人多潜能胚胎生殖细胞的分离和培养

多潜能胚胎性干细胞来源有两条途经,从植入前的早期胚胎内细胞团(inner cell mass,ICM)分离出来的称胚胎干细胞(embryonic stem cells,ES);从原始生殖细胞(primordial germ cells,PGCs)分离得到的称胚胎生殖细胞(embryonic germ cells,EG)。这两种干细胞在小鼠嵌合体实验中,都证明具有参与生殖系传递的能力。这类干细胞在体外保持     

THE LOCALIZATION OF ALBUMIN AND FIBRINOGEN IN HUMAN LIVER CELLS

Human liver sections were stained with anti-human serum albumin and/or anti-human fibrin monomer fluorescent conjugates. Approximately 10 per cent of the hepatic cells stained specifically for human serum albumin,1 per cent for fibrinogen, and 0.1 per cent for both. Approximately 18 per cent of the Kupffer cells stained specifically for human serum albumin and 33 per cent for fibrinogen. Staining of both cell types was mainly cytoplasmic, although albumin was found in the nuclei of some parenchymal cells, depending on the method of fixation. Cytoplasmic granules staining specifically for fibrinogen could be seen just inside the cell membrane facing the bile caniculi in many more parenchymal cells than the 1 per cent showing diffuse cytoplasmic staining. The technical difficulties involved in preparing fluorescent conjugates against these antigens and in the fixation of these antigens for immunofluorescent staining are discussed.     

正常人肝脏星形细胞的分离,培养及鉴定

参考Friedman等分离人肝脏星形细胞(HSC)的方法,采用校正密度的淋巴细胞分离液进行一步梯度离心,成功地分离得到了正常人HSC。HSC的收获量约为1×10~7个/10克肝脏组织,存活率在95％以上,纯度达85％以上。传代后纯度接近100％。对人HSC的标志物进行检测发现,结蛋白不宜作为鉴定初分离的及原代培养初期人HSC纯度的指标,而α-平滑肌肌动蛋白可作为鉴定激活的人HSC的可靠指标。     

PARENCHYMAL CELLS FROM ADULT RAT LIVER IN NONPROLIFERATING MONOLAYER CULTURE : II. Ultrastructural Studies

Hepatic parenchymal cells from adult rats, established in vitro as a monolayer, have been evaluated by electron microscopy. Within 24 h after the initial seeding, the incubated cells were polygonal and in close apposition with three to six neighboring cells. The ultrastructure of the monolayer cells was examined at this time and after 3 and 10 days of incubation. With the exception of a few enlarged mitochondria, organelles in both the 1- and 3-day monolayer cells were indistinguishable quantitatively and morphologically from those found in the intact liver. After 10 days of incubation, however, the rough-surfaced endoplasmic reticulum (RER) had become dilated and vesiculated. In all cells studied, portions of RER were found in a close spatial relationship to mitochondria. From its frequency, this association appeared to be more than fortuitous, and the organelle complex may represent a functional unit necessary for new membrane formation, as suggested previously. The Golgi complexes of 1- and 3-day cells contained very low density lipoprotein-sized particles, which suggests that the monolayer cells synthesize lipoproteins. These electron microscope observations demonstrate that adult hepatic parenchymal cells in monolayer retain for several days the subcellular structural elements characteristic of normally functioning hepatocytes.     

PARENCHYMAL CELLS FROM ADULT RAT LIVER IN NONPROLIFERATING MONOLAYER CULTURE : I. Functional Studies

Parenchymal cells from adult rat liver have been established in primary monolayer culture. Donor animals are subjected to a partial hepatectomy and, 4 days later, cells are prepared by collagenase perfusion of the regenerated liver. The hepatic parenchymal cells, separated from nonparenchymal material and suspended in serum-free medium, are placed in plastic tissue culture dishes, where they form a monolayer within 24 h. The monolayer cells exhibit minimal mitotic activity and demonstrate several major metabolic functions characteristic of liver in vivo; these include albumin synthesis and secretion, gluconeogenesis from 3-carbon precursors, responsiveness to insulin and glucagon, glycogen synthesis, and activity of two microsomal enzymes. These functions are present in the monolayer cells for several days at activities similar to those observed in the liver in vivo. The findings indicate that hepatic parenchymal cells in this monolayer system are viable and behave in many respects like normal adult rat liver.     

汉坦病毒在原代人胚肾与人胚肺细胞中增殖及其特性的研究

近年来研究发现,汉坦病毒能在多种细胞株及人体细胞中增殖、适应.国内学者用人胚肺二倍体细胞(2BS)体外适应该病毒成功,但有关原代人胚肺、肾细胞的报道尚少.作者选用两株病毒及两种原代人胚细胞用于体外感染、观察,应用免疫荧光间接法及胶体金包埋前染色电镜技术对宿主细胞中增殖的病毒进行了动态观察及特异性定位研究,现报告如下.1 材料和方法1.1 细胞的分离和培养1.1.1 人胚细胞:取正常孕妇5—7个月水囊引产胚肾及肺组织,按常规方法分散细胞,5—7天后长满单层.1.1.2 非洲绿猴肾上皮细胞(VeroE6):由安徽省医学科学研究所倪大石惠赠.1.2     

人胚肝发育过程中染色质组份的变化

从14-,17-,21-,27-,34-,及38-周令的人胚肝细胞核分离出柒色质,分别对其中的RNA、DNA、组蛋白(HP)及非组蛋白(NHP)进行测定。在胚胎发育过程中肝柒色质HP/DNA比值变化不大。但是,NHP/DNA与NHP/HP比值发生显著改变。人胚肝NHP量的变化一直保持在整个胚胎发育过程中,NHP量的高峰位于21-及34-周。用SDS-聚丙烯酰胺凝胶板电泳分析处于不同发育阶段的人胚肝总染色质蛋白。电泳图谱显示出染色质NHP组分在质与量上有所改变。     

人胚胎干细胞分化为神经干细胞诱导方法的对比研究

目的探讨人胚胎干细胞分化为神经干细胞过程中,经拟胚体（embryonic body,EB）法和直接分化法的不同效率。方法人胚胎干细胞常规培养消化后,分为两组：A组,经EB法分化;B组,添加noggin和ITSFn直接分化法。倒置相差显微镜观察细胞形态变化,RT-PCR检测细胞各阶段标志物,免疫荧光及流式细胞仪观察两组细胞Nestin阳性细胞率。神经干细胞继续分化,免疫荧光、RT-PCR法检测MAP2、GFAP表达。结果RT-PCR检测到OCT4、nestin表达。B组nestin阳性细胞率明显高于A组,差异有统计学意义（P〈0.01）,且诱导周期短于A组。神经干细胞继续分化,得到不同数量的神经元和胶质细胞,MAP2、GFAP分别阳性。结论在体外采用定向分化诱导,人胚胎干细胞不经EB,可直接定向分化为神经干细胞,且诱导效率比EB法高。因此直接分化法是一种经济实用的诱导方法。     

人肝癌细胞株中EGFR的表达和EGF对肝癌细胞生长的促进

本文分析了人肝癌细胞株7404,7721细胞中表皮生长因子受体(EGFR)基因表达和EGF对肝癌细胞生长的促进作用。~(125)Ⅰ-EGF对7404细胞的结合试验表明结合是可饱和的和专一的,从~(125)Ⅰ-EGF对7404、7721细胞结合浓度曲线作Scatchard作图和计算,提示每个7404和7721细胞表面分别有1.1×10~5和0.7×10~5的EGFR分子。Northern杂交分析证明EGFR基因在7404,7721细胞中的转录产物主要是5.6 kb EGFR mRNA,免疫印迹分析证明7404细胞和7721细胞的EGFR为170kd的蛋白。EGF对培养于含10%或0.5%小牛血清的RPMI-1640培液中的7404、7721细胞的贴壁依赖性生长有促进作用,促进作用的程度与培液中CS含量有一定关系,提示EGF的促生长作用可能是EGF与血清中其他成分协同作用的结果。EGF对培养于软琼脂中的7404,7721细胞的贴壁不依赖性生长也有明显促进作用。综合上述实验结果说明EGFR基因在人肝癌细胞中是活跃表达的,EGF可能是肝癌细胞生长依赖的一个重要有丝分裂原。     

CHANGES IN GLUCOSE OXIDATION DURING GROWTH OF EMBRYONIC HEART CELLS IN CULTURE

A rapid and convenient method has been utilized to investigate glucose oxidation during growth of chick embryo heart cells in tissue culture. Primary isolates of chick embryo heart cells showed exponential growth when plated at low densities and exhibited density-inhibited growth as cultures became confluent. The density-dependent growth inhibition of chick embryo heart cells is associated with a marked decrease in the specific activity of glucose oxidation to CO₂. This decrease in glucose oxidation was observed as density increased as either a function of time in culture or as related to initial plating density. The decrease in ¹⁴CO₂ production associated with density-dependent inhibition of growth is due to a marked decrease in activity of the pentose phosphate pathway.     

间隙连接蛋白Cx43在人胚肺和肺癌细胞表达的研究

细胞与细胞之间通过细胞膜上的间隙连接通道交换小分子和离子进行细胞间通讯,对细胞增殖分化调控和机体内环境稳定有重要作用。用间隙连接蛋白Ｃｘ４３ｃＤＮＡ探针Ｎｏｒｔｈｅｒｎ印迹杂交,Ｃｘ４３抗体免疫荧光染色和罗氏黄荧光染料传输方法检查,正常人胚肺细胞的Ｃｘ４３在ｍＲＮＡ和蛋白水平有高表达,Ｃｘ４３蛋白免疫荧光分布在间隙连接的部位,细胞间隙连接通讯功能明显。与正常相反,人肺癌ＰＧ系细胞Ｃｋ４３无论在ｍＲＮＡ或蛋白质水平都无表达,细胞通讯功能缺陷。结果表明Ｃｘ４３在培养的人胚肺细胞有功能性表达。人肺癌ＰＧ细胞通讯功能缺陷与Ｃｘ４３基因转录抑制有关。对Ｃｘ基因的抑癌基因性质进行讨论。     

人胚与鼠胚神经干细胞体外培养的差异

为比较人胚与鼠胚神经干细胞体外培养的差异。实验采用具有丝裂原作用的细胞生长因子。结合无血清细胞培养技术从人胚和鼠胚皮层分离神经干细胞。在连续传代过程中观察其体外培养特性,免疫荧光染色检测Nestin抗原和分化后特异性成熟神经细胞抗原的表达,并用流式细胞仪检测神经干细胞分化情况。结果表明：（1）使用单一生长因子即可从鼠胚皮层分离神经干细胞,但在人胚却需同时使用多种生长因子,协同使用bFGF,EGF和LIF是人胚神经干细胞体外培养的较佳条件;（2）鼠胚皮层神经干细胞在连续传代过程中增殖速度快于人胚,其Nestin阳性率和BrdU标记的阳性率亦高于人胚,表明其增殖能力明显高于人胚,（3）人胚神经干细胞较鼠胚更易分化为神经元。     

人肾小球系膜细胞葡萄糖转运蛋白的研究

本文采用了RT-PCR,细胞免疫荧光染色及流式细胞仪分析技术证实人肾小球系膜细胞有GLUT1(glucose transportorl)mRNA和蛋白质的表达。用2-Deoxy[~3H]-D-Glucose摄入法与根皮素抑制实验肯定了系膜细胞上GLUT1的功能及其在系膜细胞葡萄糖摄入中的作用。本项研究为进一步研究系膜细胞GLUT1在糖尿病肾病发病机理中的作用奠定了基础。     

人胚不同脑区神经前体细胞的分离培养及分化特性的比较

目的研究人胚不同脑区神经前体细胞(neural progenitor cells,NPCs)培养及增殖分化特性。方法取14-17周人胚脑区组织,分为新皮质、纹状体、间脑、中脑、后脑和延髓组,悬浮培养。鉴定细胞球巢蛋白抗原的表达,分化及自我更新能力。观察各脑区培养细胞的生长、增殖状况。新皮质、纹状体及间脑来源的神经球分化后,运用免疫荧光细胞化学法比较神经元及星形胶质细胞的比例。结果各脑区培养出的悬浮细胞球巢蛋白抗原阳性,可分化为MAP2或GFAP阳性细胞,且BrdU掺入实验阳性。体外培养第3d,纹状体及间脑组均可见大量神经球,且纹状体组明显多于间脑组;新皮质组传代后可见较多神经球;其它组仅见个别神经球。新皮质、纹状体、间脑来源的NPCs诱导分化后,MAP2或GFAP阳性细胞率各组间比较差异无显著性。结论人胚不同脑区均可培养出NPCs,从易到难依次为纹状体、间脑、新皮质及其它脑区。新皮质、纹状体、间脑来源的NPCs体外分化比例一致。     

人和大鼠胃窦部神经内分泌细胞分布和形态学的比较研究

目的探讨人和大鼠胃窦部神经内分泌细胞的分布和形态学特征。方法采用免疫细胞化学方法,检测人和大鼠胃窦部粘膜内生长抑素细胞(D细胞)、胃泌素细胞(G细胞)、5-羟色胺细胞(5-HT细胞)、嗜铬粒素A细胞(CgA细胞)的分布。结果人和大鼠的G、D细胞的特征是都具有细胞突起,但是在细胞密度及其分布上是不同的;5-HT细胞的分布在两组稍有不同,在大鼠胃窦部,大多数5-HT细胞位于腺体基部,而人的5-HT细胞主要在间质,少数位于腺上皮内;在两组中,CgA细胞几乎布满整个胃粘膜,其数量也高于其他神经内分泌细胞,CgA细胞形态多样,胞质内充满细小颗粒。结论1)人与大鼠的G、D细胞通常都伴有突起,但其分布不同。2)5-HT细胞形态多样,分布于间质和腺上皮内。3)CgA细胞特征是胞质内充满细小颗粒,细胞形态多样,几乎布满整个粘膜。     

人胸腺上皮细胞培养及其分泌产物

本文通过降低培养基中血清含量,向RPMI 1640培养基中补加三碘甲腺原氨酸而获得一种人胸腺网状上皮细胞占优势生长的培养物。在此培养基中细胞经传代培养长达90天,仍维持正常形态特征。胸腺组织在培养14天后,新生细胞的突起形成网状结构,细胞化学检查和电镜观察表明具有丰富的分泌颗粒,囊泡及张力原纤维束和桥粒等上皮细胞特征。收集合并细胞培养液,经部分纯化后检查其生物活性,表现出具有促进玫瑰花结形成和降低胸腺细胞TdT活性的作用,说明培养细胞的分泌产物具有胸腺激素活性。根据形态学,细胞化学和生物活性检测结果,我们倾向于认为该培养物主要为网状上皮细胞。