流式细胞仪富集小鼠CD90．2阳性精原干细胞

目的 寻找分离和富集小鼠精原干细胞有效方法.方法 首先使用胰酶消化幼鼠的睾丸制成单细胞悬液,联合应用30% percoll液密度梯度离心分离和流式细胞仪分选有CD90.2阳性同时CD117阴性的精原干细胞,并在体外进行原代培养尝试.结果 联合应用两种方法 获得精原干细胞的纯度高达80.4%,满足体外培养和对其进一步研究要求.结论 联合应用两种方法是获得高纯度精原干细胞的有效方法,为精原干细胞培养和研究奠定基础.     

改良消化富集法提高精原干细胞富集效率的实验研究

目的 建立一种改良的小鼠精原干细胞消化富集方法,为精原干细胞体外长期培养和移植奠定基础.方法 收集120个出生4～d BALB/c新生小鼠的睾丸,平均分为对照组和实验组,分别采用传统的两步消化法(对照组)和改良消化法(实验组)分离消化获得细胞悬液,并种植到铺有明胶的培养皿中,于种植后1h、5h、24h通过差速贴壁法去除体细胞,获得富集的精原干细胞.以Thy-1作为精原干细胞的表面标志,分别在3次差速贴壁前、后采用流式细胞仪检测Thy-1阳性细胞的比例,比较两组精原干细胞的富集效率.结果 实验组和对照组消化所获得的细胞数分别为(3.4±0.5)×105/睾丸和(3.6±0.3)×105/睾丸,无统计学差异(P＞0.05).然而,实验组细胞贴壁具有良好的活性,初次贴壁仅需40～50min,而对照组细胞需要4～5h甚至过夜培养才能贴壁.3次差速贴壁后,实验组Thy-1阳性细胞占睾丸细胞的比例为(56.3±4.7)%,而对照组仅为(32.6±4.2)%,差异具有统计学意义(P＜0.01).结论 改良消化富集法能明显提高睾丸细胞活性和富集效率,可获得大量高度纯化的精原干细胞,为精原干细胞培养和移植奠定基础.     

小鼠精原干细胞分选

目的:寻求富集小鼠精原干细胞的有效方法。方法:取6周龄雄性昆明白小鼠20只,手术制作成双侧隐睾模型,继续饲养2~3个月后,切取睾丸,用酶两步消化法制成单细胞悬液,加入FITC标记的抗α6-integrin抗体和PE标记的抗c-k it抗体冰浴20 m in后,利用流式细胞仪筛选出具有side scatterlo、α6-integrin+、c-k it-特征的细胞,苔盼兰染色监测细胞活性。另取20只小鼠作为对照组,相同条件下饲养相同时间。结果:隐睾小鼠筛选出的精原干细胞占睾丸细胞总数的2.8%,95%以上的细胞具有活性。结论:利用免疫荧光激活细胞分选术能分离出大量有活性的精原干细胞。     

精原干细胞龛境研究进展

精原干细胞是一群能够自我更新并具有多向分化潜能的永生细胞。＂干细胞龛＂理论最初是在造血系统中提出来的,龛同样也存在于睾丸组织中。精原干细胞龛在睾丸中是半开放的隔离体系,具有特定的数量调控及随年龄变化的特征。两种内源性因子nanos2和Plzf调控精原干细胞自我更新。龛细胞分泌因子胶质细胞源性神经营养因子（GDNF）也调节精原细胞的更新。     

精原干细胞龛境研究进展

精原干细胞是一群能够自我更新并具有多向分化潜能的永生细胞。"干细胞龛"理论最初是在造血系统中提出来的,龛同样也存在于睾丸组织中。精原干细胞龛在睾丸中是半开放的隔离体系,具有特定的数量调控及随年龄变化的特征。两种内源性因子nanos2和Plzf调控精原干细胞自我更新。龛细胞分泌因子胶质细胞源性神经营养因子(GDNF)也调节精原细胞的更新。     

大鼠睾丸中c-kit的表达特点与精原干细胞的分化

c-kit是干细胞因子（SCF）受体，在精原细胞的生存增值分化上具有重要作用，对于c-kit介导的信号转导引发促进生殖细胞有丝分裂的机制有了较多研究，但c-kit表达于生殖细胞的特点仍需进一步明确，这不仅有助于理解精原干细胞分化特点，还将对精原干细胞分化机制的深入探索提供帮助。     

精原干细胞的研究进展

精原干细胞是具有自我更新和多向分化潜能等特性的多能干细胞 ,可在自身基因和 /或外来信号调节下最终分化为精子 ,对其研究近年来倍受关注。本文介绍了精原干细胞的富集、初次分选技术 ,增殖调控因子以及移植技术的研究进展。     

精原干细胞微环境的生物学特性

成体干细胞的自我更新和分化与其微环境关系密切。精原干细胞(spermatogonial stem cells,SSCs)是体内自然状态下唯一能将遗传信息传至子代的成体干细胞。探讨SSCs更新和分化的调控机制有助于精子发生机理的阐明,并为探究其他成体干细胞增殖分化的调节机制提供依据。因此,SSCs系统为成体干细胞微环境研究提供了理想模型。资料表明,SSCs的更新和分化受其微环境的调控。基于本室的工作,参考最新文献,本文主要从SSCs微环境的基本特性、构成及其产生的各种调控因子等角度,评述了SSCs微环境的生物学特性及其与SSCs更新和分化间的关系,以期为本领域研究及其他成体干细胞相关研究提供借鉴。     

精原干细胞移植研究近展

在非灵长类哺乳动物睾丸中，As(Asingle)型精原细胞是精子发生的干细胞。在As型精原细胞分裂的基础上，其子代细胞彼此分开变为两个新的T细胞，或者仍停留在一起，成为通过细胞间桥连接的Aal(Aaligned)型精原细胞。Apr精原细胞是精子发牛普系中的第一代细胞，它将进入分化旁路，进一步发展     

Enrichment and transplantation of spermatogonial stem cells

Spermatogenesis is a complex, highly organized process originated from stem cell spermatogonia. Because there are very few stem cells and they can only be defined by their function, the identification and isolation of these cells has been very difficult. By using a spermatogonial transplantation assay system, we have identified α₆-and β₁-integrin expression on stem cells, and cells isolated with these antigens were significantly enriched in stem cells. This is the first demonstration of spermatogonial stem cell-associated antigens. Analysis of two infertile mouse models, Steel/Steel^Dickie (Sl/Sl^d) and experimental cryptorchidism, showed that the number of stem cells is reduced in Sl/Sl^d testis. Whereas cryptorchid testes are greatly enriched for stem cells, and one in 200 cells is a stem cell. These techniques will provide an important starting point for further purification and characterization of spermatogonial stem cells.     

JC-1单标法流式细胞术检测精子线粒体膜电位的研究

目的:探讨应用荧光染料JC-1单色标记法进行流式细胞术检测精子线粒体膜电位(MMP)的可行性及其临床意义。方法:收集63例男性精液标本,分为生育组(n=31)和不育组(n=32)。通过计算机辅助精液分析系统进行精液常规分析。精液标本洗涤处理后用JC-1染色后上流式细胞仪分析,用发橙红色荧光精子百分率(JC-1+%)表示MMP正常精子的比例。结果:生育组精子JC-1+%为(75.89±15.69)%,显著高于不育组[(54.04±22.21)%,P=0.000]。63例标本中,JC-1+%与精子活动率呈显著正相关(r=0.610,P=0.000),与(a+b)级精子百分率呈显著正相关(r=0.614,P=0.000),与d级精子百分率呈显著负相关(r=-0.504,P=0.000)。JC-1+%与已建立的罗丹明/碘化吡啶双染法检测结果(Rh123+/PI-%)呈显著正相关(r=0.938,P=0.000)。结论:应用流式细胞术JC-1单标法检测精子MMP具有可行性,精子JC-1+%可作为男性不育的辅助诊断指标。     

In vitro culture of spermatogonial stem cells isolated from adult alpaca (Vicugna pacos) testes analysed with Dolichos biflorus by flow cytometry

Spermatogonial stem cell (SSC) is known for its self‐renewal capacity. We have studied the in vitro proliferation of isolated SSC from adult alpaca (Vicugna pacos) testes. A total of 107 samples were evaluated of which 31 were evaluated at baseline, 36 were cultivated in DMEM and 40 in STEMPRO media. Half of the cultivated samples was analysed after 14 days, and the rest after 21 days. Round cell subpopulations were identified with FITC‐DBA by flow cytometry: strongly positive DBA (sDBA+) as SSC, weakly positive DBA (wDBA+) as in early differentiation and negative DBA as differentiated. At the beginning, 4.16% of the cells were SSC, 37.61% wDBA+ while 54.12% were DBA?. After 14 days, 42.28% of SSC, 44.68% wDBA+ and 11.07% DBA? were found in DMEM while 47.09% of SSC, 32.57% wDBA+ and 18.48% DBA? in STEMPRO. After 21 days 38.66% were SSC, 52.78% wDBA and 7.65% DBA? in DMEM and on STEMPRO media 47.92% SSC, 44.20% wDBA+, 4.93% DBA?. There is a significant difference between the number of initial and SSC cultivated, as well as between DBA? (p < 0.05), while there is no significant difference between the wDBA+ (p > 0.05). Our results suggest that both culture media are appropriate for the in vitro proliferation of alpacas SSC.     

Susceptibility of Liver Allografts to High or Low Concentrations of Preformed Antibodies as Measured by Flow Cytometry

Liver grafts are more resistant to damage by HLA antibodies than other organ allografts, but it is not clear if the antibodies are associated with graft rejection or graft loss, or if different antibody concentrations have different effects. To explore potential associations between antibody concentrations and outcome, preformed IgG antibodies against donor cells were quantified by flow cytometry in 465 consecutive liver transplant recipients. Antibody-positive patients were classified according to whether they had high or low antibody concentrations and analyzed for possible correlation with graft rejection or graft loss. The results showed that the incidence of rejection was not significantly different between antibody-positive and negative patients. However, patients with high antibody concentrations had a higher incidence of steroid-resistant rejections (31% at 1 year) than patients with low antibody (4%) or no antibody (8%, p < 0.0004). These effects were mainly due to T-cell (HLA class 1) antibodies. The overall incidence of rejection at 1 year was 69% for high antibody patients, 51% for patients with low antibodies and 53% for patients with no antibodies (p not significant). In an apparent paradox, antibody-positive patients underwent fewer early graft losses. Thus, the associations of preformed antibodies and outcome depend, on the one hand, on antibody concentrations, and on the other hand on whether the outcome measured is steroid-sensitive rejection, steroid-resistant rejection or graft survival. These complex interactions may explain the controversial results observed in previous studies.     

骨肿瘤DNA流式细胞术定量分析

应用流式细胞术对６２例未经化疗或放疗的原发骨肿瘤的石蜡包埋标本细胞核ＤＮＡ进行了测定，其中３例为良性肿瘤或瘤样病变，３３例为骨巨细胞瘤，２６例为其他恶性骨肿瘤。结果表明，良性骨肿瘤与瘤样病变中未见异倍体，骨巨细胞瘤中ＤＮＡ异倍体的发生率也很低，而其他恶性骨肿瘤中异倍体的发生率非常之高，故异倍体可作为恶性骨肿瘤的一个较特异的标志。且单个骨肿瘤在活检与大体标本、原发与复发肿瘤上表现了稳定的倍性，而基于病理学基础上的骨肉瘤的分型与分级在ＤＮＡ含量上并无明显的区别。     

流式细胞术在精子功能检测中的应用

目的:研究流式细胞术在精子功能评价中的作用。方法:对104例男性不育患者和10例正常生育男性的精液标本进行精液量、精子密度、活动率、畸形率等精液常规分析(CASA法),对精子存活率、染色质结构和线粒体膜电位等进行流式细胞术分析,并将检测结果通过SAS软件进行统计学分析。结果:成组U检验结果表明:不育患者的精子与正常生育男性精子相比除了具有密度低(U=2.51,P=0.014 3)、活动率差(U=3.44,P=0.001)、畸形率高(U=-5.88,P<0.000 1)等特点外,经流式细胞术测定精子结果表明不育男性的精子质膜完整率(U=4.72,P<0.000 1)、线粒体膜电位正常率(U=-2.53,P=0.030 9)和染色质完整率(αt:U=-3.82,P=0.000 3;SDαt:U=-3.98,P=0.000 1;COMPαt:U=-3.57,P=0.000 5)均显著低于正常生育男性。多元逐步回归分析结果表明:精子质膜完整性与精子活动率(t=1.66,P=0.101 6)、精子的线粒体膜电位与精子活动率(t=3.33,P=0.001 4)、精子染色质的完整性与精子活动率(t=3.24,P=0.001 9)均呈正相关,而精子的线粒体膜电位与精子颈、尾部的畸形率呈负相关(t=-3.44,P=0.001)。结论:流式细胞仪测定对精子质量评定意义显著,对男性不育的诊断和治疗也具有较高的参考价值。     

In vitro isolation of stem cells derived from human dental pulp

Agha‐Hosseini F, Jahani M‐A, Jahani M, Mirzaii‐Dizgah I, Ali‐Moghaddam K. In vitro isolation of stem cells derived from human dental pulp.
Clin Transplant 2009: DOI: 10.1111/j.1399‐0012.2009.01137.x.
© 2009 John Wiley & Sons A/S. Abstract: Stem cells are characterized by the ability to differentiate and to self‐renew. Stem cells derived from human dental pulp have been shown to differentiate into osteoblasts serving as a potential source of autologous bone produced in vitro. The purpose of the present study was to isolate mesenchymal stem cells from dental pulp. Dental pulp was gently extracted from 27 intact human permanent third molars of patients aged 18–25. Cow horn forceps were used to isolate intact dental pulp in sterilized condition. The pulps were cultured in a medium containing Dulbecco’s modified Eagle’s medium‐low glucose (DMEM)‐LG and Amphotericin 1%. The cells were subsequently expanded by passages, two passages were performed before they were stored in liquid nitrogen for further examination. DMEM + fetal bovine serum (FBS) 10% L‐Glutamin 0.1% + Trypsin 2.5% + ethylene diamine tetraacetic acid (EDTA) were used for passage. Light microscope and flow cytometry were used to study the cells. The isolated dental pulp cells expressed mesenchymal stem cell markers. The cells were negative for CD34 and CD31 and CD45 but were positive for CD13, CD44, CD90, CD166, and CD105. These results indicate that dental pulp can be use as a source of stem cells that we can isolate and culture.     

胶质细胞源性神经营养因子促进精原干细胞增殖的信号通路

胶质细胞源性神经营养因子(GDNF)是Tgf-β家族的一个新亚族,其通过以下途径促进精原干细胞(SSCs)的增殖:GDNF与GFR-α1特异性结合,活化Ret蛋白酪氨酸激酶,随后激活PI3K/Akt、Src、Ras/ERK1/2等信号通路,引起通路下游转录因子Pou3f1、Etv5、Bcl6b、Lhx1和基因H19、A-myb、N-myc、C-fos表达的上调,进而促进SSCs的增殖。同时,在该过程中还存在着信号通路间的交联现象。