人胚神经干细胞的冻存与复苏

目的：研究神经干细胞浆存复苏后的成活率及再培养生长情况。方法：利用无血清培养基短期体外培养胚胎来源的神经干细胞后，以冻存液（体积分数为70％DMEM／F12，体积分数为20％胎牛血清，10％DMSO）进行冻存，于1周，4周，8周，12周，16周，20周，24周分别复苏，计活细胞比例并进行再培养。结果：复苏后神经干细胞的成活比例分别为68％、65％、65％、62％、61％、60％、60％，再培养生长良好。结论：本实验对神经干细胞成功地进行了冻存，复苏和再培养，对临床择期进行神经干细胞移植手术和建立“神经干细胞库”具有重要的意义。     

人精液的冻贮研究

目的 研究在Ｔ－Ｇ型冷冻保护剂（Ｃｒｙｏｐｒｅｓｅｒｖａｔｉｖｅ ｍｅｄｉｕｍ,ＣＰＭ）作用下,速缓冻贮法对人精子结构和功能的影响。方法 随机双盲对照,将４５份正常精液随机分组,加与不加ＣＰＭ,以速缓冷冻法分别进行冻贮,冻贮前后分别进行常规分析、低渗肿胀试验、穿卵试验、超微结构观察及精子染色体分析。结果 未加ＣＰＭ组冻贮后的精液未见有精子复苏,经冷冻保存的精子质量明显降低,缓冻法对精子的影响显著小     

Cryopreservation and culture of the human fetal brain tissues

Summary Human embryos after 3–4.5 months of gestation were obtained with abortion. The brain tissue of the bodies was scissored up to obtain 1–3 mm³ pieces, and 7% dymethyl sulfoxide (DMSO), as a cryoprotectant, was added, and then stored at −70°C for 1–30 days or at −196°C for 1–84 days. The survival rate of stored cells was 64%–88%. During 6 days of storage with neuron culture medium, the survival rate of cells at 4°C is over 50% each day, but, as time goes on, the count of the cells is getting less and less. The cells washed out DMSO after cryopreservation and the planting fresh cells can adhere to the wall of the culture bottle, grow, display various forms of neurons and gliacytes. From the above findings, it was suggested that: 1) The fetal human brain tissue, handled properly, can endure cryopreservation with7% DMSO as a cryoprotective agent; 2) The storage time was related insignificantly to the survival rate of the tissues stored; 3)It is available for a short proservation at 4°C and 4) It is possible to set up a bank of fetal human brain tissue.     

线粒体活性对人类精子活动力的影响

目的:分析线粒体活性对人类精子活动力的影响。方法:以线粒体特异性荧光染料——mitotracker deep red 633(MTDR对不同活力的精子进行染色,然后用流式细胞术测定精子线粒体活性(即MTDR荧光强度值)。用雷帕霉素(rapamycin,RP)和羟氯喹(hydroxychloroquineon,HCQ)调控精子活性水平,测定精子活动力的变化。结果:MTDR荧光强度值与前向运动速率(progressive motility,PR)不相关(r=0.182,P=0.268)。5 nmol/L和100 nmol/L的RP均可明显降低线粒体活性,50μmol/L HCQ可明显提高线粒体活性(P<0.05),但它们对精子活动力影响都不明显(P>0.05)。结论:线粒体活性对人类精子活力影响不明显。     

原代人视网膜色素上皮细胞的冻存和复苏培养

目的 :观察对原代人视网膜色素上皮 (retinalpig mentepithelium ,RPE )细胞进行冻存和复苏培养的效果 .方法 :将 6只尸供眼 ,常规酶消化法分离、纯化的人原代RPE细胞 ,分为两组 ,实验组为即刻进行常规梯度降温液氮冷冻保存 ,1wk后行常规复苏培养 ;对照组为细胞直接进行后续实验 .台盼蓝拒染观察细胞存活率及生长状态 ;计算细胞贴壁率 ;初步计算克隆形成率 .结果 :RPE细胞存活率分别为试验组 (90± 7) % ,对照组 (87± 1 5 ) % ,两组相比无显著性差异(P >0 .0 5 ) ;两组接种细胞贴壁率从 2 4h开始分别为 (5 .0±3.1 ) %～ (6 1 .0± 7.3) %和 (1 5 .0± 6 .3) %～ (6 0 .0± 3.3) % ;培养 4 8h之前 ,两组之间存在统计学差异 (P <0 .0 5 ) ,4 8h之后组间无显著性差异 (P >0 .0 5 ) ;培养 5 ,7和 1 0d初步克隆形成率两组分别为 (1± 0 ) % ,(1± 1 ) % ,(3± 1 ) %和 (1±0 ) % ,(3± 1 ) % ,(2± 1 ) % ,组间无显著性差异 (P >0 .0 5 ,n=3) .冻存后复苏培养的RPE细胞生长状态良好 ,增生活跃 ,与对照组类似 .结论 :对原代人RPE细胞进行冻存 ,复苏培养的RPE细胞活性、存活能力、单细胞分裂增生能力不受影响 .     

精子顶体酶活性检测方法研究

本文报告检测精子顶体酶活性的方法，是以Ｎａ一苯甲酰一ＤＬ一精氨酸一ｐ一硝酰基苯胺（ＢＮＰＮＡ）为底物，检测新鲜去精浆精子顶体酶活性。文中分析了实验的温度、时间、反应体系中精子浓度等因素对实验结果的影响，提出了最佳实验条件及注意事项。     

Demonstration of specific binding of cocaine to human spermatozoa

Exposure of males to cocaine has been linked to abnormal development of their offspring. To investigate the possible role of sperm, this study examined the interaction of cocaine with human spermatozoa. Washed sperm were incubated with tritiated cocaine (6.7 nmol/L) with or without unlabeled cocaine (670 mumol/L), and the samples were filtered and the remaining radioactivity quantitated. The specific binding was optimal at 20 minutes and 23 degrees C. Competition studies with tritiated cocaine (3.4 to 66.6 nmol/L) indicated the presence of approximately 3.6 x 10(3) binding sites per cell, with a high affinity receptor dissociation constant (Kd = 12.6 nmol/L). Cocaine concentrations as high as 670 mumol/L had no detectable effect on either the motility or viability of the cells. These results support the hypothesis that the sperm may act as a vector to transport cocaine into an ovum. This novel mechanism could be involved in the abnormal development of offspring of cocaine-exposed males.     

人正常精液中早期凋亡精子的检测

目的通过细胞化学方法观察正常精液中精子早期凋亡发生的程度。方法采用荧光标记的AnnexinV和碘化丙啶对34例具有正常精液分析参数的精子进行染色和显微观察,计数凋亡精子发生率。结果精液中早期凋亡精子发生率为6.5(4.9,7.4)%,个体间存在较大的差异。凋亡精子发生率和年龄和禁欲时间等不相关。结论精液中存在少量的早期凋亡精子且个体间差异较大,荧光标记的AnnexinV和碘化丙啶染色显微镜观察可以有效地对其进行量化观察。     

人类精子蛋白质组分析的双向蛋白电泳技术研究

目的 :利用人类精子蛋白质组分析的双向蛋白电泳技术 (2 DE)建立正常精子的蛋白质图谱。方法 :比较不同蛋白质提取方法和上样量、不同的第一向等点聚焦方法所得图谱的差别 ,并初步建立人类正常精子的蛋白质图谱。结果 :用蛋白质提取方法一制得的样本电泳后有 6 70～ 70 3个蛋白质斑点 ;方法二所得样本电泳后仅有 194～ 2 10个蛋白质斑点。蛋白质提取方法一所制样本进行载体两性电解质pH梯度 SDS电泳技术 (ISO DALT)得到约 2 80～ 30 0个蛋白质斑点 ,用固相pH梯度 SDS电泳技术 (IPG DALT)得到多达 70 0个蛋白质斑点。结论 :采用蛋白质提取方法一制备精子蛋白质进行IPG DALT电泳的方法适用于建立精子全细胞蛋白质谱。     

两种精子优选法对精子活率、尾部低渗肿胀率和形态正常精子百分率的影响

目的：评价Percoll密度梯度离心和上游精子分离方法对人精子活率、尾部低渗肿胀率和形态正常精子百分率影响。方法：收集12例男性不育症患者精液样本,分别采用Percoll密度梯度离心和上游精子分离方法(简称上游法)进行精子优选,应用伊红-Y染色分析优选前后精子活率和尾部低渗肿胀率,应用改良巴氏染色法分析优选前后精子形态。结果：①Percoll法和上游法优选后精子活率和尾部低渗肿胀率均明显高于优选前（P<0.01）,两种优选法间比较精子活率和尾部低渗肿胀率差异均无显著性（P>0.05）。Percoll法和上游法优选后B-G型精子尾部低渗肿胀率均明显高于优选前（P<0.01）,两种优选法间比较B-G型精子尾部低渗肿胀率差异均无显著性（P>0.05）。②Percoll密度梯度离心法和上游法优选后形态正常精子百分率均明显高于优选前（P<0.05）,上游法优选后形态正常精子百分率明显高于Percoll法优选后形态正常精子百分率（P<0.05）。上游法优选后锥型头、无定型头和颈/中段缺陷形态异常精子百分率均明显少于优选前（P<0.05）;Percoll法优选后锥型头、无定型头和尾部缺陷形态异常精子百分率均明显少于优选前（P<0.05）;上游法优选后无定型头、颈/中段缺陷和尾部缺陷形态异常精子百分率明显低于Percoll优选后（P<0.05）。结论：Percoll法和上游法优选后精子活率、尾部低渗肿胀率和形态正常精子百分率较优选前明显改善, 此两种优选法均可广泛应用于生殖医学,其中上游法优于Percoll法。     

人类精子蛋白质组分析的双向蛋白电泳技术研究

目的：利用人类精子蛋白质组分析的双向蛋白电泳技术（2-DE）建立正常精子的蛋白质图谱。方法：比较不同蛋白质提取方法和上样量、不同的第一向等点聚焦方法所得图谱的差别,并初步建立人类正常精子的蛋白质图谱。结果：用蛋白质提取方法一制得的样本电泳后有670-703个蛋白质斑点;方法二所得样本电泳后仅有194-210个蛋白质斑点。蛋白质提取方法一所制样本进行载体两性电解质pH梯度-SDS电泳技术（ISO-DALT）得到约280-300个蛋白质斑点,用固相pH梯度-SDS电泳技术（IPG-DALT）得到多达700个蛋白质斑点。结论：采用蛋白质提取方法一制备精子蛋白质进行IPG-DALT电泳的方法租用于建立精子全细胞蛋白质谱。     

Cold—preserved human spermatozoa in electrolyte—free solution retain their penetration capacity

Objective: To evaluate penetration capacity of human sperm preserved in electrolyte-free (EF) solution at 4 ℃. Methods: The motility, acrosomal status penetration rate and fertility index of human sperm were assessed before and after cold-preservation in EF solution, respectively. Results: The motility of human sperm cold-preserved in EF solution for 1 week was significantly higher than that of human sperm cold-preserved in modified human tubal fluid ( mHTF) (43. 4% ± 7. 9% vs 9. 5% ±2. 5% , P <0. 01 ). Although acrosomal status of human sperm cold-preserved in the EF solution before reinitiation was not different from those of the fresh sperm ( capacitated sperm; 7. 6% ± 1. 8% vs6.4±1.8%; acrosome-reacted sperm; 3.0% ±1.7% vs2.4±1.1%, P>0.05), the percentage of capacitated and acrosome-reacted sperm in the EF solution significantly increased after reinitiation (capacitated sperm; 16. 0% ± 2.3%vs7.6±1.8%, acrosome-reacted sperm: 9. 4% ±2.1% vs 3. 0% ±1.7%, P<0.01). The penetration rate and fe     

Mechanism of lead induced effects on human spermatozoa after occupational exposure

Objectives: Occupational lead exposure caused several types of male reproductive impairments in different working populations. In the present study we examined the paint factory workers of active reproductive age and compared the data with the non-occupationally exposed desk job holders taken as control from Bangalore, India. Materials and methods: In the above perspective, sperm cell morphology, morphometery and motile activity were assessed. Routine seminal biochemistry, cell cycle phase analysis of sperm head DNA, estimation of serum reproductive hormones and metal levels in blood and semen were also taken into account. Result: Low sperm velocity, ATPase activity, gross and forward progressive motility with high stationary motile spermatozoa revealed lowering of cellular activity after lead exposure (p<0.001), which was supported by high seminal plasma fructose level (p<0.001). Lowering of seminal plasma total protein with concomitant rise in free amino acid level was prevalent as the exposure increased (p<0.001), suggesting disturbance in cellular nutritional support essential for cellular motility. Prolonged liquefaction time, reduced semen volume and viscosity as well as altered seminal plasma protein, fructose and cholesterol level among the workers indicated dysfunction of accessory sex glands viz. prostate and seminal vesicle after occupational lead exposure (p<0.001). Deterioration of sperm count, structural abnormality of spermatozoa and sperm head DNA hyploidy was also associated with high blood and semen lead levels in the paint factory workers (p<0.001) without interfering serum FSH, LH and testosterone level (non-significant at p<0.05). Conclusion: Therefore, the present study suggested that at the present exposure level lead might cross blood-testis-barrier and increased its value in semen of the occupationally exposed paint factory workers in Bangalore, India, thereby producing detrimental effects on semen quality and sperm characteristics. Key words: Blood/semen lead level, cell cycle, paint factory workers, semen biochemistry, sperm morphology.     

重组人透明带蛋白3诱导精子的顶体反应

目的：在体外条件下检测重组人透明带蛋白诱导的顶体反应。 方法：体外采集已生育健康男子精液,采用非连续密度梯度法分离精子,将获能的精子悬液分成阴性对照组(C组,200 μL精子悬液加1 μL DMSO)、阳性对照组(A组,200 μL精子悬液加A23187至终浓度为10 μmol•L^-1)和ZP3组(200 μL精子悬液加ZP3至终浓度为10 mg•L^-1),考马斯亮蓝染色法检测重组人ZP3蛋白诱导的顶体反应。结果：C组、A组和ZP3组发生顶体反应的精子百分率分别为(5.00±1.70)%、(46.10±7.49)%和(13.20±2.70)%,各组间比较差异有显著性(P<0.05)。 结论：重组人透明带蛋白能够诱导人精子发生顶体反应。     

龟蛇阿米巴冷冻保存

本文报道龟蛇阿米巴在含5％DMSO和10％小牛血清的RPMI1640培养液中,经长期冷冻保存后,其形态及生物学特性保持不变。