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目的:比较胎牛血清(FBS)和血清替代物——KnockoutTMSR(KSR)对睾丸组织生长发育的影响。方法:未成熟大鼠睾丸组织分别采用FBS和KSR连续培养5周,观测培养睾丸组织大体面积,苏木素伊红(HE)染色观察睾丸组织学形态及生殖细胞发育,并测量曲细精管直径。TUNEL法凋亡实验观察培养5周睾丸组织中的凋亡细胞,RT-PCR检测精子发生不同阶段的标准化标志基因Kit、Sycp3、Crisp1。结果:KSR组睾丸组织体外持续生长,曲细精管逐渐增大,培养5周观察到精原细胞、初级精母细胞、次级精母细胞、圆形精子细胞,RT-PCR检测到Kit、Sycp3、Crisp1的表达。FBS组睾丸组织随着培养时间延长逐渐萎缩,培养5周曲细精管出现明显坏死,培养睾丸组织中Sycp3、Crisp1表达不稳定。结论:KSR更有利于未成熟睾丸组织培养中生殖细胞从精原细胞发育成精子细胞,并能长期维持生殖细胞发育及睾丸组织生长。
Objective: To compare the effects of fetal bovine serum (FBS) and serum replacement - Knockout TMSR (KSR) on testicular tissue growth and development. Methods: The testis tissues of immature rats were cultured for 5 weeks with FBS and KSR respectively. The gross area of testis tissue was observed. The histopathology and germ cell development were observed by hematoxylin and eosin staining. The diameter of seminiferous tubules was measured. TUNEL method was used to observe apoptotic cells in testis tissue after 5 weeks of culture. RT-PCR was used to detect the standard marker genes Kit, Sycp3 and Crisp1 in different stages of spermatogenesis. RESULTS: The testicular tissues of KSR group grew continuously in vitro and the seminiferous tubules increased gradually. Spermatogonia, primary spermatocytes, secondary spermatocytes and round spermatids were observed after 5 weeks of culture. Kit, Sycp3, Crisp1 expression. Testicular tissue of FBS group was gradually atrophied with the prolongation of culture time. The cultured seminiferous tubules of Synechocystis sp. Conclusion: KSR is more conducive to immature testicular tissue culture of germ cells from spermatogonia into sperm cells, and long-term maintenance of germ cell development and testicular tissue growth.