小鼠精原细胞的分离和纯化

目的　探讨小鼠精原细胞的分离纯化。　方法　用组合酶消化法制备 7～ 8d小鼠的生殖细胞悬液 ;用Percoll不连续密度梯度法分离精原细胞。　结果　所获细胞悬液内活细胞、死细胞及细胞团的百分比分别为90 .0 8%、9.92 %及 8.91% ;平均每个睾丸可获得 4.136× 10 5 个细胞 ;精原细胞主要分布于位于 2 7%～ 35 %间的Percoll梯度中 ,其超微结构与 7～ 8d小鼠睾丸切片内精原细胞的超微结构一致 ,经纯化后其纯度达 6 8.76 %。　结论　用组合酶消化、Percoll不连续密度梯度法分离的 7～ 8d小鼠的精原细胞能满足体外培养的需要     

人血白蛋白对近端肾小管上皮细胞ACE2表达的影响

目的： 研究人血白蛋白对人近端肾小管上皮细胞（HK-2）血管紧张素转换酶2（ACE2）及血管紧张素转换酶（ACE）表达的影响，探讨白蛋白对肾脏损害的机制。方法: 不同浓度人血白蛋白（0、1、5、10、15 g/L）分别加入到培养的人近端肾小管上皮细胞培养48 h，用RT-PCR法和Western blotting法分别检测HK-2细胞ACE2和ACE mRNA和蛋白表达。结果: 正常培养状态下HK-2细胞（空白对照组）存在ACE2 mRNA和蛋白表达，加入不同浓度（5-15 g/L）的人血白蛋白剂量依赖抑制HK-2细胞ACE2 mRNA和蛋白表达（P<0.01）。正常培养状态下HK-2细胞ACE mRNA和蛋白表达水平较低，随着加入白蛋白浓度的增加其表达水平逐渐升高，10 g/L白蛋白显著促进了HK-2细胞ACE mRNA和蛋白表达（P<0.05）。结论: 在体外培养的HK-2细胞存在ACE及ACE2 mRNA和蛋白表达，人血白蛋白可抑制其ACE2表达而促进ACE表达,这可能是白蛋白促肾小管间质损伤及肾小球硬化、间质纤维化的机制之一。     

巴戟天对环磷酰胺诱导的大鼠睾丸生精功能的影响

目的:探讨巴戟天在环磷酰胺诱导的大鼠生精障碍动物模型中的作用,研究巴戟天对大鼠生精功能的影响。方法:检测大鼠的精子活力、精子密度、睾丸指数、附睾指数及组织结构变化;酶联免疫吸附法检测血清睾酮、卵泡刺激素(FSH)和黄体生成素(LH)含量。结果:与空白对照组相比,模型组睾丸指数、附睾指数、精子活力以及精子密度降低;治疗组相对模型组睾丸指数、附睾指数、精子活力、精子活动率均显著增高;H-E染色显示模型组生精小管直径缩小,间距增宽,生精上皮变薄,生殖细胞数量显著减少;治疗组与模型组比较生精小管壁增厚,含有精子的生精小管显著增多;模型组与空白对照组比较血清睾酮显著降低;治疗组与模型组比较其血清睾酮显著增加。结论:巴戟天对环磷酰胺诱导的生精障碍睾丸具有改善睾丸生精小管结构,促进精子发生和间质细胞分泌睾酮的功能。     

透明质酸酶提高子宫内膜容受性增加IVF-ET妊娠率的临床应用研究

目的：探讨透明质酸酶对子宫内膜容受性的影响。方法将248例自然周期下移植冻胚的患者随机分为两组,实验组和观察组,所有患者在月经周期第3d开始口服戊酸雌二醇3mg/d连续服用10d,实验组患者月经干净后第3,5,7d分别通过阴道将透明质酸酶3ml注入宫腔(80u/ml)对照组不用药,观察两组患者子宫内膜厚度差异；结果实验组在和对照组治疗后子宫内膜厚度差异具有显著统计学意义(P<0.05)两组患者临床妊娠比较差异具有显著统计学意义(P<0.05)；结论透明质酸酶通过有效的提高子宫内膜厚度,改善子宫的内膜容受性,显著提高IVF-ET的临场妊娠率     

附睾精子和睾丸精子妊娠结局比较

目的比较经皮附睾穿刺抽吸术和经皮睾丸精子抽吸术两种方法获得的精子妊娠结局。方法83例无精子症患者经皮附睾穿刺抽吸术取得附睾精子；35例无精子症患者经皮睾丸精子抽吸术（TESA）获得睾丸精子。女方进行常规超排卵。采用卵胞浆内单精子注射技术获得妊娠，比较两者的受精率、种植率和临床妊娠率。结果附睾精子组和睾丸精子组的受精率分别为75．20％和74．61％，比较其差异无显著性（P〉0．05）；两者的种植率和临床妊娠率分别为29．18％VS23．89％和52．43％VS40．21％，差异具有显著性（P〈0．05）。结论附睾是精子获能、成熟的重要部位，附睾精子优于睾丸精子，对无精子症患者行ICSI之前尽可能首先选取附睾精子。     

超声波对小白鼠生精上皮的影响

用功率为５Ｗ／ｃｍ２、频率为１．１０ＭＨｚ的超声波照射小白鼠睾丸５分钟（实验组１）、１０分钟（实验组Ⅱ），分别于处理后２４小时，４８小时及７天时间切取睾丸组织，制作石蜡切片，在光镜下观察生精上皮的组织学变化并与对照组进行比较。结果显示：（１）小白鼠睾丸经超声波照射后，曲细精管萎缩，管径变小；生精上皮变薄，精子发生时相消失；生精细胞减少，没有精子形成。（２）超声波照射１０分钟对生精上皮的损伤比照射５分钟更为严重。（３）超声波照射后２４小时，生精上皮即受到破坏，精子细胞减少；照射后４８小时，上皮受损伤的程度增大；照射后７天时间，曲细精管的组织学结构开始恢复，但仍无精子形成。（４）超声波对生精上皮的影响主要限于精母细胞、精子细胞和精子，而精原细胞与支持细胞没有明显变化。上述结果表明，超声波能够抑制小白鼠的精子发生，该抑制作用可能可逆。     

少、弱精子症的精子优选技术

目的精子优选技术用于临床治疗少、弱精子症患者。方法对3种优选技术进行优选前后比较。Ⅰ组:上游法;Ⅱ组;Percoll密度梯度离心法;Ⅲ组:血清白蛋白过滤法。结果3种优选技术对精子活动率、A级精子百分率、精子穿卵率都有非常显著的增高(P<0.01),而精子畸形率有显著下降(P<0.01)。临床上50例少、弱精子症患者采用3-4次冻贮后进行优化处理,精子密度、活动率均有提高。结论精子优选技术可以提高少、弱精子症患者的精子质量,达到受孕的目的。     

磁场穴位刺激对家兔生殖力影响的实验研究

目的:探讨磁场穴位刺激对家兔生殖力的影响.方法:选用3月龄普通级新西兰种雄性大白兔27只,随机分为3组.将对照组与实验组拟人穴位内关(双侧)、足三里(双侧)及关元穴位,分别填埋未充磁及充磁磁片,经3个月实验观测.测量一次射精的精液量、精子数量、精子存活率及畸形率.实验结束后,测量体重,取出睾丸及附睾尾分别称重.结果:实验组精液质量、附睾尾中精子数、曲细精管直径及精细胞数量均高于对照组;而精子畸形率比对照组减少.结论:磁场作用穴位可提高家兔的生精功能.     

透明质酸影响人膝骨关节炎滑膜成纤维样细胞骨桥蛋白和CD44的表达

背景：在骨关节炎病程中,透明质酸水平的改变可以使一系列细胞因子和酶表达水平改变,但是滑膜组织中骨桥蛋白、CD44的高表达是否与透明质酸水平改变相关目前尚不清楚。 目的：通过透明质酸干预体外培养的人膝骨关节炎滑膜成纤维样细胞,观察透明质酸对骨关节炎滑膜成纤维样细胞骨桥蛋白和CD44表达的影响。 方法：选取接受膝关节镜手术和膝关节置换的原发性膝关节骨关节炎患者滑膜标本10例,进行体外培养获取纯化的滑膜成纤维细胞,取第4~6代滑膜成纤维细胞,分为3组：空白对照组不进行任何干预;透明质酸组用100 mg/L透明质酸干预24 h;透明质酸酶组用300 mg/L透明质酸酶干预24 h。 结果与结论：与空白对照组相比,透明质酸组滑膜成纤维细胞骨桥蛋白mRNA表达水平上升(P < 0.05),透明质酸酶组滑膜成纤维细胞骨桥蛋白mRNA表达水平下降(P < 0.05)。透明质酸组和透明质酸酶组之间膜成纤维细胞骨桥蛋白mRNA表达水平差异有显著性意义(P < 0.05)。空白对照组、透明质酸组和透明质酸酶组滑膜成纤维细胞CD44 mRNA表达水平差异均无显著性意义(P > 0.05)。提示透明质酸可以使骨关节炎滑膜成纤维细胞骨桥蛋白mRNA表达增高,对CD44 mRNA表达没有影响。     

雷公藤多甙对睾丸附睾组织化学的影响

雄性SD大鼠20只,随机分成对照组和实验鼠各10鼠,后者灌服GTW 10mg/kg/日,8周至完全不育后,取睾丸附睾组织进行了七种组化染色,即DNA、RNA、LDH-X、ATP酶、琥珀酸脱氢酶(SDH)、非特异性脂酶和PAS反应,同时取附睾尾精子做常规分析。结果表明,服药组中受损睾丸生精细胞RNA减少,精子细胞内RNA分布不均,多聚集于胞膜上;而正常生精小管与对照组无明显不同。实验组附睾管腔中精子呈鲜绿色;睾丸附睾的SDH和精子的LDH-X活力下降;附睾尾精子计数和活动精子百分率明显低于对照(p<0.21),面畸变率显著高于对照组(p<0.01)。PAS反应、DNA含量和非特异性酯酶二组间无明显差异。结果提示所用剂量GTW主要作用于附睾精子,也影响变态期精子细胞。其原因可能是GTW干扰糖酵解和有氧氧化过程。GTW影响精子LDH-X酶活性可能是其抗生育作用的环节之一。     

Andrology: Sperm hyaluronidase activation by purified predominant and major basic human seminal coagulum proteins

This study demonstrated that semenogelin I and II, the predominantand the major basic human seminal coagulum proteins respectivelywere shown to be potent activators of sperm hyaluronidase. Theseproteins stimulated the activity of bovine testicular hyaluronidase,goat cauda sperm hyaluronidase and human ejaculated sperm hyaluronidase.Human seminal plasma protein, which predominantly contains thebasic degradation residues of the basic coagulum proteins andalbumin (pI 4.7) and which is also basic at the assay pH 3.8,also showed activation of bovine testicular hyaluronidase whileacidic pepsin (pI < 1.0) exhibited no such activation. Thefindings may be utilized as an assay method for comparativeevaluation of specific activation units of the purified basicseminal coagulum proteins or their cleavage products and forquantifying the total basic protein (pI > 3.8) units in humanseminal plasma. One unit of the coagulum protein was definedas the amount of the activator that increases 1 mIU of hyaluronidaseactivity by 50%. The specific activity of the 57 kDa and 75–79kDa coagulum proteins, and that of serum albumin against Sigmabovine testicular hyaluronidase (type IV), were 11 447, 8600and 6252 units per mg protein respectively. It was speculatedthat human seminal coagulum proteins may have an activationeffect on spermatozoal hyaluronidase activity in vivo.     

Comparison of four mechanical methods to retrieve spermatozoa from testicular tissue

To maximize the total number of spermatozoa which can be retrievedfrom a testicular biopsy, four mechanical methods for preparationwere compared. In all, 17 biopsies were divided into four equalfractions, which were weighed individually and prepared by roughshredding (control), fine mincing, vortexing and crushing (electricalPotter). After resuspension in an erythrocyte-lysing buffer,removal of the remaining tissue pieces, washing and centrifugation,the following parameters were evaluated: number of spermatozoaper 100 mg tissue, percentage motility, percentage vitalityand percentage normal morphology (strict Kruger criteria). Afterfurther purification by discontinuous Percoll centrifugation,the same parameters were assessed again. This procedure wasefficient in obtaining only spermatozoa in the final fraction.Before Percoll centrifugation, only minor differences betweenmethods were observed. Percoll centrifugation generally resultedin a pronounced loss of sperm cells, partly because of the abnormaldimensions of the eliminated cells. After Percoll centrifugation,treatment of the testicular tissue by fine mincing was the mosteffective method in terms of the total number of motile spermatozoaand percentage normal morphology.     

Acrosome intactness and seminal hyaluronidase activity: relationship with conventional seminal parameters

Seminal hyaluronidase activity was estimated after liquefaction in semen samples of 100 male partners of infertile couples including 16 azoospermic (no spermatozoon) men and 48 fertility proven men by a method based on measurement of the area of digestion of substrate (hyaluronic acid) in agar plate. Semen samples were also evaluated for Acrosomal Intactness (AI) test except the azoospermics of the studied samples. Seminal hyaluronidase activity was completely absent in azoospermic specimens confirming its cellular origin. Seminal hyaluronidase activity was found to be significantly correlated, statistically, with sperm density (r = 0.708, p < 0.001), % motility (r = 0.6478, p < 0.001) and % normal sperm morphology (r = 0.5724, p < 0.001). Acrosomal Intactness (AI) test scores were also well correlated with sperm density (r = 0.6477, p < 0.001), % motility (r = 0.5965, p < 0.001) and % normal morphology (r = 0.6237, p < 0.001). Both values were higher in semen samples with normal routine parameters (proven fertility and normozoospermic infertile groups) than those compared with abnormal routine parameters (oligozoospermic). We also found very highly significant correlation (r = 0.8442) between seminal hyaluronidase activity and Acrosomal Intactness scores, statistically (p < 0.001). This could be because; normal germinal semineferous epithelium generates abundant number of sperms with normal motility and morphology that are also having intact acrosome. Intact acrosome prevents loss of acrosomal enzymatic activity (e.g. hyaluronidase) until released after liquefaction during seminal analysis and during acrosomal reaction in female genital tract prior to fertilization. Seminal hyaluronidase activity, thus determined, is primarily dependent upon the intact status of acrosome. As each sperm contributes to the seminal hyaluronidase activity, it is directly correlated with sperm density; but at the same time it exhibits goods correlation with % motility and % normal morphology. Therefore AI score and seminal hyaluronidase activity can be considered as good indicators of sperm function.     

四种常用的人中性粒细胞分离方法的比较

目的：比较Percoll非连续密度梯度离心法、Ficoll-Hypaque 密度梯度离心法、裂解红细胞法和Dextran作用下红细胞自然沉降法四种常用的人中性粒细胞分离方法。方法：取健康人外周静脉血，分别采用以上四种方法进行中性粒细胞分离，对其细胞纯度、回收率、存活率进行比较。结果：Percoll非连续密度梯度离心法与Ficoll-Hypaque密度梯度离心法分离得到的细胞纯度均大于90%，两者间比较无统计学差异（P＞0.05）；裂解红细胞法和Dextran作用下红细胞自然沉降法分离得到的细胞纯度略低于Percoll非连续密度梯度离心法(P＜0.01)与Ficoll-Hypaque密度梯度离心法（P＜0.05）。Dextran作用下红细胞自然沉降法的回收率低于Percoll非连续密度梯度离心法（P＜0.01）、Ficoll-Hypaque密度梯度离心法(P＜0.01)和裂解红细胞法（P＜0.05）；Percoll非连续密度梯度离心法回收的中性粒细胞存活率明显高于Ficoll-Hypaque密度梯度离心法（P＜0.05），裂解红细胞法(P＜0.01）和Dextran作用下红细胞自然沉降法（P＜0.01）。结论：Percoll非连续密度梯度离心法分离中性粒细胞，纯化程度好，回收率高，是一种简单、高效的中性粒细胞分离方法，适于临床和科研中广泛应用。     

The role of hyaluronic acid as a mediator and regulator of cervical ripening

During pregnancy, hyaluronic acid (HA) concentration in the human cervix is very low, but increases rapidly at the onset of labour. HA has a high affinity for water molecules and hence can maintain tissue hydration. HA can stimulate collagenase production in rabbit cervix, and also stimulates migration and function of polymorphonuclear leukocytes in the tissues. It is an endogenous regulator of interleukin- 1 (IL-1). We hypothesized that HA plays an essential role during cervical ripening. The effect of exogenous application of HA (20 mg) on non-pregnant and pregnant (day 23) rabbit cervices was compared with controls. HA induced cervical ripening in both pregnant and non- pregnant animals, and cervical water content was significantly increased. Tissue collagen was markedly decreased. The localization and distribution of HA and HA receptor CD44 was determined in non-pregnant and pregnant human cervical connective tissue using biotinylated HA binding protein and CD44 monoclonal antibodies. Both were widely distributed in the connective tissues, especially around the blood vessels and cervical glands. The effect of IL-8 (50, 100, 150 and 200 ng/ml) on HA production and hyaluronidase (HAase) activity was investigated in cultures of lower uterine segment collected during elective Caesarean sections. HA production was stimulated in a dose- dependent manner; there was no effect on hyaluronidase activity. HA administration (0.5, 1 and 2 mg/ml) stimulated the activities of collagenase and gelatinase together with IL-8 production in the culture supernatants. Thus HA may play an important role in cervical ripening, being involved in the regulation of cervical tissue water content, collagenolytic enzymes and cytokines.      

Pregnancies achieved with testicular and ejaculated spermatozoa in combination with intracytoplasmic sperm injection in men with totally or initially immotile spermatozoa in the ejaculate

The efficacy of intracytoplasmic sperm injection (ICSI) employingtesticular and ejaculated spermatozoa was assessed in 24 coupleswith totally or initially immotile spermatozoa. No criteriawere employed in selecting which patients would be treated withtesticular or ejaculated spermatozoa. The men were chosen atrandom. Testicular spermatozoa obtained by testicular spermextraction were used in 14 and ejaculated spermatozoa were usedin 10 of these couples. In all cases, asthenozoospermia wastotal in their basal semen sample. In 12 male partners, spermatozoawere totally immotile before and after Percoll gradient fractionation(totally immotile). In the remaining 12 men, spermatozoa initiallyshowed a total absence of motility; however, some of the spermatozoahad showed very poor motility (0.1%) after Percoll gradientfractionation and a 13–2.0 h incubation period (initiallyimmotile). Of these 24 total asthenozoospermic males, 14 alsohad total terato-zoospermia. The fertilization and cleavagerates in the testicular and ejaculated sperm groups were 533and 963 and 543 and 94.4% respectively. One cycle resulted incomplete fertilization failure, and in 23 embryo transfer cyclesa total of 10 pregnancies were obtained (41.6%). Eight pregnancieswere achieved in the testicular sperm group, while only twopregnancies were obtained in the ejaculated sperm group. Fourpregnancies, two from the ejaculated sperm group and two fromthe testicular sperm group, resulted in clinical abortions inthe first trimester. Of the remaining six pregnancies, two havealready resulted in healthy births and four pregnancies arenow in the second or third trimester in the testicular spermgroup. Using testicular spermatozoa in combination with ICSIcan be an alternative mode of treatment in cases with totallyor initially immotile spermatozoa in the ejaculate. Very lowpregnancy rates have been obtained and no ongoing pregnancyhas been achieved using ejaculated spermatozoa in these cases.     

Improvement of sperm motility by the addition of progesterone to the Percoll medium during sperm purification

The Percoll centrifugation technique is widely used to selectthemost motile spermatozoa in connection with assistedre productiontechniques (ART). Progesterone is known toenhance the motilityof spermatozoa in vivo and in vitro. This study evaluates whetherthe motility of spermatozoacan be enhanced by the presence ofprogesterone simultaneously with purification using a routinePercoll centrifugation technique. Motility is measured by computer-assistedspermanalysis. The 100% Percoll medium contained aprogester one concentrationof 2 (ig/ml, which is in the range of that found in pre-ovulatoryfollicular fluid and peritoneal fluid around ovulation. Witha two-layer purification procedure (55/80% Percoll medium),exposure ofthe progesterone in the Percoll medium for only 25mincaused a highly significant improvement of motility in thespermatozoa isolated from the semen samples of 14 healthymen.It is concluded that the addition of progesterone tothe Percollpurification medium can further enhance spermmotility withoutthe need for a change in the routinely used semen purificationtechnique in most assisted reproductionprogrammes.     

Cytogenetic action of aspirin in a human lymphocyte culture

In human lymphocyte cultures treated with aspirin in concentrations of 10, 100, 200, and 300 μg/ml in the G₀ stage of the cell cycle and in concentrations of 10, 50, 100, 500, and 1000 μg/ml for 1 h in the S stage the frequency of induced chromosomal aberrations was significantly higher than in the control. After treatment with aspirin in the G₁ stage a significant difference was found only with aspirin concentrations of 50 and 500 μg/ml, and if treated in the G₂ stage, in concentrations of 10, 50, and 100 μg/ml. No linear relationship could be found between the cytogenetic effects of aspirin and its dose.     

高龄和潜在医学风险的活体供者肾移植的安全性分析

目的 探讨存在高龄及潜在医学风险的活体肾移植供者的选择和安全性问题.方法 本院亲属活体肾移植供者66例.按年龄分为<50岁组(42例)和1>50岁组(24例).按术前检查分成存在潜在医学风险组12例(A组)和一般供者组54例(B组).对供者住院时间、手术前后肾小球滤过率(GFR)、血清肌酐(Scr)、并发症等情况进行比较分析.结果 <50岁组和≥50岁组手术前后的Scr和术前总GFR差异无统计学意义(P>0.05),但术后3个月留存肾GFR分别为(74.82±17.42)、(60.34±13.32)ml/min(P<0.05).55岁以上供者术后平均住院时间长于<50岁组供者(P<0.05),而2组总并发症发生率差异无统计学意义(P>0.05).A、B 2组比较,术前总GFR,手术前后Scr,术后3个月以上留存肾GFR差异均无统计学意义(P>0.05).结论 对于存在高龄或潜在医学风险供者选择需十分谨慎.术前全面评估,严格控制纳入标准,其预后和安全性良好.     

Genetic regulation of gametogensis: The receptor encoded by the human C-MET oncogene is expressed in testicular tissue and on human spermatozoa

Because of its distinctive ability to act as a mitogen, a mitogenand a morphogen, hepatocyte growth factor/ scatter factor (HGF/SF)has all the characteristics of a molecule able to function inregulatory networks of motility, such as the spermatogenic epithelium,and this through binding of its receptor p190^MET (C-MET). Inthis study we report the expression of C-MET in the human seminiferousepithelium and on spermatozoa from men being treated for infertilityand sperm donors. The presence of C-MET was demonstrated byimmunochemistry on the cell membrane of spermatogonia, spermatocytes,spermatids and on spermatozoa, whereas Sertoli cells and Leydigcells did not show expression. Comparison of C-MET expressionon spermatozoa of the 90% Percoll layer of subfertile patientsand donors revealed clearly two distinct groups (unpaired t-test,P < 0.001), whereas comparison of C-MET expression on spermatozoain the 47% Percoll layer was not significantly different betweenpatients and donors. In addition, there was a significant inversecorrelation between sperm concentration and the C-MET expressionof spermatozoa in the 90% Percoll layer (r = –0.80, 95%confidence interval, –0.92 to –0.55; P < 0.0001),but not with the C-MET expression of spermatozoa in the 47%Percoll layer. In conclusion, the presence of C-MET was demonstratedin the seminiferous epithelium and on mature and immature spermatozoa,indicating a role for this growth factor receptor in the differentiationand/or migration that occurs during human spermatogenesis. C-MET/male infertility/spermatozoa/testis