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目的:探讨羟基脲(hydroxyurea,HU)诱导大鼠生精细胞凋亡发生的量效关系和时效关系。方法:量效关系研究中,雄性Wistar大鼠25只,分为5组,每组5只,4组为试验组,1组为对照组,4个试验组分别腹腔注射HU100mg/kg、200mg/kg、400mg/kg和600mg/kg,对照组给予磷酸盐缓冲液2ml/kg,给药后12h处死;时效关系研究中,雄性Wistar大鼠20只,分为4组,每组5只,3组均腹腔注射HU400mg/kg,1组为空白对照,分别于给药后6、12和24h处死大鼠。所有大鼠处死后称体重,取睾丸组织并称重,Bouin’s液固定后,进行HE染色、原位细胞凋亡检测(TUNEL)、糖原PAS染色,光学显微镜下观察组织结构,计数凋亡细胞并进行生精周期阶段的分析。结果:给药后6、12、24h大鼠体重、睾丸重量与给药前相比,未见明显变化。量效关系研究中,大鼠生精细胞凋亡阳性小管所占比率和凋亡指数随着HU剂量的增加而明显增加,其中以400mg/kg为最高,分别为(38.7±2.0)%和(496.4±66.8)(P<0.01)。时效关系研究中,与对照组比较,给药后12h的每管凋亡阳性生精细胞数和凋亡指数达峰值,分别为(12.9±2.10)和(496.4±66.8)(P<0.01);TUNEL阳性细胞主要存在于生精周期的Ⅰ-Ⅳ时相。结论:HU诱导的大鼠睾丸生精细胞凋亡具有细胞特异性和阶段特异性。
Objective: To investigate the dose-effect and time-effect relationship of hydroxyurea (HU) induced apoptosis of spermatogenic cells in rats. Methods: In the dose-response relationship study, 25 male Wistar rats were divided into 5 groups with 5 rats in each group. 4 rats were in the test group and 1 was the control group. The four experimental groups were injected intraperitoneally with HU100mg / kg and 200mg / kg, 400mg / kg and 600mg / kg respectively. The control group was given phosphate buffer solution 2ml / kg and sacrificed 12h after administration. In the aging relationship study, 20 male Wistar rats were divided into 4 groups The rats in the two groups were given intraperitoneal injection of HU400mg / kg, and the other one was blank control group. Rats were sacrificed at 6, 12 and 24 hours after administration respectively. After sacrificed, all rats were weighed, testicular tissues were weighed and grafted. After Bouin’s solution was fixed, HE staining, TUNEL and glycogen PAS staining were performed. The histological structure was observed under light microscope. Apoptotic cells were counted And spermatogenic cycle stage analysis. Results: At 6, 12 and 24 hours after administration, the body weight and testicular weight of rats did not change significantly compared with those before administration. In the dose-effect relationship study, the percentage of apoptotic positive tubules and apoptotic index in rat spermatogenic cells were significantly increased with the increase of HU dosage, with the highest concentration of 400mg / kg, which were (38.7 ± 2.0)% and 496.4 ± 66.8) (P <0.01). Compared with the control group, the number of apoptotic spermatogenic cells and apoptotic index reached the peak at 12h (12.9 ± 2.10) and (496.4 ± 66.8) (P <0.01), respectively. TUNEL-positive cells are mainly present in the IV phase of the spermatogenesis. Conclusion: The apoptosis of spermatogenic cells induced by HU in rat testes is cell-specific and stage-specific.