未羧化骨钙素对老年大鼠睾丸间质细胞睾酮合成功能的影响

Effects of uncarboxylated osteocalcin on testosterone synthesis of testicular stromal cells in aged rats

目的:通过建立老年大鼠模型,探究未羧化骨钙素对老年雄性大鼠睾丸间质细胞睾酮合成的影响。方法:30只3月龄成年雄性大鼠,随机对半分为正常对照组和实验组,实验组颈背部皮下注射D-半乳糖溶液300 mg·kg^-1·d^-1,对照组颈背部皮下注射等剂量生理盐水,注射时间为8周。验证衰老模型构建成功后,取大鼠的睾丸间质细胞,分别用不同质量浓度0(等量PBS溶液)、1、3 ng/mL的非羧化骨钙素刺激大鼠睾丸间质细胞,24 h后收取培养液采用睾酮ELISA检测试剂盒进行睾酮浓度检测;并在24 h后收集细胞提取RNA,然后采用荧光定量PCR法检测睾丸间质细胞睾酮合成关键酶StAR、Cyp11a、Cyp17的表达。结果:同加入等量PBS溶液的对照组大鼠睾丸间质细胞相比,加入等量PBS溶液的模型组大鼠睾丸间质细胞分泌睾酮水平下降(P<0.05);对模型组大鼠睾丸间质细胞分别进行1、3 ng/mL未羧化骨钙素处理,相较于模型组加入等量PBS溶液处理的睾丸间质细胞,骨钙素处理组睾丸间质细胞培养液上清中睾酮水平均升高(P<0.05);与加入等量PBS溶液的模型组大鼠睾丸间质细胞相比,经过1 ng/mL未羧化骨钙素处理后的模型组大鼠睾丸间质细胞的StAR、Cyp11a、Cyp17基因的表达有明显的上升;经过3 ng/mL未羧化骨钙素处理后的模型组大鼠睾丸间质细胞的StAR、Cyp11a、Cyp17基因的表达有显著上升。结论:本研究成功的构建了D-半乳糖诱导的衰老大鼠模型,证明了未羧化的骨钙素可以促进D-半乳糖诱导的老年大鼠睾丸间质细胞睾酮合成,并且该作用可能是由于未羧化骨钙素促进其睾酮合成关键酶的表达引起的。

Objective:To establish an aging rat model and explore the effect of uncarboxylated osteocalcin on testosterone synthesis in testicular stromal cells of elderly male rats.Method:Thirty 3-month-old adult male rats were randomly divided into normal control and aging model groups.The model group was injected D-galactose solution 300 mg·kg^-1·d^-1 subcutaneously in the back of the neck,while the control group was injected normal saline subcutaneously in the back of the neck.The injection cycle was 8 weeks.After verifying that the aging model was successfully constructed,the rat testicular stromal cells were taken and stimulated with different mass concentrations of 0(equal PBS solution),1,3 ng/mL non-carboxylated osteocalcin,respectively.After 24 hours,the culture broth was collected and tested for testosterone concentration using a testosterone ELISA test kit,or fluorescent quantitative PCR was used to detect the expression of key testosterone synthesis enzymes such as StAR,Cyp11 a,and Cyp17 in testicular stromal cells.Result:Compared with the testis stromal cells of the control group added with the same amount of PBS solution,the level of testosterone secretion of the testis stromal cells of the model group with the same amount of PBS solution decreased(P<0.05).The testicular stromal cells were treated with 1,3 ng/mL uncarboxylated osteocalcin respectively.Compared with the testicular stromal cells treated with the same amount of PBS solution in the model group,the level of testosterone in the supernatant of the cell culture fluid of the osteocalcin treatment group elevated(P<0.05).Compared with stromal cells of the model group added with the same amount of PBS solution,the expression of StAR,Cyp11 a and Cyp17 genes in the testicular stromal cells of the model group after 1 ng/mL uncarboxylated osteocalcin treatment was increased.The expression of StAR,Cyp11 a and Cyp17 genes in the testicular stromal cells of the model group was significantly increased after 3 ng/mL uncarboxylated osteocalcin treatment.Conclusion:This study successfully constructed a D-galactose-induced aging rat model,demonstrating that uncarboxylated osteocalcin can promote D-galactose-induced synthesis of testosterone in testicular stromal cells of elderly rats,and this effect may be caused by uncarboxylated osteocalcin promoting the expression of key enzymes for testosterone synthesis.