kallistatin表达水平与男性生精功能障碍的关系及其初步机制研究

Relationship of the kallistatin gene expression with male spermatogenic dysfunction and its possible mechanism

目的:探索kallistatin基因在男性生精功能中的作用及其可能的作用机制,为临床治疗生精功能障碍提供新的思路。方法:收集诊断为少精子症、非梗阻性无精子症患者和正常男性的精液样本以及睾丸穿刺术后的睾丸组织。首先,检测患者精浆中kallistatin表达水平差异;然后,检测睾丸组织中kallistatin、KLK1、B2R、Bcl-2、casepase-3、Bax等分子在mRNA和蛋白水平的表达情况;其次,通过Johnsen评分观察睾丸生精功能、Masson染色及天狼猩红染色检测睾丸组织间质纤维化水平;最后,通过相关性分析评价kallistatin表达水平与睾丸生精功能、细胞凋亡水平和组织纤维化的关系。结果:kallistatin高表达于精浆和睾丸组织中,与正常男性相比,非梗阻性无精子症及少精子症患者精浆中的kallistatin水平明显降低(P<0.05);少精子症组与非梗阻性无精子症组患者睾丸组织中kallistatin、KLK1、B2R表达水平显著低于正常对照组(P<0.01),而正常组、少精子症组及非梗阻性无精子症组各组内kallistatin、KLK1两者表达未见明显差异(P>0.05)。少精子症及非梗阻性无精子症组患者睾丸组织中Bcl-2表达水平显著低于正常组(P<0.01),而Bax及Casepase-3表达水平明显高于正常组(P<0.01),凋亡水平与kallistatin表达水平呈负相关趋势。Masson染色和天狼猩红染色结果显示,非梗阻性无精子症及少精子症患者睾丸组织中的纤维化水平及I/III型胶原纤维比例显著增加,非梗阻性无精子症组增加程度高于少精子症患者。结论:kallistatin表达水平降低可能与睾丸生精功能障碍有关,并且其可能通过调控睾丸组织中细胞凋亡水平、纤维化水平对生精功能发挥调控作用,具体机制有待进一步研究证实。

Objective:To explore the role of the kallistatin gene in male spermatogenesis and its possible mechanism,and provide some new ideas for the clinical treatment of spermatogenic dysfunction.Methods:We collected semen samples from the patients with oligospermia(OS)or non-obstructive azoospermia(NOAS)as well as testis tissue from testicular puncture.We detected the differential expression of kallistatin in the seminal plasma and the mRNA and protein expression levels of kallistatin,KLK1,B2R,Bcl-2,casepase-3,Bax and other molecules in the testis tissue,assessed the testicular spermatogenic function using Johnsen's scores,examined the interstitial fibrosis in the testis by Masson and Sirius staining,and analyzed the correlation of the expression level of kallistatin with spermatogenesis,apoptosis and fibrosis.Results:Kallistatin was highly expressed in the seminal plasma and testis tissue.The expression of kallistatin was significantly decreased in the seminal plasma(P<0.05)and so were those of kallistatin,KLK1 and B2R in the testis tissue of the NOAS and OS patients compared with those in the normal controls(P<0.01),but no statistically significant difference was found between the expression levels of kallistatin and KLK1 within the same group(P>0.05).The expression of Bcl-2 in the testis tissue was significantly lower(P<0.01)and those of Bax and Casepase-3 dramatically higher in the OS and NOAS patients than in the normal males(P<0.01).Cell apoptosis was negatively correlated with the expression of kallistatin.The results of Masson and Sirius staining showed that the fibrosis of the testis tissue and the ratio of type I/III collagen fibers were markedly increased in the OS and NOAS patients in comparison with the normal controls,even more significantly in the NOAS than in the OS group.Conclusion:Decreased expression of kallistatin may be related to spermatogenic dysfunction,and the kallistatin expression plays a regulatory role in the testicular spermatogenesis,probably by regulating cell apoptosis and fibrosis in the testis tissue,but the specific mechanism needs to be confirmed by further studies.