外泌体负载枸杞miR2911调控Wnt/β-catenin信号通路促进非梗阻性无精子症大鼠生精功能恢复  

Lycium barbarum miR2911-loaded exosomes promote spermatogenic function recovery in rats with non-obstructive azoospermia by regulating Wnt/β-catenin signaling pathways

作　　者：田稼[1,2] 谢治远 裴承斌 宋爱华 周岳[1,2] 马良宏[1,2,5] TIAN Jia;XIE Zhi-yuan;PEI Cheng-bin;SONG Ai-hua;ZHOU Yue;MA Liang-hong(Ningxia Human Sperm Bank,Research Institute of Medical Sciences,General Hospital of Ningxia Medical University,Yinchuan,Ningxia 750001,China;Key Laboratory of the Ministry of Education for Fertility Preservation and Maintenance,Ningxia Medical University,Yinchuan,Ningxia 750001,China;Department of Urology,Taizhou Peoples Hospital Affiliated to Nanjing Medical University,Taizhou,Jiangsu 225300,China;Day-Care Unit,General Hospital of Ningxia Medical University,Yinchuan,Ningxia 750001,China;Department of Urology,General Hospital of Ningxia Medical University,Yinchuan,Ningxia 750001,China)

机构地区：[1]宁夏医科大学总医院医学科学研究院/宁夏人类精子库,宁夏银川750001 [2]宁夏医科大学生育力保持教育部重点实验室,宁夏银川750001 [3]南京医科大学附属泰州人民医院泌尿外科,江苏泰州225300 [4]宁夏医科大学总医院日间病房,宁夏银川750001 [5]宁夏医科大学总医院泌尿外科,宁夏银川750001

出　　处：《中华男科学杂志》2024年第6期483-492,共10页National Journal of Andrology

基　　金：宁夏回族自治区重点研发计划项目(2020BFH02002)。

摘　　要：目的:研究外泌体负载的枸杞miRNA(Lb-miR2911)药物通过跨界调控Wnt/β-catenin信号通路对非梗阻性无精子症(NOA)大鼠模型生精功能恢复的影响。方法:4周龄雄性SD大鼠30只,采用白消安腹腔注射方法构建NOA模型,造模5周后将模型大鼠随机分为模型组(MC)、Lb-miR2911^(EXO)治疗组(Lb-miR2911^(EXO))、外泌体空载治疗组(Sham),每组10只。MC组NOA模型大鼠自然恢复,无特殊处理;Lb-miR2911^(EXO)组NOA模型大鼠,睾丸生精小管注射外泌体包被Lb-miR2911药物(0.1 mg/kg),1次/周,共治疗3次;Sham组NOA模型大鼠,睾丸生精小管注射外泌体空载药物(0.1 mg/kg),1次/周,共治疗3次。另取10只同龄正常健康SD大鼠作为正常对照组(NC)。治疗后第2、6周采用RNA FISH技术观察Lb-miR2911药物在睾丸组织中的摄取及代谢变化;治疗后12周采用转录组测序分析结合Western印迹、RT-PCR方法检测各组大鼠睾丸组织中细胞增殖、精子发生及Wnt/β-catenin信号通路的基因表达;睾丸组织形态学及精子质量分析比较各组大鼠生精功能恢复情况;通过组织形态学分析结合血清TNF-α、IL-1β、天冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)等指标检测评估Lb-miR2911外泌体药物对大鼠组织器官毒性。结果:治疗12周后睾丸组织形态学分析发现Lb-miR2911^(EXO)组大鼠睾丸组织各层次生精细胞排列规则,管腔中可见大量成熟精子,与Sham组比较,Johnsen评分显著增加(P<0.05),睾丸重量、睾丸指数、精子浓度和精子活力显著增加(P<0.05);转录组测序分析结合Western印迹、RT-PCR方法证实,与MC组相比,Lb-miR2911^(EXO)组DACT3基因表达下调(P<0.05),而DVL2、β-catenin表达上调,同时p-DVL2及β-catenin(nucleus)蛋白含量增加(P<0.05);细胞增殖相关基因CCND1、CCNE1、CCNE2 mRNA表达上调(P<0.05);精子发生相关基因DMC1、CCR6、JAM2、KLC3表达上调(P<0.05);组织器官毒性检测表明:治疗后Lb-miR2911^(EXO)组大鼠肺部、肝�Objective:To investigate the effect of exosomes loaded with Lycium barbarum miRNA(Lb-miR2911)on spermatogenic function recovery in non-obstructive azoospermia(NOA)rats through cross-regulation of the Wnt/β-catenin signaling pathways.Methods:We established an NOA model in 30 four-week-old male SD rats by intraperitoneal injection of busulfan.At 5 weeks after modeling,we equally randomized the rats into a model control group(MC,untreated),an Lb-miR2911~(^(EXO))group(Lb-miR2911~(^(EXO)),treated by intratesticular injection of Lb-miR2911-loaded exosomes),and a sham group(Shame,treated by intratesticular injection of exosomes-empty drug),with another 10 male SD rats taken as normal controls(NC).We observed the uptake and metabolic changes of Lb-miR2911 in the testis tissue of the rats by RNA FISH at 2 and 6 weeks after treatment,detected cell proliferation,spermatogenesis and gene expressions of the Wnt/β-catenin signaling pathways in the testis tissue by Transcriptome sequencing analysis combined with Western blot and RT-PCR at 12 weeks,evaluated the recovery of the spermatogenic function based on the testis tissue morphology and sperm quality,and assessed the organ toxicity of Lb-miR2911 in the tissue and organs of the rats based on histomorphological analysis and the levels of serum TNF-α,IL-1β,Aspartate aminotransferase(AST),Alanine aminotransferase(ALT)and other relevant indicators.Results:After 12 weeks of treatment,histomorphological analysis showed regular arrangement of spermatogenic cells at all levels in the testis tissue,with a large number of mature sperm in the tubular lumen,and with significantly higher Johnsen scores,testis weight,testicular index,sperm concentration and sperm motility in the Lb-miR2911~(^(EXO))than in the sham group(all P<0.05).Compared with the model controls,the Lb-miR2911~(^(EXO))group exhibited remarkably down-regulated gene expression of DACT3(P<0.05),up-regulated expressions of DVL2 andβ-catenin(P<0.05),elevated levels of p-DVL2 andβ-catenin(nucleus)proteins(P<0.05),increa

关 键 词：枸杞 miRNA 精子发生 DACT3 WNT/Β-CATENIN通路 大鼠 

分 类 号：Q594.434[生物学—生物化学] R321.1[医药卫生—人体解剖和组织胚胎学]