细胞因子IL-1β和TNF-α mRNA在人精子表达的变化及意义

目的：了解IL－1β和TNFα在人精子表达的变化及意义。方法应用逆转录聚合酶链式反应（RT－PCR）检测13例正常生育者、30例不育者及30例慢性及30例慢性前列腺炎患者精子IL－1β和TNFα mRNA表达。结果不育精子中IL－1β和TNFα mRNA表达增显著低于正常生育者（P＝0．018和P＝0．046）。慢性前列腺炎患者精子中TNFα mRNA的表达显著低于正常生育者（P＝0．018）。而IL－1βmRNA表达与正常生育者相比差别无显著性意义（P＝0．42）。IL－1β和TNFα mRNA－NA在不育者与慢性前列腺炎患者精子中的表达差别无显著性意义（P＝0．49和P＝0．62）。结论某些因素可能干扰不育症患者精子IL－1βTNFα合成,导致精液质量下降及不育。生殖道炎症也可能影响精子TNFα表达,其机制和意义有待进一步研究证实。     

精浆中细胞因子检测及其意义

目的　研究精浆中的细胞因子的含量及其对男性生育能力的影响。方法　用ELISA法检测了 15例证明有生育能力的健康男性及 2 7例不育男性精浆中的IL 2 .IL 6、IL 8、TNF α、sIL 2R的含量 ,同时检测了精浆中的SOD、MDA及白细胞。结果　本组男性不育症 (非感染组 )患者精浆中的IL 6、IL 8、sIL 2R水平与有生育能力的男性相比有差异 ,感染组精浆中的IL 2 .IL 6、IL 8、TNF α、sIL 2R的含量与生育组比有明显的差异 (P <0 .0 1)。结论　本研究提示生殖道感染时精浆中的细胞因子与男性不育有密切的相关     

反复自然流产病人细胞因子水平测定

目的 :探讨不明原因反复自然流产 (RSA)病人PBMC产生IFN γ、TNF α、IL 4和血清sIL 2R水平的变化及意义。　方法 :应用酶联免疫吸附法 (ELISA)测定了 36例RSA病人、15例正常妊娠、13例经产妇以及 8例配偶白细胞免疫治疗者外周血中单个核细胞经PHA刺激后培养上清中 ,IFN γ、TNF α和IL 4以及血清中sIL 2R水平。　结果 :RSA病人PBMC产生的IFN γ为 (0 .5 9± 0 .2 6 )ng/ml,TNF α为 (1.11± 0 .5 6 )ng/ml,较对照者显著增高 (P <0 .0 5 )。经配偶白细胞免疫治疗者 (成功妊娠 )的IFN γ、TNF α水平明显低于治疗前。RSA病人妊娠后再次流产血清sIL 2R显著增高。　结论 :RSA病人某些细胞因子产生异常是妊娠的不利因素 ,sIL 2R增高与先兆流产有关。     

原发性肝癌患者sIL-2R,IL-6和TNF-α测定及其临床意义

笔者采用双抗体夹心ELISA法测定 3 3例原发性肝癌 (PHC )患者血清sIL 2R ,IL 6和TNF α水平 ,并对术前、术后 2周的测定值进行对比。结果示PHC患者手术前sIL 2R ,IL 6和TNF α明显高于正常人 (P <0 .0 5 ) ;手术后 2周 ,上述三指标明显下降 (P <0 .0 5 )。AFP水平与sIL 2R ,IL 6和TNF α水平无关 ( r=0 .3 82 6,0 .5 3 0 1,0 .3 72 5 ,均 P >0 .0 5 ) ;病理类型与sIL 2R ,IL 6和TNF α水平无关 (P >0 .0 5 )。提示sIL 2R ,IL 6和TNF α可作为原发性肝癌诊断、疗效及预后评价的辅助性指标。     

重组人促红细胞生成素对大鼠脊髓损伤后肿瘤坏死因子-α表达的影响

目的 :探讨重组人促红细胞生成素 (rHuEPO)对大鼠脊髓损伤后肿瘤坏死因子 α (TNF α)表达的影响。方法 :SD大鼠 10 2只 ,随机分为 4组 :假手术组 ;脊髓损伤组 ;脊髓损伤 生理盐水 (NS)治疗组 ;脊髓损伤 rHuEPO治疗组。采用改良Allen’s脊髓损伤打击模型 ,以逆转录 聚合酶链反应 (RT PCR)法测定伤段脊髓组织TNF αmRNA的表达情况。结果 :TNF αmRNA在无损伤脊髓中即见有表达 ,脊髓损伤后 1h表达明显上调并达高峰 ;高表达持续至损伤后 2 4h ;rHuEPO治疗组脊髓伤后 6、 12、 2 4hTNF αmRNA表达明显低于NS治疗组。结论 :rHuEPO能明显抑制大鼠脊髓损伤后TNF αmRNA的表达。     

NFκB抑制剂对大鼠肝脏移植再灌注损伤的保护作用

目的　探讨大鼠肝脏移植后核因子κB(NFκB)活化对炎性介质表达和中性粒细胞浸润集聚的影响。方法　供受体鼠分别于术前 15min腹腔注射NFκB抑制剂脯氨酸二硫代氨基甲酸酯 (ProDTC 15mg/kg)。 结果　与对照组相比 ,应用ProDTC者移植后NFκBp6 5含量、肿瘤坏死因子 α(TNF α)、巨噬细胞炎性蛋白 2 (MIP 2 )、细胞间粘附分子 1(ICAM 1)mRNA表达和蛋白表达明显下降 ;髓过氧化物酶 (MPO)活性明显减低 (P均 <0 0 1)。ProDTC也显著降低天门冬氨酸转氨酶(AST)、丙氨酸转氨酶 (ALT)、乳酸脱氢酶 (LDH)水平 (P <0 0 1)。结论　ProDTC抑制移植后NFκB活化从而下调了TNF α、MIP 2、ICAM 1表达从而减轻中性粒细胞浸润及肝脏缺血再灌注损伤。     

全身炎症反应综合征患者外周血单个核细胞Toll样受体2、4基因表达及其意义

目的探讨急腹症全身炎症反应综合征(SIRS)患者外周血单个核细胞(PBMC)内Toll样受体(TLR)2、4基因表达变化及其意义。方法103例急腹症手术患者包括SIRS组65例,非SIRS组38例。采用逆转录聚合酶链反应(RT PCR)法检测TLR2、4mRNA的变化;酶联免疫吸附试验(ELISA)法检测TNF α和IL 6的表达;分析TLR2、4mRNA与TNF α和IL 6的表达量、住院时间和多器官功能障碍综合征(MODS)发生率之间的关系。结果入院第1天TLR4mRNA、TNF α和IL 6表达均升高至峰值,随后随病程下降,TLR2mRNA自入院起持续高表达至第5天,TLR2、4mRNA的表达与TNF α和IL 6、住院时间呈正相关,且TLR2、4mRNA表达越高,发生MODS的危险性越大。结论急腹症SIRS患者PBMC内TLR2、4基因表达显著增加,提示TLR在急腹症SIRS的发生发展过程中发挥了重要的促进作用。     

碳酸酐酶Ⅱ在人睾丸、精子中的表达及其临床意义

目的:观察碳酸酐酶Ⅱ(CA2)在人睾丸、精子中的表达定位及比较CA2在生育男性和弱精子症患者精子中的表达差异。方法:通过免疫组化观察CA2在人睾丸中的定位;通过免疫荧光观察CA2在人精子中的定位;收集健康生育男性和弱精子症患者的精液标本各16份,60%Percoll分离精液标本,排除生精细胞和白细胞,采用Western印迹方法,从蛋白水平检测CA2的表达。结果:免疫组化结果显示CA2在人睾丸中特异性定位于长形精子的尾部;免疫荧光结果显示CA2在人精子的尾部有较强表达。Western印迹结果显示弱精子症患者精子中CA2的表达显著高于生育男性(1.84±0.32vs1.41±0.26,P<0.05)。结论:CA2在睾丸生精周期的长形精子阶段才表达,定位于精子的尾部。CA2在弱精子症患者精子中表达显著增高,提示其可能与精子活动力下降有关。     

健康男性和弱精子症患者精液精子中GP130的表达

目的观察在正常和活动力低下的人精液精子中白细胞介素-6(IL-6)及其受体(IL-6R)和GP130的表达差异。方法收集活动力正常的人精液精子和A、B级精子低于20％的弱精子症患者的精液精子,采用逆转录-聚合酶链反应 (RT-PCR)和免疫细胞化学的方法检测IL-6及其受体和GP130的表达水平。结果与活动力正常的精液精子比较, GP130在活动力低下患者精液精子的表达显著降低(P<0．01),本实验未检测到IL-6和IL-6R在人精液精子的表达。结论精液精子GP130的表达减少与人精子活动力低下相关,提示GP130表达减少可能是精子活动力低下的原因之一。     

参附对再灌注期间肠粘膜上皮细胞凋亡的抑制作用及机制探讨

目的　探讨大鼠缺血 再灌注期间粘膜上皮细胞凋亡及参附注射液对其影响的可能机制。方法　采用大鼠肠缺血 再灌注模型 ,2 4只大鼠随机分为对照组 (C组 )、缺血 再灌注组 (I R组 )和参附治疗组 (SF组 )。在规定时间检测回肠粘膜上皮细胞Caspase 3、Bcl 2蛋白的表达、凋亡细胞数目及回肠粘膜组织肿瘤坏死因子 (TNF α)水平 ,同时观察病理形态学改变。结果　 (1)I R组凋亡细胞显著增多 ,SF组凋亡细胞数较I R组显著减少而多于C组 (P <0 0 5 )。 (2 )Caspase 3在I R组的表达显著高于SF组和C组 (P <0 0 1) ,SF组与C组差异无显著性。Caspase 3表达与凋亡细胞数目呈正相关 (r =0 914 9,P <0 0 1) ;Bcl 2的表达与Caspase 3的表达呈直线负相关 (r =- 0 9384 ,P <0 0 1)。 (3)TNF α表达 :TNF α的表达在I R组显著高于C组和SF组 (均P <0 0 1) ,SF组与C组差异无显著性。TNF α表达与凋亡细胞数目呈正相关 (r =0 9133,P <0 0 1)。 (4)SF组小肠粘膜病理损伤程度明显小于I R组 (P <0 0 1)。结论　参附注射液通过抑制TNF α、Caspase 3表达同时上调Bcl 2而抑制缺血 再灌注期间肠粘膜上皮细胞的凋亡 ,从而减轻肠粘膜缺血 再灌注损伤。     

Sperm nuclear histone to protamine ratio in fertile and infertile men: evidence of heterogeneous subpopulations of spermatozoa in the ejaculate

Sperm protamine deficiency is observed in a subset of infertile men, suggesting that the relative histone to protamine ratio may be altered in the spermatozoa of these men. We measured the ratio of nuclear histones to protamines in the spermatozoa of fertile (n = 10) and infertile men (n = 20). Sperm nuclear proteins were extracted and subsequently separated by acid-urea (AU) polyacrylamide gel electrophoresis. The relative histone (H2B) to protamine (PRM1 + PRM2) and PRM1 to PRM2 ratios were estimated by densitometric analysis of the AU gels. Immunoblotting experiments (using H2B, PRM1, and PRM2 antibodies) were conducted to confirm the specificity of the bands. The pattern and intensity of H2B staining in human spermatozoa was assessed by immunocytochemistry. Sperm samples from the infertile men in this study had a significantly higher proportion of histone H2B to protamine (PRM1 + PRM2) than did samples from the fertile men in this study (0.38 vs 0.08, P < .001). Immunocytochemistry experiments demonstrated a punctuated staining pattern (with strong, intermediate, or weak H2B staining intensity) throughout the sperm head. Infertile men had a higher proportion of spermatozoa exhibiting strong and intermediate staining than did samples from fertile men. These findings suggest that infertile men possess a higher proportion of spermatozoa with an increased histone to protamine ratio than fertile controls.     

Alterations in sperm protein phosphorylation in male infertility

Protein phosphorylation is involved in sperm capacitation, so the effect of protein phosphatase inhibitors on the capacitation of spermatozoa of males with unexplained infertility was investigated. d-mannose ligand specific receptor expression in fresh, living spermatozoa, capacitated or treated with calyculin A (an inhibitor of protein phosphatases 1 and 2A), was studied in three groups of men: pre-vasectomy (fertile) males, males in couples with male infertility, and males in couples with infertility of unknown aetiology. Flow cytometry showed significant differences between infertile couples with a male factor and fertile couples (P < 0.05), both after capacitation and after treatment with calyculin A. In the group of couples with infertility of unknown aetiology (n = 15), d-mannose receptor expression was diminished in six cases after classical capacitation. However, when the spermatozoa of these six men were treated with calyculin A, five showed an increased specific d-mannose receptor expression. From these results it is suggested that in vitro treatment of spermatozoa with inhibitors of protein phosphatases may be of great value in some cases of unexplained infertility.     

Assessment of hyperactivation, acrosome reaction and motility characteristics of spermatozoa from semen of men of proven fertility and unexplained infertility

Summary. Semen from men of proven fertility was compared with that of men with unexplained infertility to determine differences in spermatozoal functions such as hyperactivation and acrosome reaction and spermatozoal motility characteristics. The hyperactivated spermatozoa in both groups could be visualised on the monitor of the Computer Assisted Semen Analyser and they exhibited 'circling', 'thrashing', 'starspin' and 'helical' motility patterns and the mean hyperactivation rates were not significantly different. However, 20% of the men with unexplained infertility did not exhibit hyperactivation compared to only 4% in the fertile group. Furthermore, the semen from infertile men when evaluated for hyperactivation could be categorised into two groups with those having lower hyperactivation (<10% or <6% after 4 and 6 h of incubation respectively), forming the first group, and those having a higher hyperactivation rate constituting the second group. In the fertile men such distinct groups were not visible and the percentage hyperactivation ranged from 1 to 16%. No significant differences were observed in the rate of acrosome reaction of fertile and unexplained infertile men.
The non-hyperactivated spermatozoa from unexplained infertile men showed a significant increase in path velocity (VAP), curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) and a decrease in linearity (LIN) and straightness (STR) compared to spermatozoa from fertile men. Furthermore, the hyperactivated spermatozoa from infertile men also showed an increase in progressive velocity (VSL) (only after 2 h of incubation) and LIN and decrease in ALH and beat cross frequency (BCF) compared to spermatozoa from fertile men. The results are discussed in the light of the importance of the above spermatozoal functions and spermatozoal parameters in fertilization.     

Effect of varicocele on chromatin condensation and DNA integrity of ejaculated spermatozoa using cytochemical tests

Varicocele occurs in approximately 15% to 20% of the general male population and it is the most common cause of poor semen production and decreased semen quality. It has been demonstrated that patients with varicocele have a significantly higher DNA fragmentation index (DFI) and spermatozoa with nuclear anomalies than healthy fertile men. Therefore, the aim of this study was to evaluate sperm chromatin integrity in these patients. Sixty men referring to the andrology laboratory were categorised into three different groups: 20 infertile men with varicocele, 20 infertile men with abnormal semen parameters and 20 fertile men who had normal spermatogram were considered as control group. Semen analysis was performed according to WHO criteria. To evaluate sperm chromatin quality and DNA integrity, after fixation of sperm smears, aniline blue, toluidine blue, chromomycin A₃ and acridine orange staining were applied in three groups. The slides were analysed by light and fluorescent microscopy and to determine the percentage of mature or immature spermatozoa, 200 spermatozoa were counted in each slide. The results showed that the rates of aniline blue-reacted spermatozoa were significantly higher in infertile and varicocele patients than in the normal group ( P  < 0.001). In addition, with regard to chromomycin A₃, acridine orange and toluidine blue staining, there was a significant difference between the three groups ( P  < 0.001). The results showed that the varicocele samples contain a higher proportion of spermatozoa with abnormal DNA and immature chromatin than those from fertile men as well as infertile men without varicocele. Therefore, varicocele results in the production of spermatozoa with less condensed chromatin and this is one of the possible causes of infertility due to varicocele.     

正常男性精子碱性核蛋白的研究

目的　观察正常人精子碱性核蛋白的组成及含量。　方法　8名正常人精子经解聚后，用聚丙烯酰胺凝胶电泳方法，于1、4、8、12、24、36、44周检测精子碱性核蛋白。　结果　正常人总组蛋白/总精核蛋白值为0.17±0.07，P1型/P2型精核蛋白值为1.05±0.21。　结论　正常男性精子碱性核蛋白指标可供临床评价精子受孕能力。     

Apoptosis and kinematics of ejaculated spermatozoa in patients with varicocele

Increased DNA fragmentation is found in sperm from infertile men. Varicocele is an important cause of male infertility, even though it is present in 15% of men who father children. Semen analysis does not always identify infertility in these patients. Sperm motility is strongly correlated with male fertility potential. The goal of this study was to determine the correlation between apoptosis and kinematics in the ejaculated spermatozoa of patients affected by varicocele. Fresh semen samples were obtained from 30 patients with varicocele and 15 fertile controls. These samples were compared using computer-assisted semen analysis and were assayed to determine the degree of sperm apoptosis. The apoptotic index (AI) was calculated by dividing the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine-5'-triphosphate nick end labeling (TUNEL) stained spermatozoa by the total number of Hoechst 33258-stained sperm cells for 300 sperm. Five microscopic fields were analyzed to obtain 5 AIs for each individual. Results demonstrated no significant difference in semen quality and sperm motion characteristics; however, a significantly higher AI (23.05% +/- 4.07%: mean difference +/- SE, 95% CI, 15.06%-31.03%, P <.0001) was identified in the varicocele group than in the fertile controls. We concluded that sperm apoptosis does not seem to correlate with semen quality and sperm kinematics and that apoptosis is increased in ejaculated spermatozoa in patients with varicocele compared to normal fertile men.     

Localisation and quantification of alkali‐labile sites in human spermatozoa by DNA breakage detection–fluorescence in situ hybridisation

The localisation and quantification of constitutive alkali‐labile sites (ALSs) were investigated using a protocol of DNA breakage detection plus fluorescence in situ hybridisation (DBD–FISH) and alkaline single‐cell gel electrophoresis (SCGE or comet assay), in spermatozoa of infertile and fertile men. Semen samples from 10 normozoospermic patients undergoing infertility treatment and 10 fertile men were included in this study. ALSs were localised and quantified by DBD–FISH. The region most sensitive to alkali treatment in human spermatozoa was located in the basal region of the head. ALSs were more frequent in spermatozoa of infertile men than in those of fertile men. These results were confirmed by SCGE comet assays. In conclusion, the most intense localisation of hybridisation signals in human spermatozoa, representing the highest density of constitutive ALSs, was not randomly distributed and was predominantly located in the base of the head. Moreover, infertile men presented with an increase in ALS frequency. Further studies are necessary to determine the association between ALS, sperm chromatin organisation and infertility.     

Assessment of MUSASHI 1 and MUSASHI 2 expression in spermatozoa and testicular tissue

MUSASHI (MSI) family plays the main role in the spermatogenesis process. The purpose of this study was the assessment of sperm MSI1 and MSI2, and sperm functional tests in infertile men (n = 30) with varicocele and fertile men (n = 30). Furthermore, MSI1 and MSI2 proteins were assessed in testicular tissue of azoospermic men (n = 9) as well as epididymal spermatozoa and testis of mice. Expression of MSI1 and MSI2 was assessed at RNA and protein levels in human spermatozoa. Sperm concentration and motility were significantly lower, while abnormal sperm morphology, lipid peroxidation, DNA fragmentation and protamine deficiency were significantly higher in men with varicocele compared to fertile individuals. Any significant difference was not observed in the expression of MSI1 and MSI2 mRNA between the two groups. Unlike MSI1 protein that was not detectable in humans, the relative expression of MSI2 protein was similar in varicocele and fertile individuals. The expression level of both Msi1 and Msi2 proteins was also observable in mouse spermatozoa. No significant relationship was observed between sperm functional parameters with expression of these genes. The data of this study demonstrated that although MSI1 and MSI2 play important roles during spermatogenesis, their relative expression in spermatozoa was not affected by varicocele.     

Reassessment of the accuracy of traditional sperm characteristics and adenosine triphosphate (ATP) in estimating the fertilizing potential of human semen in vivo

Receiver operating characteristic curves and accuracy parameters were computed for traditional sperm characteristics (concentration, motility, morphology) and the number of peroxidase negative cells, and the concentration of adenosine triphosphate (ATP) in semen from populations of fertile and infertile men, and men who achieved a pregnancy after varicocele treatment. The percentage and concentration per millilitre of spermatozoa with rapid linear progressive motility, and the ATP concentration, provided the best discrimination between fertile and treated fertile from infertile men. The misclassification rate was higher for sperm morphology, total progressive motility and viability, whereas sperm concentration and the total sperm count per ejaculate had the worst discriminating power. The number of peroxidase negative cells per 100 spermatozoa was highly specific in identifying men who achieved pregnancy after varicocele treatment. The lower limit of normality of sperm characteristics was remarkably different between fertile men and men achieving pregnancy after treatment or during infertility work-up.