血睾屏障组分在γ射线致不育症小鼠中的变化及影响

Role of bood-testis barrier on male infertility induced by γ-ray irradiation in mice

目的 探讨γ射线损伤致不育症小鼠中血睾屏障重要组分Claudin-3、Claudin-11表达的变化. 方法 2011年7月至2011年10月我们选取42只雄性昆明小鼠,6～8周龄,体质量20～25g.随机分为7组,每组6只,分别为空白对照A组；接受60Coγ射线单剂量射线下腹局部照射的B、C、D组,照射剂量分别为2、6、10 Gy;接受6 Gy剂量照射后应用雌二醇1、2、4μg/d干预4周的E、F、G组.于末次给药后2周,处死各组小鼠并收集睾丸,计算各组小鼠睾丸曲细精管分化指数（tubule differentiation index,TDI）,应用逆转录-荧光定量聚合酶链式反应检测睾丸组织抑制素βB、Claudin-3、Claudin-11 mRNA的水平,蛋白质印迹法检测睾丸Claudin-11蛋白的表达情况. 结果 与A组（100.0）比较,B、C、D组睾丸TDI显著下降（68.5±6.4、35.0±6.1、16.3±5.7）,差异均有统计学意义（P＜0.05）,且D组睾丸组织抑制素βB mRNA显著降低（0.5±0.2比1.0±0.1）,差异有统计学意义（P＜0.05）；C、D组Claudin-3 mRNA水平分别为A组的2.17倍和3.49倍,Claudin-11蛋白水平分别为A组的2.18倍和2.23倍.与C组（35.0±6.1）比较,F、G组TDI显著增高（61.7±7.2、55.8±11.9）,差异均有统计学意义（P＜0.05）,抑制素βB mRNA虽升高,但差异无统计学意义（均P＞0.05）；F、G组Claudin-3 mRNA水平分别为1.3±0.2和1.6±0.3,低于C组（2.2±0.2）,差异均有统计学意义（P＜0.05）,且F组Claudin-11 mRNA水平（1.2±0.2）和蛋白表达水平（1.5±0.5）均显著低于C组（1.79±0.2和2.17±0.3）,差异均有统计学意义（P＜0.05）.TDI与Claudin-3 mRNA、Claudin-11 mRNA水平均呈负相关（rs=-0.884,P＜0.05;rs=-0.758,P＜0.05）. 结论 γ射线照射可使小鼠血睾屏障组分Claudin-3、Claudin-11表达水平升高.

Objective To investigate the changes of Claudin-3 and Claudin-ll, two key compo- nents of blood-testis harrier （BTB） on male infertility induced by y-ray irradiation. Methods Forty- two KunMing male mice （20- 25 g） were divided into one control group, three y-ray irradiation groups and three estrodiol （E2） intervention groups randomly： Group A, sham controlled; the lower abnominal and scrotal area of the mice in Group B, C, D were irradiated with single dose of 2, 6 or 10 Gy 60 Co y-ray after anaesthetizd 17-estradio1 intervention were initiated in Group E, F, G after 6 Gy y-ray irradiation via hypodermic injection for 4w at the dose of 1, 2, 4μg/d, respectively. Mice were sacrificed 2 w after the last E2 administration. The tubule differentiation index （TDI） was counted in testis sections. Inhibinl3B, Claudin-3 and Claudin-ll transcription levels were assayed with semiquanti- tative real time PCR. Claudin-ll protein levels in testis were generated by western blot. Results Compared with sham control group, TDI in three y-ray irradiation groups were markedly reduced（68.5=k6.4, 35.0±6.1, 16.3±5.7 vs 100.0, all P〈0.05）. Inl/ibin^B mRNA expression level in testis of gourp D was markedly decreased （0.5 ± 0.2 vs 1.0 ± O. 1, P 〈0.05）. Claudin-3 mRNA levels of group C and D were up-regulated to 2.17 and 3.49 times, respectively. Claudin-ll protein levels were significantly increased to 2.18 and 2.23 times. Compared with group C, TDI in three E2 intervention groups were improved, which were obvious in group F and G （61.7±7.2, 55.8±11.9 vs 35.0±6.1, P〈0.05）. The Inhibin^B mRNA levels were increased, though there were no significant differences （all P ± 0.05）. Claudin-3 mRNA levels in group F and G were down-regulated （1.3 ±0.2, 1.6 ± 0.3 vs. 2.2±0.2, all P〈0.05）. In group F significantly reduced mRNA level and protein level of Claudin-ll were observed （mRNA： 1.2±0.2 vs. 1.8=t=0.2, P〈0.05~ Protein： 1.5±0.5 vs. 2.2±0.3, P〈0.05）. It was negatively correlated TDI with mRNA expression levels of Claudin-3 and Claudin-ll in the irra- diated testis （r, = --0.884, P〈0.05; rs=0.758, P 〈0.05, respectively）. Conclusions Irradia- tion could elevated the expression of claudin-3 and claudin-ll.