5s/次),再灌注3d；E组：联合处理组,进行缺血再灌注前夹闭精索3次(缺血5min/次+灌注5min/次),夹闭右侧精索致缺血60min后,进行3次缺血再灌注(再灌注5s/次+再缺血5s/次),再灌注3d。造模完成后,采用3%巴比妥钠麻醉兔子后采集血液并分离血清测定血清睾酮含量；接着分离睾丸组织,按照缺血侧及健侧分组后一部分进行裂解后按照MDA、PC、NO, SOD、 MPO、GSH-Px酶联免疫吸附检测试剂盒所述步骤进行检测分析表达水平变化,一部分组织样本采用Western blot法进行检测分析Bcl-2,Bax表达水平,其余组织样本采用10%中性福尔马林固定后进行HE染色和TUNEL细胞凋亡检测。结果与正常对照A组相比,缺血B组、缺血预处理C组、缺血后处理D组及联合处理E组后血清睾酮表达水平显著降低(p0.01)；缺血B组、缺血预处理C组、缺血后处理D组及联合处理E组术前与术后比较发现,血清睾酮表达水平明显降低(p0.05)。与正常对照A组及缺血B组健侧相比,缺血B组缺血侧MDA、PC、 NO、SOD、MPO、GSH-Px表达水平显著升高(p＜0.01)；与缺血B组缺血侧相比,缺血预处理C组、缺血后处理D组及联合处理E组缺血侧MDA、PC、NO、SOD、 MPO、GSH-Px表达水平显著降低(p0.01),且三种处理方式之间无统计学差异。病理染色结果显示,与正常对照A组及缺血B组健侧相比,缺血B组缺血侧睾丸组织内大部分生精小管结构被破坏,生精小管内各级生精细胞结构消失,可见凋亡的生精细胞,间质及管腔内有淡伊红色水肿液渗出,凋亡指数明显升高p0.01),Johnsen评分显著降低(p0.01)；与缺血B组缺血侧相比,缺血预处理C组、缺血后处理D组及联合处理E组缺血侧睾丸组织生精小管结构完整,细胞层次清楚,排列整齐,大部分管腔内有精子生成,部分小管管腔内有少量淡伊红色水肿液,凋亡指数明显降低(p0.01),Johnsen评分显著增加(p0.01),且三种处理方式之间无统计学差异。同样地,与正常对照A组和缺血B组健侧相比,缺血B组缺血侧Bcl-2/Bax比值显著降低(p0.01)；与缺血B组缺血侧相比,缺血预处理C组、缺血后处理D组及联合处理E组缺血侧Bcl-2/Bax比值明显增加并趋近正常水平p0.01)。结论缺血预处理、后处理及联合处理可通过调控氧化应激指标及细胞凋亡水平明显改善兔睾丸缺血再灌注损伤,且三种处理方式对睾丸缺血再灌注损伤的保护作用相同。不仅为睾丸缺血再灌注损伤的潜在保护机理研究提供了重要方法参考,而且为睾丸缺血再灌注损伤的临床治疗提供了潜在开发药物,具有重要的临床应用价值。
[Abstract]:Objective to establish a rabbit testis ischemia-reperfusion injury model based on testicular torsion and to explore the protective effect and potential regulation mechanism of testis ischemic preconditioning, post-processing and combined treatment on testis ischemia reperfusion injury combined with oxidative stress index and apoptosis factor. Methods 30 male New Zealand white rabbits were selected and divided into 5 randomly selected rabbits. Group A: the control group, the control group, exposed to the right spermatic cord, without ischemic treatment and closure; group B: ischemia reperfusion group (I/R), using noninvasive blood vessels to close the right spermatic cord to -60min and reperfusion 3D; group C: preconditioning group, 3 times before ischemia and reperfusion (ischemia 5min/ + perfusion 5min/), and clipping the right spermatic cord ischemia 60min, reperfusion Group 3D, group D: post treatment group, after occlusion of the right spermatic cord ischemia 60min, 3 times of ischemia reperfusion (reperfusion 5s/ + re ischemia 5s/), and reperfusion 3D, E group: combined treatment group, before ischemia-reperfusion of the clipping spermatic cord (ischemia 5min/ + perfusion 5min/), clipping the right spermatic ischemia 60min, 3 times ischemia reperfusion (RE) After perfusion of 5s/ + ischemia 5s/) and reperfusion of 3D. after reperfusion, the blood was collected and serum testosterone content was measured by 3% barbiturate sodium anaesthetized rabbits. Then the testicular tissue was separated and a part of the ischemic side and the healthy side were divided according to the MDA, PC, NO, SOD, MPO, and GSH-Px enzyme immunoadsorption kit. Western blot method was used to detect the expression level of Bcl-2 and Bax in some tissue samples. The other tissue samples were stained with 10% neutral formalin for HE staining and TUNEL cell apoptosis detection. The results were compared with the normal control A group, ischemic B group, ischemic preconditioning C group, and post ischemic place. Serum testosterone expression levels in group D and group E were significantly decreased (P0.01); ischemic B group, ischemic preconditioning C group, ischemic postconditioning group D group and combined treatment group E group before and after operation showed that serum testosterone expression level decreased significantly (P0.05). The expression level of GSH-Px was significantly higher (P < 0.01). Compared with ischemic side of ischemic B group, ischemic preconditioning C group, D group after ischemic post-processing and combined treatment of E group MDA, PC, NO, SOD, MPO, GSH-Px expression level were significantly reduced, and the three treatments were no difference between the methods. Pathological staining results showed that with normal control group and ischemic group In the ischemic B group, most of the spermatogenic tubule structure in the ischemic testicular tissue was destroyed. The structure of spermatogenic cells in the seminiferous tubules disappeared, the apoptotic spermatogenic cells were seen, the interstitial and the endoplasma and the oedema fluid exuded in the lumen, the apoptosis index was significantly increased by P0.01), the Johnsen score was significantly decreased (P0.01), and the ischemic side of the ischemic B group was in the ischemic side phase. Compared with the ischemic preconditioning group C, the structure of the seminiferous tubules in the ischemia side testis tissue of group D and E group was complete, the cell level was clear and the arrangement was neat, most of the lumen had spermatogenesis, a small amount of light red edema was found in the lumen of the part of the tubules, the apoptosis index decreased significantly (P0.01), and the Johnsen score increased significantly (P0.01), and three Compared with the normal control group A and the ischemic B group, the ischemic side Bcl-2/Bax ratio in the ischemic B group was significantly lower than that in the ischemic B group (P0.01), and compared with the ischemic side of the ischemic group, the ischemic preconditioning C group, the ischemic postconditioning group and the combined treatment of the E group were significantly increased and approached the normal level of the normal level in the E group. 1) conclusion. Conclusion ischemic preconditioning, post-processing and combined treatment can obviously improve the ischemia reperfusion injury of rabbit testis by regulating the oxidative stress index and the level of cell apoptosis, and the protective effect of the three treatments on testis ischemia-reperfusion injury is the same. It not only provides an important study on the potential protection mechanism of testis ischemia reperfusion injury. This method provides a potential development medicine for clinical treatment of testicular ischemia reperfusion injury, and has important clinical application value.

【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R697.22

【参考文献】