摘要
目的
糖尿病(diabetes mellitus, DM)是一种慢性终身性疾病,随着人们平均寿命的延长,生活水平的提高及饮食结构的变化,糖尿病的患病率不断增加。糖尿病已成为继肿瘤、心血管之后的第三大严重威胁人类健康的慢性非传染性疾病。生殖系统功能障碍是糖尿病并发症之一,其发病率是非糖尿病病人的5-10倍,给患者的身心健康带来了严重的压力和困扰,近年来越来越受到重视。糖尿病生殖系统功能障碍主要包括勃起功能障碍(erectile dysfunction, ED)及男性不育等。
OLETF鼠是一种自发性2型糖尿病大鼠,本实验以OLETF鼠作为2型糖尿病模型,以LETO鼠和摄入罗格列酮的OLETF鼠作为其对照,通过观察睾丸微观病理变化和生精细胞凋亡情况及附睾中精子的受精力,并运用免疫组化方法对各级生精细胞与凋亡相关的Bcl-xL和Fas蛋白表达情况进行检测,来探讨糖尿病生殖系统功能障碍的发生机制和罗格列酮的干预情况。
方法
大鼠在无特定病原体级(SPF级)条件下单笼饲养,饲以标准饲料,12/12h光照黑暗循环,自由获取食物和饮水。定期行口服葡萄糖耐量试验(OGTT)监测血糖,血糖峰值>16.7mmol/L和负荷后120 min血糖>11.1mmol/L诊为糖尿病,只具备上述1条为糖耐量减低。至30周时,共有成模OLETF大鼠12只,随机分为糖尿病对照(DM)组和罗格列酮(RGZ)组(每组6只),8只LETO鼠为正常对照(NC)组。罗格列酮以蒸馏水溶解稀释后为RGZ组大鼠灌胃给药,剂量为3mg/kg·d。DM组与NC组以等量蒸馏水灌胃,各组均每日灌胃1次,至12周。12周后称重大鼠体重,然后脱臼处死大鼠,下腹正中切开腹壁,取出大鼠睾丸,用冷生理盐水冲洗,滤纸拭干,准确称重。然后置于10%中性福尔马林溶液中固定,常规石蜡包埋切片,HE染色。光镜下观察组织结构的改变,并测量有关形态学指标。吖啶橙荧光染色评价附翠中精子的受精能力。ISEL原位末端切口平移法检测生精细胞的凋亡。Bcl-xL的测定应用SABC (strept avidin-biotin complex)法,以胞浆或(和)胞膜中出现棕黄色颗粒为阳性表达,以PBS代替抗作为阴性对照。Fas蛋白表达变化检测按照SP法进行。图像用Motic Images Advanced3.2版图像分析软件进行分析。
结果
1.造模情况：OLETF大鼠较LETO大鼠明显肥胖、多饮、多食、多尿,与糖尿病临床表现相似。
2.睾丸大体形态及称重：肉眼观察,模型组睾丸有明显的萎缩现象,严重者睾丸塌瘪松软,颜色暗红,纹理不清,少数睾丸白膜破裂。罗格列酮组睾丸与模型组睾丸肉眼观察变化不大。睾丸重量变化,与模型组比较,对照组睾丸重量大于模型组,罗格列酮组睾丸重量和模型组差别无统计学意义。
3.睾丸组织HE染色后镜下观察：模型组睾丸组织较对照组有明显的病理变化。罗格列酮组生精小管间距增宽,生精细胞排列紊乱,管腔内精子少见。与对照组相比,模型组精原细胞和间质细胞计数降低；罗格列酮组与模型组相比,并无统计学差异。
4.睾丸生精细胞凋亡变化：与糖尿病组相比,对照组睾丸凋亡的生精细胞数少于糖尿病组,而罗格列酮组与糖尿病组并无统计学差异。
5.生精细胞Bcl-xL蛋白表达变化：与模型组相比,对照组生精细胞Bcl-xL蛋白阳性表达平均灰度值降低,罗格列酮组生精细胞Bcl-xL蛋白阳性表达平均灰度值与模型组无统计学差异。
6.生精细胞Fas表达变化：Fas蛋白在对照组以弱阳性表达为主,在模型组以强阳性表达为主,在罗格列酮组以中度阳性表达为主。
结论
1. OLETF大鼠表现与糖尿病相似,可作为研究2型糖尿病的理想动物模型。
2. OLETF大鼠睾丸明显萎缩,甚至塌陷。睾丸系数减小。光镜下病理改变明显,表现为生精小管数量减少,萎缩扭曲变形,基膜有破损,生精细胞数量减少,间质结缔组织增生,而间质细胞减少。
3. OLETF大鼠睾丸生精细胞凋亡增多,附睾中精子的受精能力减弱。说明凋亡机制可能参与了2型糖尿病生精障碍的过程。
4.罗格列酮虽然可降低2型糖尿病的血糖,但对2型糖尿病引起的生精障碍并无改善作用。
Objective
Diabetes (diabetes mellitus, DM) is one kind of chronic lifelong disease. Along with the people life expectancy extension, living standard's enhancement and diet structure's change, diabetes' prevalence rate increases unceasingly. Diabetes has become the third-largest serious threat to human health of chronic non-communicable diseases after tumor, cardiovascular. Reproductive system dysfunction is one of the complications of diabetes. Its incidence rate is non-diabetes patient's 5-10 times. It has brought the serious pressure and puzzle for patients' physical and moral. In recent years, it has been received more and more attention. Diabetes reproductive system function barrier mainly includes the erection function barrier (erectile dysfunction, ED), the male sterility and so on.
OLETF rats are a spontaneous type 2 diabetic model. OLETF rats are as type 2 diabetes model in this experiment, LETO rats and OLETF rats intake of rosiglitazone are as its controls. Through observing the testicle microscopic pathological change and spermatogenic cell apoptosis, and using the immunohistochemical method for all levels of spermatogenic cell Bcl-xL and Fas protein expression circumstance, diabetes reproductive system dysfunction happens and rosiglitazone intervention are to be discussed.
Methods
Rats are raised in specified-pathogens free level (SPF) by placing in a cage and feeding standard feed. Blood glucose was determined by oral glucose tolerance test (OGTT). The diabetes was confirmed with blood glucose peak level>16.7 mmol/L or blood glucose level>11.1 mmol/L taken glucose 20 minutes later. After 30 weeks,12 T2DM OLETF rats were randomly divided into rosiglitazone(RGZ) group and diabetes mellitus control(DM) group (n=6 per group). Eight male LETO rats were used as normal control (NC) group. DM group and NC group irrigate stomach with distilled water. RGZ group were given rosiglitazone(3mg/kg·d). Give medicine 12 weeks, daily 1 time. After weighing, the rats were sacrificed, and taken out the testes rapidly. The testes were flushed in the cold physiological saline, weighed; then fixed in 10% neutral formalin, embedded, sliced, and stained with HE. Under the light microscope was observed the change of organizational structure, and measured morphological index. The testes were stained by acridone orange fluorescence for the evaluation of sperm energy in epididymis. The Bcl-xL determination made use of SABC (StreptAvidin-Biotin Complex) according to the immunohistochemical reagent box. PBS instead of Primary antibody was as negative control. Detection of Fas expression changed according to the reagents instructions (SP process). The results were analysised by Motic Images Advanced 3.2 image analysis software.
Results
1. Observation of the general conditions of rats
OLETF rats were obviously obese, drank much, ate much, had polyuria, the performance was resemble to diabetes clinical manifestations.
2. Testicular general shape and weighing
The model group testes atrophied obviously, even collapsed and shriveled, became dark red. The minority of albuginea was breakaged. The rosiglitazone group testes had little change through microscopic observation. In the control group, the testes' weight was bigger than the model group. The rosiglitazone group testes' weight was non-statistics difference from model group's.
3. Observe the testicular tissue microscopically after HE staining
The light microscope observation of testicle structure demonstrated that model group compared to control group was obviously pathological changes in testes. In the rosiglitazone group, spermatogenic cell arranging was disordered. Sperms in lumen were rare. After the model group compared with the control group, the spermatogonia and the intersititial cell counting was reduce; but there was non-statistics difference between the rosiglitazone group and the model group.
4. Apoptotic spermatogenic cell change:
Compared with the model group, the apoptotic spermatogenic cells in the control group were reduced. However, there was no-statistically different between the rosiglitazone group with the model group.
5. Bcl-xL protein expression changes in spermatogenic cell:
Compared with the model group, Bcl-xL positive express average gray values in spermatogenic cell were reduced; there was non-statistically significant difference between the rosiglitazone group and the model group.
6. Fas protein expression changes in spermatogenic cell: Fas proteins in the control group were expressed with weak positive primarily. The ones in model group were given priority to strong positive expression. Those in the rosiglitazone group were expressed to moderate positive primarily.
Conclusion
1. OLETF rats can be as an ideal animal model for research.
2. OLETF rats testes are obvious atrophy and even failures. Testicular coefficient decreases. The number of seminiferous tubule is decreased, and seminiferous tubules are atrophic and distorted. Their basement membranes are damaged. Spermatogenic cells and intersititial cells are reduced. But interstitial are hyperplasia.
3. OLETF rats testes spermatogenic cell apoptosis increase. The energy of sperm becomes weaken in epididymis. Apoptosis mechanisms may be involved in the spermatogenesis obstacle of type 2 diabetes.
4. Although the rosiglitazone can reduce blood sugar of type 2 diabetes, but the cause of spermatogenesis obstacle is not improving efficiently.
引文
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