睾丸特异性含溴结构域的蛋白作为潜在男性抗生育药物靶标的研究进展

哺乳动物的四种含溴结构域蛋白(BET)基因在精子发生不同阶段均有表达,其中睾丸特异表达的bromodomain-containing protein(BRDT)对精子发生必不可少。BRDT在核染色质重组中起关键作用,也参与基因转录和共转录调节。BRDT的第一个溴结构域缺失导致圆形精子细胞错误伸长、精子形态严重缺陷,而圆形精子细胞的错误伸长与减数分裂之后染色质结构异常相关。另外,BRDT是形成圆形精子细胞染色质核仁所必需的;精母细胞和精子细胞缺乏全长的BRDT将会导致转录和可变剪接过程发生改变。动物实验证实一种小分子的BRDT抑制剂(JQ1),具有良好的抗生育作用,这为进一步开发可逆的男性抗生育药物开辟了新思路。     

溴结构域蛋白4通过Noxa诱导骨肉瘤细胞凋亡的机制

目的:探讨溴结构域蛋白4(BRD4)通过Noxa诱导骨肉瘤细胞凋亡的机制。方法:采用小分子干扰RNA(siRNA)沉默骨肉瘤细胞中BRD4的表达,通过蛋白质印迹法(Western blot)和实时荧光定量聚合酶链反应(RT-qPCR)检测Noxa及凋亡相关分子表达;应用BRD4降解剂ARV-825处理骨肉瘤细胞,通过W...     

溴结构域蛋白4在转录调节与肿瘤形成中的作用及其抑制剂的研究进展

溴结构域蛋白4(BRD4)作为溴域及超末端结构家族中的一员,通过调节细胞基因转录调节细胞周期,在正常细胞以及肿瘤细胞的生理活动中扮演着重要角色。作为一种转录和表观遗传调控因子,BRD4基因的过度表达以及基因重排和基因突变常与多种疾病、尤其是恶性肿瘤的发生有关。笔者对BRD4及其抑制剂在转录调节中的功能及其与肿瘤细胞的相互作用进行综述与分析,从而为相应的临床治疗提供新的思路。     

一个新的人睾丸特异性高尔基蛋白NYD-SP12的确定及其特性

本研究以含有9216个克隆的人cDNA微点阵(基因芯片)对成年人睾丸或胎儿睾丸杂交确定了一个新的人睾丸特异性基因—NYD-SP12。成年人睾丸NYD-SP12 mRNA表达水平较胎儿睾丸高30倍。同样地,半定量RT-PCR分析显示NYD-SP12同源基因在成年小鼠和幼年小鼠也具有差异性表达。对多种人组织的 NYD-SP12 mRNA行PCR扩     

多梳蛋白4与含FERM结构域蛋白4A在胃癌组织中的表达及与意义

目的:探讨胃癌组织中多梳蛋白4(Cbx4)和含FERM结构域蛋白4A(FRMD4A)表达及其与胃癌患者临床病理参数及预后的关系。方法:收集2010年2月—2013年11月收治的112例胃癌患者的手术标本(癌组织及癌旁组织)及临床病理资料,用qRT-PCR和免疫组化法中检测标本中Cbx4、FRMD4A mRNA与蛋白表达;分析胃癌组织Cbx4和FRMD4A蛋白表达水平与临床病理参数及生存率的关系,并分析影响胃癌预后的因素。结果:与癌旁正常组织比较,胃癌组织中Cbx4和FRMD4A mRNA与蛋白表达水平均明显升高(均P0.05)。Cbx4及FRMD4A蛋白表达水平与胃癌患者T分期、N分期、分化程度及远处转移明显有关(均P0.05);Cbx4、FRMD4A蛋白阳性表达组胃癌患者5年生存率均明显低于各自阴性表达组患者(χ~2=15.42,P=0.000;χ~2=21.55,P=0.000)。Cox多因素分析显示,Cbx4(HR=2.754,95%CI=1.827～4.151,P=0.000)和FRMD4A(HR=3.129,95%CI=2.282～4.290,P=0.000)蛋白表达水平以及T分期(HR=1.432,95%CI=1.241～1.652,P=0.005)、远处转移(HR=1.257,95%CI=1.208～1.308,P=0.032)是影响胃癌患者预后的独立危险因素。结论:Cbx4及FRMD4A在胃癌组织中高表达,且与患者的不良临床病理特征及预后密切相关,可能对胃癌病情评估及预后判断有一定意义。     

结直肠癌组织中溴结构域蛋白4表达水平与病人临床病理特征及预后的相关性

目的 分析结直肠癌(CRC)组织中溴结构域蛋白4(BRD4)的表达,探讨其与病人临床病理特征及预后的关系。方法 选取2014年11月～2017年5月本院收治的CRC病人,从病人根治切除术中切取CRC组织及癌旁组织。采用实时荧光定量PCR(qRT-PCR)法、免疫组化法分别检测CRC组织及癌旁组织中BRD4 mRNA及BRD4蛋白表达情况;采用Kaplan-Meier生存曲线分析BRD4表达与病人预后的关系;采用Cox回归分析影响CRC病人预后的危险因素。结果 与癌旁组织相比,CRC组织BRD4 mRNA表达水平及BRD4蛋白阳性表达率均升高(P<0.05)。BRD4 mRNA表达水平与CRC病人区域淋巴结转移(N)、远处转移(M)相关(P<0.05),BRD4蛋白表达与CRC病人组织分化程度、N、M相关(P<0.05)。Kaplan-Meier法分析显示,BRD4 mRNA高表达、BRD4蛋白阳性表达病人5年无进展生存率分别显著低于BRD4 mRNA低表达(P<0.05)、BRD4蛋白阴性表达(P<0.05)病人;BRD4 mRNA高表达、BRD4蛋白阳性...     

重组人睾丸精子结合蛋白对人精子运动参数的影响

目的:探讨重组人睾丸精子结合蛋白(TSBP)对体外培养人精子运动参数的影响。方法:将22例生育男性的精液经Percoll密度梯度离心后,分别与0.01mg/ml及0.1mg/ml的重组His6-TSBP在体外共同孵育1h或3h,同时设立对照组,Western印迹检测重组His6-TSBP与精子膜的结合情况,计算机辅助精液分析(CASA)系统测定重组His6-TSBP对精子运动参数的影响。将12例弱精子症患者的精液按同样方法处理,检测重组His6-TSBP对弱精子症患者精子运动参数的影响。结果:0.1mg/ml重组His6-TSBP与生育男性精子作用1h可以提高体外培养精子的前向运动百分率(a+b级精子百分率),培养3h后前向运动百分率和活率均有所提高,差异具有显著性(P<0.05);0.01mg/ml重组His6-TSBP对检测各指标均无显著性影响。0.1mg/ml重组His6-TSBP与弱精子症患者精子作用3h可以提高精子前向运动百分率(P<0.05),但对活率无显著影响。结论:0.1mg/ml的重组His6-TSBP在体外可以提高生育男性精子的前向运动百分率和活率及弱精子症患者精子的前向运动百分率。     

前列腺特异性膜抗原人源Fab抗体可变区基因的筛选与鉴定

目的:筛选、鉴定抗中国人前列腺特异性膜抗原(PSMA)的人源Fab抗体可变区的编码基因,为PSMA蛋白质生物学功能的研究及前列腺癌的基因治疗研究开辟新途径。方法:采用噬菌体表面展示技术,以PSMA原核表达重组子pET30 a(+)-PSMA诱导表达并纯化后的PSMA为固相抗原,从噬菌体Fab可变区抗体库中经过5轮“吸附-洗脱-扩增”筛选过程,获得抗原结合活性和特异性较强的PSMA人源Fab可变区抗体基因片段的阳性克隆,并对其进行免疫检测及序列测定。结果:筛选得到Fab抗体克隆的Fd段基因核苷酸序列为696 bp,编码由232个氨基酸残基组成的多肽,与人免疫球蛋白γ链恒定区的同源性为98%;轻链κ基因核苷酸序列为630 bp,编码由210个氨基酸残基组成的多肽,与κ链恒定区同源性为93%。结论:利用噬菌体抗体库技术,成功获得了PSMA人Fab抗体可变区编码基因克隆,该克隆抗体基因具有典型的免疫球蛋白轻链和重链可变区的结构特点,基因编码产物具有与PSMA反应的免疫学活性和特异性。     

淀粉样蛋白A在人下肢闭塞性动脉硬化的表达及其激活Nod样受体家族包含pyrin结构域蛋白3炎性体的机制

目的 观察血清淀粉样蛋白A( SAA)、Nod样受体家族包含pyrin结构域蛋白3( NLRP3)、白细胞介素-1β(IL-1β)在人下肢闭塞性动脉硬化症(ASO)中的表达,并通过SAA激活NLRP3炎性体的体外实验分析在ASO中NLRP3炎性体激活的可能机制.方法 免疫组织化学方法检测SAA、NLRP3、IL-1β在ASO血管壁中的表达.SAA处理巨噬细胞,Western blot检测成熟型IL-1β和裂解型Caspase-1,检测细胞中IL-1β前体蛋白、Caspase-1前体蛋白的表达.RNA干扰技术下调单核细胞(THP-1)细胞中NLRP3的表达.免疫荧光技术检测SAA对人巨噬细胞中核因子(NF) -κB p65入核的影响.结果 ASO下肢血管壁中SAA、NLRP3、IL-1β的表达较正常动脉壁上调倍数分别为10.19 ±0.29、7.11 ±0.14、5.06 ±0.15,与正常动脉比较差异有统计学意义(P＜0.01),各蛋白表达呈现出共区域化;SAA处理巨噬细胞,SAA各剂量组(1、2、5、10 mg/L)较对照组比较,NLRP3上调倍数相应为(2.49±0.34、3.19 ±0.22、3.61 ±0.29、4.46±0.20,P＜0.01),Caspase-1p20上调倍数相应为(1.32 ±0.11、4.59±0.28、17.55±1.40、25.69±1.71,P＜0.01),IL-1β p17上调倍数相应为(1.24±0.09、2.48 ±0.22、15.59±0.96、23.57±0.80,P＜0.01),下调THP-1巨噬细胞中的NLRP3表达后,SAA诱导的IL-1β分泌明显减低.SAA可以上调人巨噬细胞中IL-1β前体蛋白的表达,而且被NF-κB特异性阻滞剂Bay1 1-7082所抑制,上调倍数分别为17.64 ±2.07、6.45±1.78,两组比较差异有统计学意义(P＜0.01).免疫荧光显示SAA促进巨噬细胞中NF-κB p65入核.结论 SAA在ASO血管壁中广泛沉积,并上调了NLRP3和IL-1β的表达.SAA可以激活NLRP3炎性体,引起IL-1β的分泌.SAA可以上调IL-1β前体蛋白的表达,从而起到NLRP3炎性体激活致IL-1β分泌的预启动功能.因此在ASO中SAA可能通过激活NLRP3炎性体而促进动脉硬化炎症的发生、发展.     

邻苯二甲酸二(2-乙基)己酯对小鼠胎鼠睾丸及睾丸引带的影响

目的:研究邻苯二甲酸二(2-乙基)己酯(DEHP)对小鼠胎鼠睾丸与睾丸引带形态结构及功能的影响,探讨隐睾发生的可能机制。方法:30只健康KM孕鼠随机均分为3组:空白对照组、玉米油对照组、DEHP组。DEHP组以剂量500mg/(kg.d)的DEHP灌胃作用于怀孕12～19d(GD12～GD19)的KM母鼠,观察DEHP对每胎胎鼠数、雌雄性胎鼠比例、雄性胎鼠体重、睾丸重量、睾丸引带形态结构、位置、睾丸到膀胱颈之间的相对距离(TBD)、颅侧悬韧带残留情况、引带内雄激素受体(AR)、雌激素受体(ER)、肌动蛋白、增殖细胞核抗原(PCNA)表达水平的影响。结果:DEHP对每胎胎鼠数、雌雄性胎鼠比例、雄性胎鼠体重无明显影响;DEHP可影响胎鼠睾丸重量及TBD;DEHP组睾丸均有一定程度的下降不全,睾丸引带形态结构无明显差异;光镜下见DEHP组睾丸生精小管、支持细胞存在明显的发育障碍,睾丸Leydig细胞明显增生;DEHP组睾丸引带AR阳性表达率降低(P<0.01)。结论:DEHP可通过抗雄激素效应导致睾丸引带功能障碍,使睾丸下降发生异常而诱导小鼠隐睾;同时造成睾丸Ser-toli细胞、睾丸Leydig细胞和生精细胞的发育障碍和功能改变。     

Medical treatment of male infertility

The majority of male infertility is idiopathic. However, there are multiple known causes of male infertility, and some of these causes can be treated medically with high success rates. In cases of idiopathic or genetic causes of male infertility, medical management is typically empirical; in most instances medical therapy represents off-label use that is not specifically approved by the FDA. Understanding the hypothalamic-pituitary-gonadal (HPG) axis and the effect of estrogen excess is critical for the assessment and treatment of male infertility. The use of certain medical treatment has been associated with an increase in sperm production or motility, and primarily focuses on optimizing testosterone (T) production from the Leydig cells, increasing follicle-stimulating hormone (FSH) levels to stimulate Sertoli cells and spermatogenesis, and normalizing the T to estrogen ratio.     

Genetics of CFTR and male infertility

Cystic fibrosis (CF) is a rare autosomal-recessive disorder manifested as multisystem organ dysfunction. The cystic fibrosis transmembrane conductance regulator (CFTR) protein functions as an ion transporter on the epithelium of exocrine glands, regulating secretion viscosity. The CFTR gene, encoded on chromosome 7, is required for the production and trafficking of the intact and functional CFTR protein. Literally thousands of human CFTR allelic mutations have been identified, each with varying impact on protein quality and quantity. As a result, individuals harboring CFTR mutations present with a spectrum of symptoms ranging from CF to normal phenotypes. Those with loss of function but without full CF may present with CFTR-related disorders (CFTR-RDs) including male infertility, sinusitis, pancreatitis, atypical asthma and bronchitis. Studies have demonstrated associations between higher rates of CFTR mutations and oligospermia, epididymal obstruction, congenital bilateral absence of the vas deferens (CBAVD), and idiopathic ejaculatory duct obstruction (EDO). Genetic variants are detected in over three-quarters of men with CBAVD, the reproductive abnormality most classically associated with CFTR aberrations. Likewise, nearly all men with clinical CF will have CBAVD. Current guidelines from multiple groups recommend CFTR screening in all men with clinical CF or CBAVD though a consensus on the minimum number of variants for which to test is lacking. CFTR testing is not recommended as routine screening for men with other categories of infertility. While available CFTR panels include 30 to 96 of the most common variants, complete gene sequencing should be considered if there is a high index of suspicion in a high-risk couple (e.g., partner is CFTR mutation carrier). CF treatments to date have largely targeted end-organ complications. Novel CFTR-modulator treatments aim to directly target CFTR protein dysfunction, effectively circumventing downstream complications, and possibly preventing symptoms like vasal atresia at a young age. Future gene therapies may also hold promise in preventing or reversing genetic changes that lead to CF and CFTR-RD.     

初治获得性免疫缺陷综合征男性患者的糖脂代谢特点

目的总结未接受高效抗反转录病毒治疗（HAART）的初治获得性免疫缺陷综合征（AIDS）男性患者的糖脂代谢特点。 方法比较未行HAART的初治男性AIDS患者（病例组）与同期健康男性（对照组）的体重指数（BMI）、空腹血糖、血脂、血压、尿酸、胰岛素抵抗指数（HOMA-IR）和胰岛β细胞功能（HOMA-β）等糖脂代谢参数的差异。 结果与对照组相比,病例组患者的空腹血糖显著升高（5.54 mmol/Lvs 4.97 mmol/L,t = 4.980、P = 0.000）,BMI和总胆固醇显著降低,且伴有明显的HOMA-β降低（20.46% vs 99.48%,t = 5.011、P = 0.000）,差异均具有统计学意义。Pearson分析显示,HIV病毒载量与HOMA-IR呈负相关（r =-0.474,P = 0.03）,与HOMA-β有一定负相关关系,但差异无统计学意义（r =-0.368,P = 0.077）。 结论初治男性获得性免疫缺陷综合征患者常伴有空腹血糖升高和胰岛β细胞功能明显减退,且与HIV RNA载量有一定相关性,需定期监测血糖水平,警惕糖尿病的发生。     

男性型秃发头发移植术后性激素受体的变化

目的探讨头皮性激素受体在男性型秃发(male pattern baldness，MPB)发生机制中的作用。方法2001年7月至2003年6月，采用荧光组织化学方法，对13例接受过自体头发移植术患者的供、受区头皮及头发对应组织中雌、雄激素受体进行检测，并分别进行对比研究。结果头发移植术后，各实验对象供区与受区相应移植组织中，其性激素受体的含量对比，差异无统计学意义。结论供体毛胚移植到受区(原脱发区)后，其局部组织性激素受体的含量不会发生改变。从另一角度佐证了头皮及头发性激素受体的含量异常，在MPB发生中起重要作用。     

先天性双侧输精管缺如患者睾丸组织中CFTR蛋白的表达及意义

目的通过观察中国CBAVD患者CFTR蛋白在睾丸组织中的表达水平,探讨其与睾丸生精功能之间的关系。方法免疫组化检测66例中国先天性双侧输精管缺如患者睾丸组织CFTR蛋白表达水平并对睾丸生精功能进行评分,探讨先天性双侧输精管缺如患者CFTR蛋白的表达情况以及其与睾丸生精功能的关系。结果免疫组化结果显示CFTR蛋白可表达于睾丸内支持细胞及生精上皮细胞,以细胞膜和细胞浆为主;CFTR蛋白在CBAVD患者睾丸生精上皮及支持细胞的表达以阳性和弱阳性为主,比例分别为50%(33/66)和37.87%(25/66),部分患者可见阴性表达,比例为4.54%(3/66);睾丸生精Johnsen评分7~10分的比例分别为4.45%(3/66)、40.9%(27/66)、42.42%(28/66)和12.23%(8/66)。部分患者存在生精功能障碍。CFTR蛋白在睾丸组织中的表达与生精功能强正相关性,相关系数为0.785(P<0.01)。结论BAVD患者睾丸内的CFTR蛋白表达以阳性和弱阳性为主;部分CBAVD患者存在生精功能障碍,CFTR蛋白在睾丸组织中的表达水平与生精功能存在强正相关性。     

Non-invasive high-risk screening for Fabry disease hemizygotes and heterozygotes

The most appropriate time for screening for Fabry disease (FD) is school age. For this reason, we developed non-invasive methods for measuring urinary alpha-galactosidase A (alpha-gal A) protein, using enzyme-linked immunosorbent assay (ELISA), and for globotriaosylceramide (GL-3), using tandem mass spectrometry (MS/MS). We measured these two biomarkers in the urine of previously diagnosed FD hemizygotes and heterozygotes, and in controls. All the classic FD hemizygotes were clearly distinguished from controls by either method alone, and combining the two assays produced 96% sensitivity for detecting heterozygotes. To assess the utility of these methods for screening school children and adults at high risk of FD, a pilot study was conducted. To distinguish FD from 432 controls, cut-off values for alpha-gal A protein and GL-3 were set at the 5th and 95th centile values of the controls, respectively. Among the high-risk patients, the measurements exceeded the cut-off values for both biomarkers in male and female subjects and were strong indicators for Fabry hemizygotes and heterozygotes. However, we recommend that if the results of the first measurements exceed the cut-off values for only one of these biomarkers, another urine sample should be requested for re-assay to confirm the result.     

Significance of screening for Fabry disease among male dialysis patients

Background Fabry disease is an X-linked disorder resulting from a deficiency of the lysosomal enzyme alpha-galactosidase A(α-Gal A). Renal insufficiency is a very important manifestation and affects the prognosis of patients. Recently, a renal variant type that is characterized by low plasma α-Gal A activity and a milder phenotype, but which progresses to end-stage renal failure, has been reported. In this study, we clarified the incidence of this atypical variant of Fabry disease in hemodialysis patients. Methods We measured plasma α-Gal A activity in 450 male dialysis patients who had never been diagnosed with Fabry disease. Results The mean of the α-Gal A activity of the patients was 9.75 ± 3.20 nmol/h/ml, while the controls with classical Fabry (n = 3) were 0.52–1.04 nmol/h/ml. Among the patients, one patient was found to exhibit low α-Gal A activity in plasma (3.18 nmol/h/ml) and in leukocytes (0.639 nmol/h/mg). This patient was a 43-year-old Japanese man who had been on regular dialysis since the age of 23. He did not present typical clinical signs of classical Fabry, such as acroparesthesias or hypohidrosis, but did present renal insufficiency and severe left ventricular hypertrophy which had developed only recently, suggesting a variant form of Fabry disease. Sequencing of the DNA of this patient revealed a deletion of a single amino acid of valine in 10252. Conclusions A case of an atypical variant of Fabry among 450 male dialysis patients (0.22%) was found in the survey. This indicates the potential for undiagnosed Fabry disease among dialysis patients. The results of this study indicate the significance of screening for Fabry disease among male dialysis patients.     

Anabolic effect of prostaglandin E2 on cortical bone of aged male rats comes mainly from modeling-dependent bone gain

In this study, prostaglandin E₂ (3 mg/kg per day) was administered to 20-month-old male Wistar rats for 10 and 30 days. Histomorphometric analyses were performed on double-fluorescent-labeled undecalcified tibial shaft sections. Thirty days of prostaglandin E₂ (PGE₂) administration increased bone formation rate/total bone surface from undetectable levels to 0.6 μm/day at the periosteal surface and from 0.5 to 2.1 μm/day at the endocortical surface. Endocortical osteoid surface area increased from 2% to 67% at day 10 and decreased to 6% at day 30; woven and lamellar bone formation started at day 0, but was most obvious at day 30, resulting in a 12% increase of total bone mass. The red to yellow marrow ratio was 0.2 in pretreatment controls, and increased to 1.6 by day 10 and 2.4 by day 30 with PGE₂ administration. Intracortical cavity number and area increased after 10 days of PGE₂ treatment, but with forming osteon number and area far exceeding those of resorption cavities at day 30. Endocortical modeling surface/endocortical surface was only 1.5%, and remodeling was 11.1% in pretreatment controls. PGE₂ treatment increased modeling to 24.5% in the 10 day group and 93.7% in the 30 day group, whereas remodeling remained unchanged at 10 days, and decreased to 6.2% at 30 days. Osteoprogenitor cells and osteoblasts could not be detected in pretreatment controls, but increased by day 10, and returned almost to control levels by 30 days. Our data indicate that PGE₂ induced periosteal and endocortical bone formation mainly by modeling-dependent bone gain, accompanied by increases in intracortical remodeling and red bone marrow, and a transient increase in the osteoprogenitor cells adjacent to the endocortical surface. These findings suggest that 20-month-old male Wistar rats were very responsive to the anabolic action of PGE₂ in the tibial shaft, a site consisting mainly of cortical bone and yellow marrow.