高脂和糖尿病SD大鼠辜丸组织中Hsp60的表达变化研究

发布时间：2018-11-14 09:14

【摘要】：目的:复制雄性Sprague-Dawlay大鼠血脂异常以及2型糖尿病模型,并研究以上两种疾病对中年SD大鼠生殖系统的影响,通过对睾丸内生精小管微结构变化和热休克蛋白60(HSP60)表达差异,以及曲细精管中生精细胞凋亡数量的改变,研究热休克蛋白60在大鼠睾丸中的表达情况与大鼠生精细胞凋亡数量变化间的联系以及其可能的影响机制。方法:挑选60只无特定病原体(SPF级)健康雄性Sprague-Dawlay大鼠,每只通过统计软件编号后随机划编成三个实验组:对照组(n=15)、高脂组(n=15)、糖尿病组(n=30)。对照组全程使用基础饲料喂养;高脂组前期食用高脂饲料,24 W后在原有饲料基础上添加丙硫氧嘧啶(PTU),加重脂代谢异常;糖尿病组前期进食高脂饲料,24 W末腹腔注射链脲佐菌素进行糖尿病造模(对照组和高脂组采用柠檬酸注射液代替),完成注射后糖尿病大鼠更换为普通饲料。36周末大鼠尾静脉采血检测大鼠随机血糖、血脂四项等指标,并剔除未成模大鼠。确定造模成功后处死大鼠,迅速分离大鼠一侧睾丸组织,放入Bouin固定液中室温下浸泡48小时后送病理实验室对标本包埋、切片;另一侧睾丸组织离体后立即塞入冻存管后置于液氮罐中暂时保存,取材结束后统一移至负80度冰柜中长时间存放。通过免疫组化(SABC)检测热休克蛋白60蛋白表达情况及所在位置;光镜分析HE染色切片,观察大鼠睾丸曲细精管构造变化;蛋白质免疫印迹法(Western Blot)分析大鼠睾丸组织中HSP60蛋白表达程度;通过TUNEL染色了解凋亡细胞分布情况以及睾丸生精组织细胞凋亡水平。一切实验数据搜集整理后经过spss16.0统计分析软件进行数据处理,以P0.05作为判别数据间具有显著统计学差异的标准。结果:通过随机血糖以及血脂四项的检测结果提示高脂和糖尿病SD大鼠模型成功建立。HE染色在镜下观可见对照组生精细胞按发育成熟顺序由管壁向管腔逐层排列,数量较多。高脂及糖尿病大鼠睾丸曲细精管中细胞排布较对照组松散、混乱,且各级精母细胞数量出现下降,成熟精子细胞在管腔中央的分布数量显著下降,部分睾丸组织切片中可呈现生精细胞团块与生精上皮分离;免疫组化显示高脂组和糖尿病组大鼠睾丸HSP60表达高于对照组;Western Blot实验发现高脂组和糖尿病组大鼠睾丸组织HSP60表达水平都出现显著升高,对照组的目的蛋白表达水平表达则远低于其他两组。Tunel检测可观察到高脂组和糖尿病组睾丸组织凋亡细胞与对照组相比数量较多,糖尿病组细胞凋亡多于高脂组。结论:糖尿病和高脂血症可以使睾丸生精细胞凋亡数目上升,精子发育成熟数量减少,对睾丸生精结构产生影响。HSP60蛋白在高脂血症及糖尿病大鼠睾丸中明显增加。糖尿病与高脂血症所致生精细胞凋亡可能与睾丸组织中的HSP 60蛋白表达改变以及相关的凋亡因子表达改变有关。
[Abstract]:Objective: to establish a model of dyslipidemia and type 2 diabetes mellitus in male Sprague-Dawlay rats and to study the effects of these two diseases on the reproductive system of middle-aged SD rats. The changes of the microstructures of testicular seminiferous tubules, the expression of heat shock protein 60 (HSP60) and the number of spermatogenic cells apoptosis in seminiferous tubules were studied. To study the relationship between the expression of heat shock protein 60 (HSP60) and the apoptosis of rat spermatogenic cells and its possible mechanism. Methods: sixty healthy male Sprague-Dawlay rats without specific pathogen (SPF grade) were randomly divided into three experimental groups: control group (n = 15), hyperlipidemia group (n = 15) and diabetes group (n = 30). The control group was fed with basic diet for the whole course, and the high-fat group was fed with high-fat diet at the beginning of the experiment. After 24 weeks, the abnormal lipid metabolism was aggravated by the addition of prothiouracil (PTU),) on the basis of the original diet. In the diabetic group, the diabetic model was established by intraperitoneal injection of streptozotocin at the end of 24 weeks after eating high fat diet (citric acid injection was used instead of citric acid injection in the control group and hyperlipidemia group). At the end of the 36th week, the blood samples were collected from the tail vein of the rats to detect the random blood glucose, blood lipid and other indexes, and to eliminate the non-model rats. The rats were killed after the establishment of the model, and the testis of the rats were quickly separated. The rats were immersed in the Bouin fixative solution at room temperature for 48 hours and then sent to the pathology laboratory to bury and slice the specimens. The testicular tissue on the other side of the testis was immediately inserted into a cryopreservation tube and stored in a liquid nitrogen tank for a short time, and then transferred to a negative 80 degree freezer for a long time. The expression and location of heat shock protein 60 (HSP60) were detected by immunohistochemical (SABC), and the changes of testicular seminiferous tubules were observed by HE staining. Western blot (Western Blot) was used to detect the expression of HSP60 protein in rat testis and the distribution of apoptotic cells and the apoptosis level of testicular spermatogenic tissue were studied by TUNEL staining. After all the experimental data were collected and sorted, the data were processed by spss16.0 statistical analysis software, and P05 was taken as the criterion of significant statistical difference among the discriminant data. Results: the results of random blood glucose and blood lipids showed that the SD rat model of hyperlipidemia and diabetes mellitus was successfully established. HE staining showed that the spermatogenic cells in the control group were arranged from the wall to the lumen in the order of development and maturation, and the number of spermatogenic cells in the control group was more than that in the control group. In hyperlipidemic and diabetic rats, the distribution of cells in the seminiferous tubule of testis was looser and confused, and the number of spermatocytes decreased, and the number of mature sperm cells in the center of the tubules decreased significantly. Spermatogenic cell mass and spermatogenic epithelium could be separated in some testicular sections. Immunohistochemistry showed that the expression of HSP60 in testis of hyperlipidemia group and diabetic group was higher than that of control group. Western Blot assay showed that the expression of HSP60 in testis of both hyperlipidemia group and diabetic group was significantly increased. The expression of target protein in the control group was much lower than that in the other two groups. Tunel assay showed that the number of apoptotic cells in testis of hyperlipidemia group and diabetic group was more than that of control group, and that of diabetic group was more than that of hyperlipidemic group. Conclusion: diabetes and hyperlipidemia can increase the number of testicular spermatogenic cells apoptosis and decrease the number of spermatozoa maturation. HSP60 protein in the testis of hyperlipidemia and diabetic rats is obviously increased. The apoptosis of spermatogenic cells induced by diabetes mellitus and hyperlipidemia may be related to the expression of HSP 60 protein and related apoptosis factors in testis.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R58;R698.2

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