论文部分内容阅读
目的建立D-半乳糖(D-gal)致小鼠衰老模型,探讨睾丸结构与功能变化及相关机制。方法 C57BL/6J小鼠随机分为衰老组和对照组。衰老组小鼠皮下注射D-gal;对照组等时等量注射生理盐水。模型复制完成第2d,采集小鼠内眦静脉血测定血清睾酮水平;取小鼠睾丸测定睾丸指数;石蜡切片HE染色观察睾丸组织形态学;衰老相关β-半乳糖苷酶(SA-β-Gal)染色检测生精细胞衰老;TUNEL法检测生精细胞凋亡;制备睾丸组织匀浆上清,ELISA检测TNF-α、IL-1β、和IL-6的水平;硫代巴比妥酸法检测丙二醛(MDA)含量;酶学法检测超氧化物歧化酶(SOD)活性;ABTS法检测总抗氧化能力(TAC);Western blotting检测衰老相关蛋白p21与p53表达水平。结果衰老模型组小鼠血清睾酮水平显著下降,睾丸脏器指数无明显差异,生精小管结构紊乱,生精细胞病理损伤明显,SA-β-Gal染色阳性的生精细胞百分率上升,细胞凋亡指数上升,睾丸匀浆上清中TNF-α、IL-1β、IL-6水平显著上升,SOD活性、TAC能力显著降低,MDA含量上升,睾丸组织中衰老相关蛋白:p21与p53表达水平显著提高。结论 D-gal建立的衰老动物模型可见睾丸形态结构与功能出现衰老的生物学表现,其机制可能与氧化应激损伤及p21/p53通路激活有关。
Objective To establish a mouse model of aging induced by D-galactose (D-gal) and to explore the changes of testicular structure and function and related mechanisms. Methods C57BL / 6J mice were randomly divided into aging group and control group. The aging mice were subcutaneously injected with D-gal; the control group was injected with the same amount of normal saline at the same time. Serum testosterone levels were measured at the 2nd day after model duplication. Serum testosterone was collected from the mice. The testis index was determined by the testis in mice. The morphology of testis was observed by HE staining. The expression of the aging-related β-galactosidase (SA-β-Gal ); The apoptosis of spermatogenic cells was detected by TUNEL method; The supernatant of testis homogenates was prepared and the levels of TNF-α, IL-1β and IL-6 were detected by ELISA; The content of malondialdehyde (MDA) was measured. The activity of superoxide dismutase (SOD) was detected by enzymology method. The total antioxidant capacity (TAC) was measured by ABTS. The expression of p21 and p53 were detected by Western blotting. Results Serum testosterone levels were significantly decreased in senescent model mice, while there was no significant difference in testicular organ index. The structure of seminiferous tubules was disorganized and the pathological damage of spermatogenic cells was obvious. The percentage of spermatogenic cells with SA-β-Gal staining increased, The levels of TNF-α, IL-1β and IL-6 in testis homogenate supernatant increased significantly, the activities of SOD and TAC decreased significantly, MDA content increased, the expression of p21 and p53 in testis significantly increased . Conclusion The aging animal model established by D-gal shows the biological appearance of senescence in morphology and function of testis. The mechanism may be related to the oxidative stress injury and the activation of p21 / p53 pathway.