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目的探讨脂多糖(LPS)处理后的小鼠睾丸支持细胞(SCs)中胶质细胞源性神经营养因子(GDNF)表达的变化及该变化对精原干细胞(SSCs)的影响。方法取5～6周龄昆明小白鼠睾丸组织,分离纯化并培养SCs,将其分为4组:正常对照组(Con组),添加LPS处理1 d组(1 d组)、2 d组(2 d组)和3 d组(3d组),收集4组培养液作为条件培养液,采用RT-PCR和Western blot检测各组SCs GDNF表达的变化;取5～7 d龄昆明小乳鼠睾丸组织,筛选SSCs后分为4组:Con组、Ⅰ组、Ⅱ组和Ⅲ组,分别用前面的4组培养液培养96 h。通过免疫荧光染色和RT-PCR检测细胞中PLZF、oct4、PCNA和c-kit、sohlh2的表达变化,并通过RT-PCR检测SSCs中Gfrα1、c-ret和Bcl6b、Etv5的表达变化。结果 RT-PCR与Western blot结果显示,SCs GDNF表达按Con、1 d、2 d、3 d组的顺序降低;RT-PCR和免疫荧光染色结果显示,SSCs PLZF、oct4和PCNA的表达按Con、Ⅰ、Ⅱ、Ⅲ组顺序降低,而c-kit和sohlh2的表达按Con组、Ⅰ、Ⅱ、Ⅲ组顺序升高;SSCs中Gfrα1、c-ret、Bcl6b与Etv5的表达Ⅰ、Ⅱ和Ⅲ组均低于Con组。结论 LPS处理导致小鼠SCs GDNF表达减少,该变化可使SSCs分化加速而其自我更新受到抑制,其机制可能与Bcl6b和Etv5的表达下调有关。
Objective To investigate the changes of glial cell line-derived neurotrophic factor (GDNF) expression in mouse testis supportive cells (SCs) treated with lipopolysaccharide (LPS) and the effect of these changes on spermatogonial stem cells (SSCs). Methods The testicular tissues of Kunming mice were obtained from 5 to 6 weeks old. The SCs were isolated and purified. The SCs were divided into four groups: normal control group (Con group), LPS group for 1 d (1 d group) and 2 d group 2 d group) and 3 d group (3d group). Four groups of culture medium were collected as conditioned medium. The expression of GDNF in SCs of each group was detected by RT-PCR and Western blot. After being screened for SSCs, the cells were divided into 4 groups: Con group, Ⅰ group, Ⅱ group and Ⅲ group, which were cultured in the former four groups for 96 h. The expression of PLZF, oct4, PCNA and c-kit, sohlh2 were detected by immunofluorescence staining and RT-PCR. The expression of Gfrα1, c-ret, Bcl6b and Etv5 in SSCs were detected by RT-PCR. Results The RT-PCR and Western blot results showed that the expression of GDNF in SCs was decreased in the order of Con, 1 d, 2 d and 3 d. The expression of PLZF, oct4 and PCNA in SSCs was detected by RT-PCR and immunofluorescence staining. The expression of c-kit and sohlh2 increased in the order of Con, Ⅰ, Ⅱ and Ⅲ. The expression of Gfrα1, c-ret, Bcl6b and Etv5 in SSCs Ⅰ, Ⅱ and Ⅲ All lower than Con group. CONCLUSIONS: LPS treatment resulted in a decrease of GDNF expression in SCs in mice. This change accelerated the differentiation of SSCs and inhibited their self-renewal. The mechanism may be related to the down-regulation of Bcl6b and Etv5.