人胚胎胰发育中巢蛋白和转化生长因子β1表达的变化

目的:观察巢蛋白和转化生长因子β1(TGFβ1)在人胚胎胰发育过程中的表达变化.方法:应用免疫组织化学EnVision法,检测30例人胚胎胰组织中巢蛋白和TGFβ1的表达及分布变化.结果:各胎龄段(10～37周)胰均有巢蛋白和TGFβ1表达,巢蛋白阳性细胞存在于胰岛、间充质和血管中,在腺泡、导管中未见阳性细胞;TGFβ...     

巢蛋白在人胚胎消化管的表达

目的　通过研究不同时期人胚胎消化管巢蛋白的表达及其变化探讨巢蛋白在消化管发育过程中的作用。 方法　取3~8月人胚胎食管、胃和小肠,常规石蜡切片,采用免疫组织化学法检测巢蛋白在胚胎消化管中的表达及分布。 结果　各胎龄段（3~8月）消化管均有巢蛋白的表达。巢蛋白在消化管主要表达在固有层、黏膜下层和肌层内的微血管和毛细血管壁上,且巢蛋白的表达随胎龄的增加而减少。与发育早期组相比,发育中期、晚期胚胎食管、胃和小肠组织中巢蛋白的表达显著减少（P<0.05）,差异有统计学意义。而在发育中期和发育晚期巢蛋白的表达无显著性差异（P>0.05）。 结论　巢蛋白在胎儿消化管壁的微血管和毛细血管的管壁上表达,且随着胎儿发育,其表达逐渐减少,提示巢蛋白在这些器官组织的血管形成过程中起作用。     

微管相关蛋白2和巢蛋白在人胚胎脊髓的表达

目的 探讨微管相关蛋白2(MAP-2)和巢蛋白在人胚胎脊髓发育阶段的分布规律及其表达意义.方法 应用免疫组织化学法,检测第2、3、4月龄段共16例人胚胎脊髓前角、中央管及后角中MAP-2和巢蛋白的表达、分布状况.结果 第2～4个月龄段,人胚胎脊髓内均可见巢蛋白表达阳性的神经纤维分布,随着胎龄的增大,脊髓前角处巢蛋白阳性...     

巢蛋白及神经元素3在胚胎早期发育中的表达与分布

目的:比较巢蛋白(Nestin)与神经元素3(Ngn3)在胚胎早期发育各组织中的表达与分布.方法:采用免疫组织化学SABC法对30例5～10 周人胚胎发育中各器官组织中Nestin与Ngn3的表达与分布进行定位研究.结果:5周时,肝造血干细胞Nestin、 Ngn3阳性,胰芽原始导管上皮细胞、皮肤上皮细胞也呈Ngn3阳性反应;8周后,胚胎皮肤、椎间盘及肾上腺组织Nestin阳性,而Ngn3表达为阴性.结论: Nestin、 Ngn3在胚胎早期发育不同组织细胞的表达有不同的时空性,确切意义及机制有待进一步研究.     

生长抑素在胚胎早期胰及胃肠道发育中的表达及分布

目的:探讨人胚胎早期发育中生长抑素在胰及胃肠道的表达及分布.方法:采用免疫组织化学SABC法对30例7～14周人胚胎胰及胃肠生长抑素阳性细胞进行定位.结果:胚胎7周时胰导管上皮及小肠均未见有生长抑素阳性细胞;9周时,胰导管上皮可见较少的生长抑素阳性细胞,14周时生长抑素阳性细胞增多并开始聚集参与形成胰岛;11周时小肠内才出现生长抑素阳性细胞,阳性细胞分布在小肠黏膜上皮及固有层内,14周时生长抑素阳性细胞明显增多.结论:胚胎早期发育中,分布于胰导管上皮的D细胞其生长抑素的表达早于小肠的D细胞;生长抑素不仅参与胰岛细胞的分化发育,还与胃肠道的内分泌功能形成密切相关.     

人胎肺细胞增殖与凋亡及其与发育分化的关系

<正> 目的:探讨增殖与凋亡这两种细胞学行为在同一个胚胎器官中的发生时间、表达规律以及它们对胚胎器官发育的影响,以了解这两种细胞行为在胚胎器官正常发生中各自的作用及相互的的联系.方法:采用原位核酸末端缺口杂交方法(TUNEL法)和增殖细胞核抗原(PCNA)免疫组织化学技术,分别观察了28例胎龄16-32周的人胎肺组织在不同胎肺发育阶段细胞凋亡与增殖的表达率、表达部位及其随发育时间的变化趋势.结果:发现支气管粘膜上皮细胞在胎肺发育的早、中期(即假腺期和小管期),细胞的凋亡与增殖均明显较晚期(原始囊泡期)旺盛,而且两者均于胎肺发育的第24周达到最高峰(阳性细胞表达率分别为0.5263±0.0085及0.3684±0.0238),但凋亡与增殖细胞在不同发育时期表达部位有动态的变化:早期以间质细胞为主,中期以各级支气管粘膜上皮为主,晚期以支气管树远端的单扁上皮为主,而且增殖与凋亡在分布部位上的变化与胎肺发育过程中支气管树的形成发生的需要相一致.结论:细胞的凋亡     

胎儿胸腺发育过程中T细胞抗原受体的表达

目的:检测不同胎龄胎儿胸腺组织内T细胞抗原受体(TCR)的表达及发育规律.方法:18～40周胎儿胸腺组织,采用免疫组织化学SP法检测TCRαβ和TCRγδ的表达. 结果:胚胎21周,胸腺组织开始出现TCRαβ和TCRγδ阳性细胞.TCRαβ阳性细胞主要分布被膜下及小叶间隔中,围绕血管成群分布.TCRγδ阳性细胞的分布与TCRαβ阳性细胞相似, 但细胞数量较TCRαβ阳性细胞少.38周以后胎儿胸腺内TCR阳性细胞主要分布在皮髓质交界处,被膜下及小叶间隔中的TCR阳性细胞较早期少.结论:TCR阳性细胞在胚胎早期开始出现,其分布部位提示局部微环境有利于这些细胞的分化发育.     

巢蛋白在不同发育阶段人胚胎脊髓中的表达变化

目的:研究人胚胎脊髓生长发育过程中巢蛋白(nestin)的变化趋势。方法:采用免疫组织化学方法对不同发育时期(3周～8个月)的人胚胎脊髓中巢蛋白(nestin)的表达及变化进行了研究。结果:Nestin阳性细胞在神经上皮贯穿整个发育过程,5周基板、翼板形成后即可见到阳性物,而边缘层6周出现。中央管、基板、翼板、边缘层内nestin阳性细胞达高峰的时间分别是6周、7周、8周、9周,而表达明显减少的时间分别是8周、9周、10周、11周。随着脊髓的发育成熟,灰、白质内nestin阳性产物逐渐减少,而中央管的阳性细胞数至3月后较为稳定。结论:在脊髓不同部位神经干细胞出现时间和达到高峰的时间均不同,提示在脊髓的不同部位神经干细胞增殖、分化并不同步。     

人胎肺发育过程中生存素表达及生物学意义

目的检测生存素(survivin)和caspase-3在人胎肺发育过程的表达特征,探讨两者在胎肺发育中的意义。方法采用16～35周人胎肺组织,用免疫组化SP法检测survivin和caspase-3的表达。结果在胎肺的假腺期和小管期,survivin主要表达于远端支气管上皮,在原始肺泡期,survivin阳性表达于原始肺泡上皮,到35周,近端支气管上皮细胞中出现散在的survivin阳性细胞。caspase-3在支气管上皮的表达,以小管末期最强,主要定位于近端支气管上皮、Ⅱ型肺泡细胞。结论 胎肺发育早期,survivin对支气管树形态构筑过程中上皮细胞的发育与分化具有重要的保护意义,caspase-3参与支气管树的重建。胎肺发育晚期,Survivin和caspase-3协同作用,调控着Ⅰ型和Ⅱ型肺泡细胞的成熟和分化。     

穹隆海马伞损伤诱导巢蛋白在隔-海马通路表达

目的:探讨穹隆海马伞横断后巢蛋白在隔-海马通路表达的情况。方法:在立体定位仪上横切SD大鼠大脑皮质、胼胝体和穹隆海马伞,于术后1～30 d处死动物。用免疫组化染色方法,观察损伤后巢蛋白在隔-海马通路的表达。结果:穹隆海马伞损伤后,第1天,隔区和海马未见巢蛋白阳性细胞;第3天巢蛋白阳性细胞开始出现在损伤近侧背外侧核区和海马CA3区及齿状回;第5天巢蛋白阳性细胞范围扩大,数量增加,出现在远离损伤平面嘴侧端的隔区和尾侧端的海马以及损伤对侧。第7天起,巢蛋白阳性细胞在损伤平面区减少而在远离区增加;第15天损伤对侧多于损伤侧;第20天消失。结论:穹隆海马伞横断引起隔-海马通路巢蛋白表达的改变,巢蛋白阳性细胞具有修复中枢神经系统病变的潜能。     

微管相关蛋白2和巢蛋白在人胚胎小肠中的表达

目的: 探讨微管相关蛋白2(MAP-2)和巢蛋白(nestin)与人胚胎小肠壁组织发育的关系。 方法: 应用免疫组织化学PV法和图像分析(NIS-DR)软件检测第2、3、4个月龄段,人胚胎小肠组织内MAP-2和nestin的表达分布规律,数据的组间比较采用单因素方差分析。 结果: 第2、3、4个月龄段,MAP-2主要表达于人胚胎小肠壁黏膜下和肌间神经丛内神经细胞和纤维,随胎龄的增大,肌间神经丛内神经细胞的阳性强度增强, 两两比较, 差异有统计学意义(P<0.05)。Nestin蛋白在小肠壁各层均有阳性表达,其黏膜下层和肌间神经丛内阳性细胞数量随胎龄增大而呈先增高再降低的趋势,两两比较, 差异有统计学意义(P<0.05)。 结论: MAP-2和nestin参与调节人胚胎小肠壁组织的生长发育过程。     

Human bronchial fibroblasts exhibit a mesenchymal stem cell phenotype and multilineage differentiating potentialities

Mesenchymal stem cells (MSCs) are multipotent cells able to differentiate along different pathways including chondrogenic, osteogenic and adipogenic lineages. MSCs with a fibroblast-like morphology have been identified in human fetal lung. However, their frequency and characterization in human adult lung have not been yet evaluated. Therefore, we analyzed the mesenchymal phenotype and differentiation ability of cultured human adult bronchial fibroblast-like cells (Br) in comparison with those of mesenchymal cell progenitors isolated from fetal lung (ICIG7) and adult bone marrow (BM212) tissues. Surface immunophenotyping by flow cytometry revealed a similar expression pattern of antigens characteristic of marrow-derived MSCs, including CD34 (-), CD45 (-), CD90/Thy-1 (+), CD73/SH3, SH4 (+), CD105/SH2 (+) and CD166/ALCAM (+) in Br, ICIG7 and BM212 cells. There was one exception, STRO-1 antigen, which was only weakly expressed in Br cells. Analysis of cytoskeleton and matrix composition by immunostaining showed that lung and marrow-derived cells homogeneously expressed vimentin and nestin proteins in intermediate filaments while they were all devoid of epithelial cytokeratins. Additionally, alpha-smooth muscle actin was also present in microfilaments of a low number of cells. All cell types predominantly produced collagen and fibronectin extracellular matrix as evidenced by staining with the monoclonal antibodies to collagen prolyl 4-hydroxylase and fibronectin isoforms containing the extradomain (ED)-A together with ED-B in ICIG7 cells. Br cells similarly to fetal lung and marrow fibroblasts were able to differentiate along the three adipogenic, osteogenic and chondrogenic mesenchymal pathways when cultured under appropriate inducible conditions. Altogether, these data indicate that MSCs are present in human adult lung. They may be actively involved in lung tissue repair under physiological and pathological circumstances.     

胎鼠胰腺干细胞原位移植治疗糖尿病的实验研究

目的：原位移植胎鼠胰腺干细胞，探讨其在糖尿病鼠胰腺微环境中转分化为胰岛样细胞团的可行性。方法：分离纯化SD大鼠胎鼠胰腺干细胞；荧光原位杂交（fluorescent in situ hybridization, FISH）检测SRY上的性别决定区（sex determining region on Y, SRY）以鉴别雄雌；免疫细胞化学检测巢蛋白（Nestin）、胰十二指肠同源异型盒基因（PDX-1）的表达及流式细胞术测定Nestin细胞含量以鉴定胰腺干细胞；分胰腺实质内移植组、实验对照组及空白对照组，10只/组；监测各组大鼠血糖及血浆胰岛素含量，8周后取大鼠胰腺组织切片观察，FISH检测SRY，逆转录-聚合酶链反应（RT-PCR）观察各组大鼠胰腺内Nestin、PDX-1及胰岛素等mRNA的表达情况，免疫印迹（Western blotting）检测PDX-1及胰岛素的蛋白表达水平。结果：有5只胎鼠经FISH检测为雄性。免疫组化示存在Nestin 和PDX-1阳性细胞，流式细胞术测定Nestin阳性细胞含量占74.1%。胰腺实质内移植组大鼠于移植后第3周血糖开始下降，血浆胰岛素水平逐渐升高；第5周血糖及血浆胰岛素均达到正常水平并维持。取第8周大鼠胰腺组织切片HE染色可见外源性细胞团，FISH检测SRY阳性。RT-PCR示胰腺实质内移植组胰腺组织内胰岛素的mRNA表达明显高于实验对照组（P<0.05），而Nestin及PDX-1的mRNA表达量高于实验对照组及正常组（P<0.05）。Western blotting示干细胞移植组胰腺组织内胰岛素含量接近正常大鼠（P>0.05），而PDX-1的含量高于正常大鼠(P<0.05)。结论：胎鼠胰腺干细胞原位移植后可在体内转分化为胰岛样细胞团且具有良好的功能，移植后可使血糖降至正常，胰腺干细胞原位移植可能为1型糖尿病的治疗提供了一个新的策略。     

微管相关蛋白2、巢蛋白与人胚胎食管肌层发育的关系

目的: 探讨微管相关蛋白2(MAP-2)和巢蛋白(nestin)与人胚胎食管肌层发育关系。方法: 应用免疫组织化学PV法检测第2、3、4月龄段人胚胎食管组织内MAP-2和nestin的表达分布规律。结果: 第2、3、4月龄段,MAP-2在食管肌层的肌细胞呈阴性表达,肌间神经丛内神经细胞处呈阳性表达;nestin在第2-3月龄食管肌层的肌细胞处呈阳性表达,到第4月龄转阴性表达;而在食管肌间神经丛内大部分神经细胞,第2-4月龄段呈弱阳性或阴性表达。结论: MAP-2和nestin可能参与调节人胚胎食管肌层的形成过程。     

Expression of tubulin beta II in neural stem/progenitor cells and radial fibers during human fetal brain development

Recent studies revealed that the "radial glia" in fetal rodent brains are dividing neuronal precursor cells. However, in fetal primate brains, this issue remains unclear, with previous reports indicating that radial glia are a specialized form of astroglia. To investigate the relationship between radial fibers (RFs) and neural stem/progenitor cells in the fetal human brain, we generated polyclonal antibodies to human nestin protein and developed a new mAb, KNY-379, by screening for antibodies that immunostained RFs on paraffin-embedded human fetal brain specimens (12 gestational weeks). The immunostaining for KNY-379 antigen and nestin was seen over the RFs in brains at 8 gestational weeks. Furthermore, KNY-379 antigen and nestin were also detected in human neural stem/progenitor cells in neurosphere cultures. At 12 to 15 gestational weeks, the KNY-379 immunostaining of RFs remained in the periventricular zone and the deep part of the intermediate zone, but it also appeared in outgrowing axons in the cortical plate, in the superficial portion of the intermediate zone, and in apical dendrites in the molecular layer. In the later stages of fetal development (18-40 gestational weeks), this antigen remained in the outgrowing axons and dendrites, but was no longer associated with RFs. Expression cloning and immunoblot analysis demonstrated the antigen to be tubulin beta II, which would thus be a good marker for studying RFs and neural stem/progenitor cells in the early developing human brain.     

Expression of Epithelial and Mesenchymal Differentiation Markers in the Early Human Gonadal Development

Expressions of cytokeratin 8 (CK8), vimentin, nestin, and alpha‐smooth‐muscle‐actin (alpha‐SMA) were analyzed in the developing gonads of 12, 5–9 week old (W) human conceptuses by immunohistochemistry and immunofluorescence. During the investigated period, the number of CK8 positive cells increased from 56% to 92% in the gonadal surface epithelium, from 50% to 60% in the stroma, and from 23% to 42% in the medulla. In the early fetal period, the cell expression of CK8 increased in all gonadal parts, whereas primordial germ cells (PGC) remained negative. The expression of vimentin increased in the gonad stroma (gs) from 73% to 88%, and in the surface epithelium from 18% to 97% until ninth W. The medulla had the highest expression of vimentin in the seventh to eighth W (93%). Vimentin and CK8 colocalized in the somatic cells, while some PGCs showed vimentin expression only. Initially, nestin was positive in the gonad surface epithelium (8%) and stroma (52%), however during further development it decreased to 1% and 33%, respectively. In the early fetal period, the nestin positive cells decreased from 44% to 31% in the gonad medulla. Alpha‐SMA was positive only in the blood vessels and mesonephros. The described pattern of expression of intermediate filaments (IF) in developing human gonads suggests their role in the control of PGC apoptosis, early differentiation of gs cells and cell migration. Both epithelial and mesenchymal origins of follicular cells and possible epithelial‐to‐mesenchymal transition of somatic cells is proposed. Lastly, IF intensity expression varies depending on the cell type and developmental period analyzed. Anat Rec, 300:1315–1326, 2017. © 2017 Wiley Periodicals, Inc.     

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases during normal human pulmonary development

AIMS : Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are thought to be involved in lung development because they play an important role in the turnover of the extracellular matrix. Although limited data on MMP and TIMP expression are available from animal studies during prenatal pulmonary development, little is known about their expression during human fetal lung development. The aim of this study was to investigate the expression of MMP-1, -2, -9, TIMP-1, -2 and -3 in human fetal lungs from 9 to 42 weeks of gestation. METHODS AND RESULTS : Forty-five normal human fetal lung samples were analysed by immunohistochemistry. MMP-1, -9, TIMP-1, -2 and -3, but not MMP-2, were expressed in the epithelium at all gestational ages. The endothelium of all vessels and the arterial smooth muscle cells expressed MMP-1, -2, -9, TIMP-2 and -3, but not TIMP-1, at all developmental stages. CONCLUSION : The extensive distribution of MMPs and TIMPs throughout all stages of human lung development suggests that they play a significant role in the remodelling that occurs in the interstitium and epithelial basement membrane during lung development and in pulmonary vascular development. These data will serve as a base line for comparison with neonatal lung pathology, including pulmonary hypertension.     

Nestin在高温致畸神经管的表达及多种维生素和微量元素对神经管畸形的保护作用

本研究旨在探讨高温致神经管畸形(NTD)中巢蛋白(nestin)的表达规律及多种维生素和微量元素对NTD的保护作用。孕鼠随机分为实验组和对照组。实验组又进一步分为3组,A组:孕前第8d开始灌服多种维生素和微量元素;B组:孕后第1d开始灌服;C组:不灌服。孕鼠分别于孕第8d高温处理,对照组不灌服多种维生素和微量元素,也不进行高温处理。对以上各组胎鼠进行形态学观察,计算神经管畸形发生率;利用免疫荧光染色观察nestin在胚胎不同发育时期的神经上皮细胞中的表达规律。结果显示,在实验组中,C组神经管畸形发生率最高,为77.5%;各组鼠胚神经上皮细胞胞质中nestin呈阳性染色,实验C组的染色明显弱于其它组;不同发育阶段各组染色强度不同。结果提示,nestin低表达是神经管畸形发生的重要因素,多种维生素和微量元素对高温致神经管畸形有保护作用。