kallistatin表达...障碍的关系及其初步机制研究_汪玉.pdf

NJA中华男科学杂志National Journal of AndrologyZhonghua Nan Ke Xue Za Zhi2022，28(11):976 984http:/www androl cn论著Clinical esearch(临床研究)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汪玉，陈鑫月，尚坤，胥琴，李云秀，杜愿，转黎(昆明理工大学附属医院/云南省第一人民医院生殖医学科，云南 昆明 650032)【摘要】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、B2、Bcl-2、casepase-3、Bax 等分子在 mNA 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、B2 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 kal-listatin 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。【关键词】kallistatin 基因;精子发生;生精功能障碍;男性不育症中图分类号:698文献标志码:Adoi:10 13263/j cnki nja 2022 11 003elationship of the kallistatin gene expression with male spermatogenicdysfunction and its possible mechanismWANG Yu，CHEN Xin-yue，SHANG Kun，XU Qin，LI Yun-xiu，DU Yuan，ZHUAN LiDepartment of eproductive Medicine，The Affiliated Hospital of Kunming University of Science and Technology/TheFirst Peoples Hospital of Yunnan Province，Kunming，Yunnan 650032，China【Abstract】Objective:To explore the role of the kallistatin gene in male spermatogenesis and its possible mechanism，and pro-vide some new ideas for the clinical treatment of spermatogenic dysfunctionMethods:We collected semen samples from the patients679基金项目:国家自然科学基金项目(82260301，81460234，81760470，81660247);云南省生殖妇产疾病临床医学中心 开放课题(2020LCZXKF-SZ01);云南省卫生健康委员会医学学科带头人培养计划项目(D-2019018，D-2017022);云南省卫生健康委员会医学学科后备人才培养计划项目(H-2017025);云南省科技计划项目-昆医联合专项 2017FE467(-155)作者简介:汪玉(1996-)，女，云南昭通市人，硕士研究生，从事生殖医学与人类辅助生殖技术研究。陈鑫月(1997-)，女，云南昆明市人，硕士研究生，从事生殖医学与人类辅助生殖技术研究。汪玉和陈鑫月对本研究有同等贡献，为共同第一作者。通讯作者:转黎，Email:zhuanli yunnan163 comwith oligospermia(OS)or non-obstructive azoospermia(NOAS)as well as testis tissue from testicular puncture We detected the dif-ferential expression of kallistatin in the seminal plasma and the mNA and protein expression levels of kallistatin，KLK1，B2，Bcl-2，casepase-3，Bax and other molecules in the testis tissue，assessed the testicular spermatogenic function using Johnsens scores，exam-ined the interstitial fibrosis in the testis by Masson and Sirius staining，and analyzed the correlation of the expression level of kallistatinwith spermatogenesis，apoptosis and fibrosisesults:Kallistatin was highly expressed in the seminal plasma and testis tissue Theexpression of kallistatin was significantly decreased in the seminal plasma(P 0 05)and so were those of kallistatin，KLK1 andB2 in the testis tissue of the NOAS and OS patients compared with those in the normal controls(P 0 01)，but no statistically sig-nificant difference was found between the expression levels of kallistatin and KLK1 within the same group(P 0 05)The expressionof Bcl-2 in the testis tissue was significantly lower(P 0 01)and those of Bax and Casepase-3 dramatically higher in the OS andNOAS patients than in the normal males(P 0 01)Cell apoptosis was negatively correlated with the expression of kallistatin Theresults of Masson and Sirius staining showed that the fibrosis of the testis tissue and the ratio of type I/III collagen fibers were markedlyincreased in the OS and NOAS patients in comparison with the normal controls，even more significantly in the NOAS than in the OSgroupConclusion:Decreased expression of kallistatin may be related to spermatogenic dysfunction，and the kallistatin expressionplays a regulatory role in the testicular spermatogenesis，probably by regulating cell apoptosis and fibrosis in the testis tissue，but thespecific mechanism needs to be confirmed by further studies【Keywords】kallistatin gene;spermatogenesis;spermatogenic dysfunction;male infertilitySupported by grants from National Natural Science Foundation of China(82260301，81460234，81760470 and 81660247)，Open Project of Yunnan Pro-vincial Clinical Medical Center of eproduction，Obstetrics and Gynecology(2020LCZXKF-SZ01)，Training Program of Yunan Province Health Commis-sion for High-Level Medical Talents(D-2019018 and D-2017022)，Training Program of Yunan Province Health Commission for eserve Medical Talents(H-2017025)and Joint Foundation of Yunnan Provincial Science and Technology Program and Specialized Project of Kunming Medical University(2017FE467-155)Correspondence to:ZHUAN Li，email:zhuanli yunnan163 comeceived:May 27，2022;accepted:September 15，2022WHO 将不育症定义为男女双方有规律、无保护性生活 1 年后无法成功受孕1。大约 30%不育症是由男性因素导致的，不育症严重影响了 1/20 左右育龄期男性(青春期至 40 岁)生殖健康2-3，其被认为是由遗传因素、有毒化学品和炎症因素及内分泌异常导致的精子参数异常4。研究表明，氧化应激通过调节生殖细胞自噬、衰老、凋亡、微血管生成和组织纤维化对精子发生进行调控5-6。组织激肽释放酶结合蛋白(kallistatin)是一种独特的丝氨酸蛋白酶抑制剂，是激肽释放酶 激肽系统(Kallikrein-kinin system，KKS)的重要成员之一，其广泛分布