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目的:检测SD大鼠实验性隐睾早期不同时点精原细胞c-kit和PI3K(P110α)的表达及其细胞周期的变化,探讨睾丸受热作用早期,温度对精原细胞增殖的影响。方法:育龄期SD雄性大鼠32只,按随机数字表法分为实验组(n=24)和对照组(n=8)。实验组建立隐睾模型,建模后1～3周末三个时点,随机8只取睾丸标本。对照组不干预取睾丸标本。SP免疫组化法观察睾丸形态学变化,非连续性Percoll密度梯度离心结合差速贴壁的方法纯化富集各组精原细胞,逆转录多聚酶链反应(RT-PCR)和蛋白免疫印迹(Westernblot)检测各组精原细胞c-kit和PI3K的mRNA及蛋白表达;流式细胞仪检测各组精原细胞的细胞周期。结果:对照组c-kit主要表达于精原细胞,而精母细胞、精子细胞有少量表达。实验组生精细胞明显减少,到第3周时仅见单层散在于基膜强表达c-kit的精原细胞,实验组各时点和对照组精原细胞大部分处于G0/G1期,S期及G2/M期均较少,实验组组间及与对照组比较无统计学差异性(P>0.05)。结论:实验性隐睾可导致精原细胞的增殖信号增强,但并未诱导精原细胞处于增殖期的细胞增加,即体腔温度对体内精原细胞的增殖影响不大。
OBJECTIVE: To detect the expression of c-kit and PI3K (P110α) and the cell cycle of spermatogonia in experimental early cryptorchidism in SD rats, and to investigate the effect of temperature on the proliferation of spermatogonial cells in early stage of testicular heat. Methods: 32 SD male rats of reproductive age were divided into experimental group (n = 24) and control group (n = 8) by random number table. The experimental group was established cryptorchidism model, 1 to 3 weeks after modeling at three time points, 8 were randomly selected testicular specimens. The control group did not intervene to take testicular specimens. SP immunohistochemistry was used to observe the morphological changes of testis. Non-continuous Percoll density gradient centrifugation combined with differential adherent method was used to purify and enrich the spermatogonia. RT-PCR and Western blot ) Were used to detect the mRNA and protein expression of c-kit and PI3K in each group. Flow cytometry was used to detect the cell cycle of spermatogonia in each group. Results: The c-kit of the control group was mainly expressed in spermatogonia, while the spermatids and spermatids were slightly expressed. The spermatogenic cells in the experimental group were significantly reduced. Only the monolayer spermatogonia were scattered in the basement membrane strongly expressing c-kit by the third week. Most of the spermatogonia in the experimental group and the control group were in G0 / G1 phase, S Stage and G2 / M phase were less, no significant difference between the experimental group and the control group (P> 0.05). CONCLUSIONS: Experimental cryptorchidism can lead to enhanced spermatogonial proliferation signal, but it did not induce the proliferation of spermatogonia in proliferating phase. That is, body cavity temperature had little effect on the proliferation of spermatogonia in vivo.