基于GR/ERK/CX43研究母代肾精亏虚诱发子代原发性睾丸生精功能减弱的宫内编程机制  

GR-Mediated Intrauterine Programming Regulation CX43 to Study the Mechanism of Induced Primary Spermatogenic Dysfunction in Offspring with Deficiency of Renal Essence of Female Rat

作　　者：萧闵[1] 江晓翠 刘祺[2] 田代志[1] 姜兴宇 龚健 陈思易 曹继刚 XIAO Min;JIANG Xiao-cui;LIU Qi;TIAN Dai-zhi;JIANG Xing-yu;GONG Jian;CHEN Si-yi;CAO Jigang(Laboratory Animal Center,Hubei University of Chinese Medicine,Wuhan 430065,China;Hubei Hospital of Traditional Chinese Medicine,Wuhan 430061,China;College of Basic Medical Sciences,Hubei University of Chinese Medicine,Wuhan 430065,China)

机构地区：[1]湖北中医药大学实验动物中心,武汉430065 [2]湖北中医药大学附属省中医院,武汉430061 [3]湖北中医药大学基础医学院,武汉430065

出　　处：《时珍国医国药》2024年第2期494-499,共6页Lishizhen Medicine and Materia Medica Research

基　　金：国家自然科学基金青年基金(82104704);湖北省中医药管理局2023~2024年度中医药重点项目(ZY2023Z024);2022年度湖北省教育厅科学研究计划项目(D2022006);湖北省中医药管理局2023~2024年度中医药指导性项目(ZY2023F134);湖北省武汉中青年名中医项目{武卫[2019]17号};2021年湖北名师工作室项目;2022年校级科研平台项目:中医药防治生殖系统疾病研究中心项目(中医校[2022]80号);湖北中医药大学“青苗计划”资助项目(2022ZZXQ033)。

摘　　要：目的明确母代肾精亏虚导致子代原发性睾丸生精功能减弱的宫内编程机制。方法制备孕鼠24只,数字随机法均分为空白组,模型组,补肾组;除空白组以外,余下各组孕期采用慢性应激复制母鼠肾精亏虚模型。酶联免疫吸附法(ELISA)法检测母鼠血清甲状腺素(T4)、糖皮质激素(GC)、胎产数评估母鼠模型。从孕0天开始,补肾组给予左归丸灌胃填补肾精;孕期20天时,比较母鼠一般情况及体重变化,雄性胎鼠睾丸体质比,ELISA法检测其血清T4、GC、促卵泡生长激素(FSH)含量,并通过苏木素-伊红染色评估睾丸生殖细胞发育状况,免疫荧光双染检测胎鼠睾丸缝隙连接蛋白43(Cx43)、SYR-盒包含蛋白9(Sox9)表达,实时荧光定量聚合酶链式反应检测胎鼠睾丸细胞外调节蛋白激酶(ERK1/2)、Cx43基因表达,蛋白免疫印迹法检测胎盘组织GR、胎鼠睾丸ERK1/2、Cx43、促卵泡生长激素受体(FSHR)蛋白表达。结果与空白组母鼠比较,模型组母鼠,产后体重减轻、胎产数减少、血清T4含量降低、血清GC含量升高(P<0.01);与模型组母鼠比较,补肾组母鼠,产后体重增加、胎产数增加、血清T4含量升高、GC降低、(P<0.01)。与空白组雄性胎鼠比较,模型组雄性胎鼠血清T4含量降低、GC、FSH含量均升高、睾丸体质比降低、支持细胞数量、精原细胞数量及曲精小管个数均降低、睾丸Sox9、Cx43蛋白降低,ERK、Cx43 mRNA表达降低、FSHR表达升高(P<0.01或P<0.05),胎盘组织GR蛋白升高(P<0.05);与模型组比较,补肾组胎鼠血清T4含量升高、GC、FSH含量均降低、睾丸体质比升高、支持细胞数量、精原细胞数量及曲精小管个数均升高、睾丸ERK、Cx43 mRNA表达升高、Sox9、Cx43蛋白升高、FSHR表达降低(P<0.01或P<0.05),胎盘组织GR蛋白降低(P<0.05)。结论母鼠孕期肾精亏虚是原发性睾丸生精功能障碍的诱因之一,其机制可能与GR/ERK/CX43宫内编程调控支持细胞数量相关�Objective To clarify the mechanism of primary testicular spermatogenic dysfunction in offspring induced by deficiency of renal essence in Maternal generation.Methods 24 pregnant rats were prepared and randomly divided into blank group,model group,and kidney tonifying group;In addition to the blank group,the remaining groups used chronic stress to replicate the maternal model of kidney essence deficiency during pregnancy.Enzyme-linked immunosorbent assay(ELISA)was used to detect maternal serum thyroxine(T4),glucocorticoid(GC),and fetal number to evaluate the maternal model.Starting from the 0th day of pregnancy,the kidney tonifying group was given Zuogui Pill(0.39 mg/Kg)by gavage to fill the kidney essence;At the 20th day of pregnancy,compare the general situation and weight changes of female rat.Take male fetal rat,and detect their serum T4,GC,and FSH levels by ELISA.Evaluate the development of testicular germ cells by hematoxylin eosin staining.Immunofluorescence double staining is used to detect the expression of Cx43 and Sox9 proteins in the testicular tissue of fetal rat.Real-time fluorescence quantitative polymerase chain reaction is used to detect the expression of ERK and Cx43 genes in the testicular tissue of fetal rat.Protein immunoblotting is used to detect GR Expression of ERK1/2,Cx43,and FSHR proteins in fetal rat testis.Results Compared with the blank group,the maternal weight in the model group decreased,the number of fetal births decreased,the serum T4 content decreased,and the serum GC content increased(P<0.01);Compared with the model group,the mothers in the kidney tonifying group had increased postpartum weight,increased fetal number,increased serum T4 content,and decreased GC(P<0.01).Compared with the male fetal rats in the blank group,the serum T4 content in the model group decreased,the GC and FSH content increased,the testicular body mass ratio decreased,the number of sertoli cells,spermatogonia,and the number of seminiferous tubules decreased,the Sox9 and Cx43 proteins in the testicles decr

关 键 词：母代 肾精亏虚 睾丸发育 宫内编程 糖皮质激素 缝隙连接蛋白43 

分 类 号：R2-03[医药卫生—中医学]