缺氧诱导因子α在特发性非梗阻性无精子症生精功能障碍中的作用研究

发布时间：2018-06-07 03:52

本文选题：特发性非梗阻性无精子症 + 缺氧诱导因子α　；参考：《青岛大学》2017年博士论文

【摘要】：研究目的:特发性非梗阻性无精子症(idiopathic azoospermia,IA)是男性不育中病因不明、治疗困难的一种病症,主要病理机制是睾丸生精功能障碍,一直是该领域的研究热点。缺氧诱导因子(hypoxia-inducible factors,HIFs)是一类在氧稳态失衡状态下激活的重要转录因子,而睾丸在正常生理状态下系低氧器官,有研究显示,在HIF-α基因敲除的小鼠中会出现睾丸生精功能衰竭。本研究拟探讨HIF-α在IA患者睾丸生精功能障碍中的作用。研究方法:本研究按照WHO标准的诊断流程进行病史采集和各项检查,制定严格的入组筛选流程,共募集30例IA患者作为实验组,30例OA患者作为对照组,所有入组患者均实施经皮睾丸穿刺抽吸术(testicular sperm aspiration,TESA)。观察TESA标本HE染色病理变化,分别计数实验组和对照组的睾丸支持细胞,并应用免疫组化方法检测支持细胞异常增生情况。应用western blotting方法检测两组TESA标本的抗氧化蛋白超氧化物歧化酶(Superoxide Dismutase,SOD)、血红素加氧酶(Hemeoxygenase-1,HO-1)、HIF-1α/2α、紧密连接蛋白Zonula occluden-1(ZO-1)、Claudin和Occludin的表达变化,并进一步检测HIF-α的靶点调节分子铁代谢相关蛋白二价金属离子转运体1(Divalent metal transporter 1,DMT1)、ferroportin1(FPN1)和铁调节蛋白1(Iron regulatory protein1,IRP1)的表达变化。然后在体外培养的小鼠睾丸支持细胞(TM4)系中,应用western blotting方法检测不同浓度双酚A、氮芥处理后上述蛋白的表达变化,并评价了抗氧化剂姜黄素和N-乙酰半胱氨酸(Nacetyl-L-cysteine,NAC)的保护作用。研究结果:1、参考WHO 2000年《男性不育标准化诊疗手册》(2000 WHO Manual for the Standardized Investigation,Diagnosis and Management of the Infertile Male)所述标准的OA和IA诊断流程,经过严格筛选,对招募的220位无精子症志愿者进行分类,将符合上述分类诊断标准者分别纳入实验组(IA组,共30例)和对照组(OA组,共30例)。两组病人在年龄、体重指数、就诊时不育时间方面均无统计学差异。2、IA和OA组患者病因分析表明,IA组病人均无明确病因,且无其他适用的疾病诊断,为特发性无精子症;OA组病人无精子症病因则包括附睾炎、附睾结核、先天性发育异常、医源性病因等多种类型。3、IA和OA组患者体格检查表明:两组患者全面体格检查未发现明显异常,第二性征发育良好,阴茎发育正常;IA组30例患者双侧睾丸、附睾均可于阴囊内扪及,但IA组患者睾丸容积较OA组患者变小,且质地明显变软,OA组患者双侧睾丸查体未及异常,所有OA患者的附睾均可扪及明显膨胀、饱满,另有5例OA患者未能触及输精管结构。阴囊超声检查辅助估算睾丸容积大小,IA组患者睾丸容积明显减小,仅不到OA组患者睾丸容积的一半(6.2m L vs.15.1m L,P0.001)。4、血清卵泡刺激素(Follicle Stimulating Factor,FSH)、黄体生成素(luteinizing hormone,LH)、催乳素(Prolactin,PRL)和睾酮(Testosterone,TT)水平检测表明:IA组患者血清FSH(16.40±5.60 IU/L vs.3.64±1.53 IU/L,P0.001)、LH(10.48±2.20 IU/L vs.4.49±1.59 IU/L,P0.001)水平较OA组均显著升高,而IA和OA两组患者的血清PRL(10.59±2.00 IU/L vs.10.55±1.91 IU/L,P0.05)、TT(11.33±2.26 nmol/L vs.10.65±2.10 nmol/L,P0.05)水平无明显差异。5、IA组和OA组患者睾丸穿刺组织的HE染色病理分析表明:IA组典型的病理改变包括:(1)生精小管内生殖细胞数量显著减少,且往往精子发生紊乱,几乎不见成熟的精子细胞;(2)生精小管腔内充满脱落的细胞,含有少量生殖细胞的生精小管与仅有支持细胞的生精小管同时存在,支持细胞数量明显增多;(3)生精小管管腔明显缩小;(4)睾丸间质细胞未见明显异常改变。OA组患者睾丸穿刺组织的典型病理改变包括:(1)精子发生的各个阶段都存在,可见成熟的精子细胞,但各阶段生殖细胞正常的排列顺序消失;(2)生精小管结构、管腔直径正常,但是中心腔消失并充满脱落的细胞;(3)睾丸间质细胞未见明显异常改变。6、IA组和OA组患者睾丸穿刺组织中支持细胞计数表明:IA组生精小管每单位横截面的睾丸支持细胞数量要明显多于OA组(38.0±3.5 vs.19.7±2.6,P0.001),IA组生精小管周长与OA组无明显差异(422.15±18.33μm vs.433.17±25.61μm,P0.05);IA组患者睾丸组织中的支持细胞分布密度(Sertoli cell density,SCD)显著高于OA组(0.093±0.008 vs.0.044±0.005,P0.001)。7、IA组和OA组患者睾丸支持细胞增殖活性评价:IA组和OA组的睾丸支持细胞均呈现胞浆内支持细胞标记物抑制素α(Inhibinα)染色强阳性,而且在连续组织切片上,可观察到部分睾丸支持细胞呈现细胞核细胞增殖活性标记物Ki-67染色强阳性;进一步计算Ki-67阳性指数(Ki-67 Labelling Index,Ki-67 LI),结果提示IA组睾丸支持细胞的Ki-67 LI高于OA组3倍以上(48%±6%vs.13%±5%,P0.001)。8、免疫印记法检测IA组和OA组患者睾丸穿刺组织中应激反应蛋白HO-1的表达变化,结果提示IA组与OA组之间无明显变化;但IA组的抗氧化蛋白SOD表达水平明显下降,IA组比OA组降低了75.2%±16.2%(P0.05)。提示IA患者睾丸组织内存在着抗氧化能力不足。9、免疫印记法检测IA组和OA组患者睾丸穿刺组织中HIF-1α的表达变化:IA组明显上调,升高达OA组的2.44±0.94倍(P0.01);而IA组的HIF-2α表达水平显著下调,比OA组降低了49%±11.3%(P0.01)。10、免疫印记法检测IA组和OA组患者睾丸穿刺组织中铁代谢相关蛋白的表达变化:IA组的铁转入蛋白DMT1+IRE表达水平要比OA组高出29%±8.3%(P0.05);IA组的铁转出蛋白FPN1表达水平相对比OA组存在升高趋势,但是两组间FPN1的表达水平差异无统计学意义;铁调节蛋白IRP1在IA组患者睾丸穿刺组织中表达水平较OA组升高22%±5.2%(P0.05)。11、免疫印记法检测IA组和OA组患者睾丸穿刺组织中紧密连接蛋白的表达变化:IA组ZO-1和Claudin蛋白表达水平较OA组均显著降低,分别为OA组的62.3%和43.5%(P0.05);Occludin蛋白表达水平降低最明显,IA组只有OA组的7.2%(P0.05)。12、体外培养小鼠睾丸支持细胞(TM4)系,分别应用不同浓度(10-7、10-6、10-5、10-4、10-3mol/L)的双酚A(Bisphenol A,BPA)和氮芥(nitrogen mustard,NM)处理后,在药物浓度低于10-5mol/L时TM4细胞存活率均没有明显变化,但在药物浓度高于10-4mol/L时TM4细胞存活率明显下降,BPA处理组TM4细胞存活率分别仅为对照组的57.8%、24.9%,氮芥处理组分别是对照组的62.2%、38.6%(P0.05)。13、BPA和氮芥分别处理TM4细胞后,免疫印迹法观察HIF-1α和HIF-2α的表达变化:较低浓度10-6mol/L BPA处理TM4细胞24h后,BPA处理组细胞裂解液中的HIF-2α表达水平明显上调,是对照组的1.41倍(P0.05),而HIF-1α的表达有升高趋势,但无统计学意义;10-6mol/L氮芥处理TM4细胞24h后,氮芥处理组的HIF-1α和HIF-2α表达水平均明显上调,分别是对照组的2.71倍和1.72倍(P0.05)。较高浓度10-5mol/L BPA处理TM4细胞24h后,BPA处理组细胞裂解液中的HIF-1α和HIF-2α表达水平均明显下调,分别仅为对照组的39%和32.2%(P0.05);较高浓度10-5mol/L氮芥处理TM4细胞24h后,氮芥处理组的HIF-1α和HIF-2α表达水平也均明显下调,分别为对照组的33.6%和37.7%(P0.05)。14、BPA和氮芥分别处理TM4细胞后,免疫印迹法观察铁代谢蛋白的表达变化:10-6mol/L和10-5mol/L BPA处理TM4细胞24h后,BPA处理组细胞裂解液中铁转入蛋白DMT1+IRE表达水平均明显上调,分别为对照组的1.83倍和1.72倍(P0.05);10-6mol/L和10-5mol/L氮芥处理TM4细胞24h后,氮芥处理组细胞裂解液中的DMT1+IRE表达水平也分别上调1.75倍和1.82倍(P0.05)。上述不同浓度的BPA和氮芥分别处理TM4细胞24h后,药物处理组的铁转出蛋白FPN1表达水平虽有上升趋势,但差别无统计学意义。铁调节蛋白IRP1在BPA和氮芥处理组表达水平均明显上调,10-6mol/L和10-5mol/L BPA处理组的IRP1表达水平分别是对照组的1.92倍和1.5倍(P0.05),10-6mol/L和10-5mol/L氮芥处理组的IRP1表达水平分别是对照组的2.28倍和1.87倍(P0.05)。15、10-6mol/L和10-5mol/L BPA或氮芥分别处理TM4细胞24h后,免疫印迹法检测TM4细胞裂解液中的应激反应蛋白HO-1和抗氧化蛋白SOD表达水平均没有明显变化。16、分别用1、2、5、10μmol/L姜黄素处理TM4细胞24h后,在5μmol/L时细胞存活率略有下降,为对照组的94.5%,但无统计学意义;10μmol/L姜黄素可引起TM4细胞存活率明显下降,为对照组的81.9%(P0.05)。应用5μmol/L姜黄素与BPA共孵育,未能观察到姜黄素对TM4细胞存活率的保护作用;强抗氧化剂N-乙酰半胱氨酸(N-acetyl-L-cysteine,NAC)与BPA共孵育,也未能观察到其对细胞存活率的保护作用。研究结论:1.特发性非梗阻性无精子症患者睾丸穿刺组织中存在支持细胞异常增生,抗氧化能力下降,提示特发性非梗阻性无精子症患者睾丸支持细胞存在着数量和功能异常。2.特发性非梗阻性无精子症患者睾丸穿刺组织中,以及药物处理后的体外培养小鼠睾丸支持细胞中,均观察到了缺氧诱导因子-α蛋白表达异常。3.缺氧诱导因子α的下游调控分子紧密连接蛋白和铁代谢相关蛋白表达异常,提示疾病状态下血-睾屏障破坏和铁稳态失衡,这可能是缺氧诱导因子α调控异常参与特发性非梗阻性无精子症患者睾丸生精功能障碍的发病机制。
[Abstract]:Objective: idiopathic non obstructive azoospermia (idiopathic azoospermia, IA) is a disease with unknown etiology and difficult treatment in male infertility. The main pathological mechanism is testicular spermatogenesis dysfunction, which has always been a hot topic in this field. Hypoxia inducible factor (hypoxia-inducible factors, HIFs) is a class of imbalance in oxygen homeostasis. The important transcription factors are activated and the testicles are hypoxic organs in normal physiological state. Studies have shown that testicular spermatogenesis failure in HIF- alpha knockout mice. This study intends to explore the role of HIF- alpha in the Gao Wansheng sperm dysfunction in patients with IA. Methods: This study was conducted in accordance with the diagnostic process of the WHO standard. A total of 30 IA patients were recruited as the experimental group and 30 patients with OA were used as the control group. All the patients were treated with percutaneous testicular puncture aspiration (testicular sperm aspiration, TESA). The pathological changes of HE staining in TESA specimens were observed, and the testicular support of the experimental and control groups was counted. Western blotting method was used to detect the antioxidant protein superoxide dismutase (Superoxide Dismutase, SOD), heme oxygenase (Hemeoxygenase-1, HO-1), HIF-1 alpha /2 alpha, and tightly connexin Zonula occluden-1 (TESA). Expression changes, and further detection of HIF- alpha targets to regulate the expression of molecular iron metabolism related protein two valence metal ion transporter 1 (Divalent metal transporter 1, DMT1), ferroportin1 (FPN1) and iron regulatory protein 1 (Iron regulatory protein1, IRP1), but then in the mouse testis support cells (TM4) in vitro Tern blotting method was used to detect the changes of the expression of the above protein after the treatment of different concentrations of bisphenol A and nitrogen mustard, and the protective effects of the antioxidant curcumin and N- acetylcysteine (Nacetyl-L-cysteine, NAC) were evaluated. 1, reference WHO 2000 < standard treatment manual for male infertility > (2000 WHO Manual for the Standardized) Ion, Diagnosis and Management of the Infertile Male) the standard OA and IA diagnostic process. After strict screening, the recruited 220 azoospermia volunteers were classified into the experimental group (IA group, 30 cases) and the control group (OA group, a total of 30 cases). The two groups of patients were in age, body mass index, There was no statistical difference in the time of infertility at the time of diagnosis.2. The cause analysis of the patients in group IA and OA showed that the patients in group IA had no definite cause, and no other suitable diagnosis of the disease, and it was idiopathic azoospermia. The causes of azoospermia in group OA include epididymitis, epididymal tuberculosis, congenital dysplasia, iatrogenic etiology, and other types of.3, IA, and OA. The physical examination showed that there were no obvious abnormalities in the physical examination of the two groups, the second sex sign developed well, and the penis developed normally. In group IA, 30 cases of testis and epididymis were palpable in the scrotum, but the volume of testicle in group IA was smaller than that in group OA, and the texture of the patients was softer than that in the group of OA. All the patients in group OA had no abnormalities in the bilateral testis body, all O The epididymis of the patients with A could be palpably inflated and plump, and 5 patients with OA failed to touch the vas deferens. The scrotal ultrasonography assisted the estimation of the size of the testis, and the testicular volume in the IA group decreased significantly, only half of the OA patients' testicular volume (6.2m L vs.15.1m L, P0.001).4, and serum follicle stimulating hormone (Follicle Stimulating). FSH), luteinizing hormone (LH), prolactin (Prolactin, PRL) and testosterone (Testosterone, TT) level test showed that the serum FSH (16.40 + 5.60 IU/L vs.3.64 + 1.53) in the IA group was significantly higher than that in the group of two groups (10.59 + 2. (10.59 + 2.). There was no significant difference between 00 IU/L vs.10.55 + 1.91 IU/L, P0.05), TT (11.33 + 2.26 nmol/L vs.10.65 + 2.10 nmol/L, P0.05),.5. The pathological analysis of testicular biopsy in IA group and OA group showed that the typical pathological changes included: (1) the number of germ cells in the seminiferous tubules decreased significantly, and the sperm was often disorganized. Mature spermatids; (2) the cells filled with exfoliated tubules were filled with shedding cells, the seminiferous tubules containing a small amount of germ cells existed at the same time as the seminiferous tubules only supporting cells, and the number of supporting cells increased significantly; (3) the cavity of the spermatogenic tubule was obviously reduced; (4) the testicular cells did not significantly alter the canon of the testicular tissue in the.OA group. Pathological changes include: (1) all stages of spermatogenesis exist, mature spermatocyte can be seen, but the sequence of normal arrangement of germ cells in each stage disappears; (2) spermatogenic tubule structure, the diameter of the lumen is normal, but the central cavity disappears and fills the shedding cells; (3) there is no obvious abnormal change in.6, IA and OA patients. The count of support cells in the pellet puncture tissue showed that the number of testis supporting cells per unit cross section of the IA group was more than that of the OA group (38 + 3.5 vs.19.7 + 2.6, P0.001). There was no significant difference between the perimeter of the seminiferous tubules in the IA group and the OA group (422.15 + 18.33 mu m vs.433.17 + 25.61 m, P0.05), and the support cells in the testicular tissue of the IA group were densely distributed. Sertoli cell density (SCD) was significantly higher than that of OA group (0.093 + 0.008 vs.0.044 + 0.005, P0.001).7, and the proliferation activity of testis support cells in the IA group and OA group was evaluated. Ki-67 positive index (Ki-67 Labelling Index, Ki-67 LI) of Ki-67 positive index (Ki-67 Labelling Index) was further calculated. The results suggested that the Ki-67 LI of testis support cells in IA group was more than 3 times higher than that of OA group (48% + 5%). The expression of stress response protein HO-1 expression in the tissue showed no significant changes between the IA group and the OA group, but the expression level of the antioxidant protein SOD in the IA group decreased obviously, and the IA group decreased by 75.2% + 16.2% (P0.05) than the OA group. It suggested that the testicular tissue in the IA patients was less than the antioxidant capacity.9, and the immunoimprint method was used to detect the testicles of IA group and OA group. The changes in the expression of HIF-1 alpha in the puncture tissue: the IA group was obviously up-regulated and up to 2.44 + 0.94 times (P0.01) in the OA group, while the HIF-2 alpha expression level in the IA group was significantly lower than that in the OA group by 49% + 11.3% (P0.01).10. The expression of iron metabolism related proteins in the testis tissue of the IA and OA groups of the IA and OA groups was detected by the immuno imprint method: the iron transfer of the IA group to the protein The expression level of 1+IRE was 29% + 8.3% higher than that of the OA group (P0.05), and the expression level of iron transfer protein FPN1 in the IA group was higher than that in the OA group, but the expression level of FPN1 in the two groups was not statistically significant; the expression of iron regulatory protein IRP1 in the testis tissue in the IA group was 22% + 5.2% (P0.05).11, immune imprinting method The expression of tight connexin in IA group and OA group was detected. The expression level of ZO-1 and Claudin protein in group IA was significantly lower than that in group OA, 62.3% and 43.5% (P0.05) in group OA, respectively, and the expression level of Occludin protein was the most obvious. The IA group was only 7.2% of OA group (P0.05), and in vitro culture of mouse testis support cells After the treatment of Bisphenol A (Bisphenol A, BPA) and nitrogen mustard (nitrogen mustard, NM) with different concentrations (10-7,10-6,10-5,10-4,10-3mol/L), there was no significant change in the survival rate of TM4 cells when the drug concentration was lower than 10-5mol/L, but the survival rate of TM4 cells decreased significantly when the drug concentration was higher than that of 10-4mol/L. Only 57.8% of the control group, 24.9%, nitrogen mustard treatment group was 62.2% of the control group, 38.6% (P0.05).13, BPA and nitrogen mustard treated TM4 cells respectively. The expression of HIF-1 A and HIF-2 alpha was observed by immunoblotting: after the low concentration 10-6mol/L BPA treated TM4 cell 24h, the expression of HIF-2 alpha in the cell lysate of BPA treatment group was obviously up. The expression of HIF-1 alpha was 1.41 times (P0.05), but the expression of HIF-1 alpha was higher, but it was not statistically significant. After 24h of 10-6mol/L mustard treated TM4 cells, the expression level of HIF-1 alpha and HIF-2 alpha in the treatment group of nitrogen mustard were obviously up, which were 2.71 times and 1.72 times of that of the control group (P0.05). The expression level of HIF-1 alpha and HIF-2 alpha in the liquid was obviously down, only 39% and 32.2% of the control group (P0.05), while the high concentration of 10-5mol/L nitrogen mustard treated TM4 cells 24h, and the expression level of HIF-1 A and HIF-2 alpha in the treatment group of nitrogen mustard were also down significantly, respectively, 33.6% and 37.7% (P0.05).14 of the control group, and BPA and nitrogen mustard treated TM4 cells respectively. The expression of iron metabolic protein was observed by the immunoblotting method: after 10-6mol/L and 10-5mol/L BPA treated TM4 cell 24h, the expression level of iron to protein DMT1+IRE in the cell lysate of BPA treatment group were up to 1.83 and 1.72 times (P0.05), respectively. 10-6mol/L and 10-5mol/ L nitrogen mustard treated the cell cracking of the nitrogen mustard treatment group. The expression level of DMT1+IRE in the liquid was also up up 1.75 and 1.82 times (P0.05). The expression level of iron transferred protein FPN1 in the treatment group was up, but the difference was not statistically significant. The expression level of iron regulatory protein IRP1 in BPA and nitrogen mustard treatment group was up obviously up, 10, 10. The expression level of IRP1 in -6mol/L and 10-5mol/L BPA treatment group was 1.92 times and 1.5 times as much as that of the control group (P0.05). The IRP1 expression level of 10-6mol/L and 10-5mol/L nitrogen mustard treatment group was 2.28 times and 1.87 times (P0.05).15,10-6mol/L and 10-5mol/L BPA or nitrogen mustard respectively. The expression level of stress response protein HO-1 and antioxidant protein SOD in the liquid did not significantly change.16. The cell survival rate of 1,2,5,10 mu mol/L curcumin in TM4 cell 24h decreased slightly at 5 u mol/L, which was 94.5% of the control group, but no statistical significance. The survival rate of TM4 cells decreased significantly by 10 u mol/L curcumin, which was the control group. 81.9% (P0.05). The protective effect of curcumin on the survival rate of TM4 cells was not observed with 5 mu curcumin and BPA, and the strong antioxidant N- acetylcysteine (N-acetyl-L-cysteine, NAC) was incubated with BPA, and the protection of cell survival was not observed. Conclusion: 1. idiopathic non obstructive azoospermia patients There are abnormal proliferation of support cells and decreased antioxidant capacity in the testicular tissue, suggesting that the testicular support cells in the patients with idiopathic non obstructive azoospermia exist in the testicular tissue of the patients with.2. idiopathic non obstructive azoospermia, and the mouse testis support cells in vitro after the drug treatment. The abnormal expression of close connexin and iron metabolism related protein in the downstream regulator of hypoxia inducible factor alpha protein expression.3. hypoxia inducible factor alpha was observed, suggesting the destruction of blood testosterone barrier and iron homeostasis under the condition of disease, which may be the abnormal involvement of hypoxic inducible factor alpha in idiopathic non obstructive azoospermia patients. The pathogenesis of testicular spermatogenesis dysfunction.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R698.2

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