分离MEF和ES细胞的差速贴壁法研究(简报)

胚胎干细胞的分化控制是胚胎干细胞研究的一个重要方面。由于常规的拟胚体诱导途径是在形成拟胚体后才开始进行诱导分化,受多种胚层的共同作用使得我们无法简便探索诱导分化的机制。而且用这种方法进行的诱导分化试验的结果检测比     

维甲酸和EGF对大鼠脑胚胎神经干细胞增殖和分化的影响

目的 观察全反式维甲酸(RA)和表皮生长因子(EGF)对大鼠胚胎神经干细胞增殖和分化的影响。方法 从大鼠胚胎脑中分离神经干细胞,经RA和EGF处理后,用台盼蓝确定细胞数量,BrdU标记分析细胞生长能力,采用免疫细胞化学法鉴定神经干细胞和分化的神经细胞。结果 20ng／ml EGF和1μmol／LRA处理的培养细胞均显示增殖效应,但EGF处理组增殖速度明显高于RA组,悬浮细胞中有大量nestin和BrdU阳性细胞。用EGF和EGE／RA诱导的神经元分化率分别为17％和31％,而RA处理的神经元分化率显升高至89％。由EGF、EGF／RA和RA诱导的星形胶质细胞分化率分别为83％、69％和11％。结论 EGF主要促进神经干细胞增殖并主要诱导星形胶质细胞的生成,RA主要诱导神经干细胞向神经元分化,二无明显协同效应。     

DIFFERENTIATION AND PROLIFERATION OF EMBRYONIC MAST CELLS OF THE RAT

Histochemical reactions and radioautography were used to investigate the sequence of mast cell development in rat embryos. Mast cells arise ubiquitously in and are confined to the loose connective tissue in the embryo. The alcian blue-safranin reaction distinguishes between weakly sulfated and strongly sulfated mucopolysaccharides by a shift from alcian blue to safranin staining. Based on this reaction and morphologic characteristics, four stages were identified. Stage I mast cells are lymphocyte-like cells with cytoplasmic granules which invariably stain blue with the alcian blue-safranin reaction. In Stage II cells the majority of granules are alcian blue-positive, but some safranin-positive granules have appeared. Stage III mast cells are distinguished by a majority of safranin-positive cytoplasmic granules; some alcian blue-positive granules still remain. Stage IV cells contain only safranin-positive granules. Thymidine-H³ uptake and identification of mitotic figures indicates that mast cells in Stages I and II comprise a mitotic pool while those in Stages III and IV are mitotically inactive. The pattern of S³⁵O₄ incorporation and the sequence of appearance of histochemically identifiable mast cell constituents corroborates division of the proliferation and differentiation of embryonic mast cells into the four stages described above. The process of formation of mast cell granules is interpreted as reflecting the synthesis and accumulation of a heparin precursor in alcian blue positive granules followed by the synthesis and accumulation of highly N-sulfated heparin along with mast cell chymase and finally histamine in safranin-positive granules.     

小鼠胚胎干细胞分化为血管内皮细胞的永生化研究

本文探讨了小鼠胚胎干细胞(ES细胞)诱导分化的血管内皮细胞永生化。在体外培养系统中,以维甲酸(RA)和转化生长因子-β1(TGF-β1)诱导小鼠胚胎干细胞(ES细胞)的拟胚体(EB)分化为“圆形细胞”和由这些“圆形细胞”组成的血管样结构。经光学和扫描电镜及免疫荧光等法分析检测,证明组成血管样结构的细胞具有专一性vWF荧光染色,表明是血管内皮样细胞。利用脂质体将人端粒酶催化亚基逆转录酶(hTERT)基因转染诱导分化中的“圆形细胞”。应用Dot-blot,RT-PCR,Western blot及免疫组织化学等方法分析、观察和证明了诱导分化的组成血管样结构的园形细胞和被hTERT基因转染的“圆形”细胞的形态和生物学特性。结果表明,携带hTERT基因的从ES细胞分化来的圆形细胞在体外可大量增殖,持续传代,95%具有血管内皮细胞的一些特有标志和管道化生长特性。因此,通过人端粒酶基因的转染途径可解决由ES细胞诱导分化而来的内皮细胞扩增和永生化问题,为构建组织工程化血管及其它人工血管的内皮化提供种子细胞来源打下基础。     

从大鼠肝染色质富集具有转录活性的DNA

采用DNase消化法从大鼠肝染色质分离得到富有转录活性的DNA(sDNA)。sDNA琼脂糖凝胶电泳,在0.5—6Kb范围内背景有一片荧光,小于1Kb范围内,出现明显区带。sDNA为探针与大鼠正常肝核RNA杂交百分数(29.5%),为以总核DNA为探针杂交百分数(8.2%)的3.6倍,并高于sDNA与大鼠肝癌核RNA杂交百分数(16.4%).     

CELL CONTACT REFORMATION OF DISSOCIATED CELLS FROM EMBRYONIC CELLS OF STRONGYLOCENTROTUS PURPURATUS

Disaggregated single cells from the gastrula of Strongylocentrotus purpuratus were studied as they reaggregated and reformed quasi-normal embryos. In this investigation emphasis was placed on the structural events involved during the reformation of cell contacts vis-a-vis cell migration. The early cell contacts are non-junctional cell appositions, which are characterized by non-parallel apposing membranes. Between post-migratory epithelial cells, there is a shift from non-parallel to parallel apposing membranes. These cell appositions are found between the overlapping lamellapodia along the apical margins of the epithelial cells during blastocoel enlargement. Incipient continuous junctions are formed by the deposition of an electron dense material in the intermembrane space. As the junction develops, electron dense plaques form in the cytoplasm immediately subjacent to the junction and septa form between the apposing membranes.     

HORMONE SECRETION BY CELLS DISSOCIATED FROM RAT ANTERIOR PITUITARIES

A new procedure has been developed for dissociating anterior pituitary tissue and producing a viable suspension of single cells. The procedure involves incubation of small tissue blocks in 1 mg/ml trypsin (15 min), followed by incubation in 8 µg/ml neuraminidase and 1 mM EDTA (15 min), followed by mechanical dispersion. Cell yields are ~55%, based on recovered DNA. By electron microscopy five types of secretory cells (somatotrophs, mammotrophs, thyrotrophs, gonadotrophs, and corticotrophs) plus endothelial and follicular cells can be identified and are morphologically well preserved up to 20 h after dissociation. Throughout this period, the cells incorporate linearly [³H]leucine into protein for up to 4 h at a rate 90% greater than hemipituitaries, and they synthesize, transport intracellularly, and release the two major pituitary secretory products, growth hormone and prolactin. Immediately after dissociation the cells' ability to respond to secretogogues (high K⁺ and dibutyryl cyclic AMP) is impaired, but after a 6–12-h culture period, the cells apparently recover and discharge 24% and 52%, respectively, of their content of prelabeled growth hormone over a 3-h period in response to these two secretogogues. This represents a stimulation of 109% and 470% over that released by cells incubated in control medium. The results demonstrate that function and morphologic integrity are preserved in this cell system. Therefore it is suitable for the study of various aspects of pituitary secretion and its control.     

ULTRASTRUCTURAL CHANGES IN EMBRYONIC ECTODERMAL CELLS OF THE NEURULATING RAT EMBRYO

Embryonic ectodermal cells of rat embryos were examined by light and electron microscopy during the early stage of neurulation. Before the onset of neurulation (day 9–6 hr embryos), the cells underwent certain characteristic ultrastructural changes; that is, apical cytoplasmic protrusions and free spherules appeared, numerous vacuoles were formed in the cytoplasm, mitochondria showed ballooning, and the endoplasmic reticulum became dilated. The amniotic cells derived from the embryonic ectoderm exhibited the same ultrastructural changes, but those from the extraembryonic mesoderm did not. Embryonic mesodermal cells and neuroectodermal cells also did not show these changes. In the middle stage of neurulation (day 9–12 hr embryos), the embryonic ectodermal cells and the amniotic cells derived from the embryonic ectoderm assumed a flat squamous shape. None of the ultrastructural changes observed in day 9–6 hr embryos were noted in these cells. The functional significance of the production of apical cytoplasmic protrusions and free spherules in the embryonic ectodermal cells and amniotic cells is discussed in relation to similar phenomena reported to occur in other cell types.     

用DNA过量核酸分子杂交法对大鼠肝和大鼠肝癌细胞核RNA的比较研究

用DNA过量核酸分子杂交法观察到,大鼠肝癌细胞中重复频率在10~4以上的Poly(A)-核RNA的频率和种类,均比大鼠正常肝细胞有所增加。大鼠肝癌细胞中重复频率在1—4×10~2的Poly(A)-核RNA和Poly(A)~+核RNA的重复频率,比大鼠正常肝细胞减少,而RNA种类则增加。大鼠肝癌细胞中,接近单拷贝的Poly(A)-核RNA和Poly(A)~+核RNA,其频率比大鼠正常肝细胞略有增加,而种类则减少。用总细胞核RNA得类似结果。     

人胚胎干细胞程序降温保存的实验研究

本文采用升降式程序降温仪对人胚胎于细胞进行了程序降温保存,并探讨和比较了降温速率、置核温度、保护剂和投入液氮前温度对冻存复苏后胚胎干细胞的存活率、活力及分化特性的影响。结果表明:采用Me_2SO 血清 DMEM(体积比为1∶3∶6)的保护剂,从0℃开始,以0.5℃/min的速率对细胞悬液降温;至-10℃时对其进行置核,并于-35℃时将其快速投入液氮中保存,复温后效果最佳。冻存复温后细胞存活率可达81.8%,复苏后的胚胎干细胞形态和集落生长方式都与冻前的生长形态相同,且胚胎干细胞标志之一碱性磷酸酶(AKP)反应阳性,同时染色体组型仍正常。     

INTRACELLULAR ACCUMULATION OF PROTOCOLLAGEN AND EXTRUSION OF COLLAGEN BY EMBRYONIC CARTILAGE CELLS

The synthesis of collagen can be interrupted, after the assembly of proline-rich and lysine-rich polypeptide chains called protocollagen, by incubating connective tissues anaerobically. Under these conditions the proline and lysine residues in protocollagen are not hydroxylated to hydroxyproline and hydroxylysine, and protocollagen molecules accumulate intracellularly. Chemical data and radioautographs at the level of the light and electron microscopes indicated that in tissues labeled with proline-3,4-³H under nitrogen, there appeared to be an accumulation of radioactivity over the ground cytoplasm. When the inhibition of protocollagen hydroxylase was reversed by exposing the tissue to oxygen, the accumulated protocollagen-³H was converted to collagen-³H and there was a rapid transfer of label from the ground cytoplasm to the extracellular matrix. There was no significant change in distribution of label over either the Golgi vacuoles or the cisternae of the endoplasmic reticulum. The failure to find a significant change in distribution of label over the Golgi vacuoles or the cisternae does not completely exclude the possibility that these two compartments are involved in the extrusion, but the data are consistent with the simpler notion that the completed collagen molecules pass directly from the ground cytoplasm to the extracellular matrix.     

小鼠胚胎神经干细胞的分离培养及其鉴定

且的探索小鼠胚胎神经干细胞的体外培养方法,并获取高纯度的神经干细胞,为神经干细胞的深入研究提供实验材料。方法无菌条件下分离E15天小鼠胚脑皮质,制成单细胞悬液,在bFGF和B27存在的培养基中培养扩增,通过免疫细胞化学染色鉴定神经干细胞及其子代细胞的分化方向。结果培养的部分细胞在B27和bFGF存在的无血清培养基中可以在体外分裂增殖,同时表达神经干细胞特异性抗原nestin,并在撤出B27和bFGF的有血清培养基中向神经细胞和胶质细胞分化。结论小鼠胚脑皮质存在具有多向分化潜能的神经干细胞,这些细胞可以在体外稳定培养、传代并自然分化,为细胞替代治疗提供了理想的细胞来源。     

DIRECT ISOLATION OF NATIVE THIN FILAMENTS FROM EMBRYONIC MUSCLE CELLS*

A method is presented for the release of “native” thin filaments from 13-day old embryonic chick muscle without tryptic digestion or desoxycholate (DOC) solubilization of Z bands. The isolated filaments were 50–60 Å in diameter, of variable length, and formed “arrowhead-like” complexes with heavy meromyosin (HMM). In addition, the filaments interacted with purified myosin to form actomyosin as effectively as action extracted from an acetone powder of muscle. The Mg⁺⁺-dependent ATPase activity and extent of superprecipitation of the synthetic actomyosin required a low concentration of Ca⁺⁺, strongly suggesting the presence of troponin and tropomyosin on the thin filaments isolated from muscle at this stage of embryogenesis. The native thin filaments were more sensitive to trypsin than synthetic F-actin prepared from an acetone powder based on measurements of flow birefrengence, viscosity and the ability to activate myosin ATPase.     

成年大鼠雪旺细胞的快速扩增

采用接种雪旺细胞的可降解导管修复外周神经损伤是一种有望替代自体神经移植的方法。如何在短期内利用病人少量的神经碎片获得大量雪旺细胞是该方法用于临床的关键。以大鼠坐骨神经为模型,利用雪旺细胞增殖的内在机制,探索出一种快速增殖成年雪旺细胞的方法。采用预变性7d的坐骨神经,用酶消化分离出雪旺细胞,接种在层粘连蛋白包被的培养瓶中,经过7d的培养,获得纯度为96％、细胞密度为600个／mm^2的雪旺细胞,雪旺细胞的纯度和密度明显高于对照的新鲜神经。未使用霍乱毒素、毛喉素等促有丝分裂剂和抑制成纤维细胞的基因毒素,符合临床使用要求。结果表明,可以利用少量的损伤神经碎片在短期内获得大量可用于临床的雪旺细胞。     

UNSTIMULATED SECRETION OF PROTEIN FROM RAT EXOCRINE PANCREAS CELLS

Our earlier work demonstrated that the rate of protein synthesis in the exocrine cells of the rat pancreas is constant in different physiological states, including prolonged fasting. In this study we have followed the fate of the protein in the pancreatic cells of the fasting animal in vivo as well as in vitro. The data were obtained by quantitative radioautography and by biochemical determinations. In nonanesthesized, fasting rats, without cannulated pancreatic duct, some 80% of the proteins synthesized at a given time leaves the cell within 12 hr by way of secretion, intracellular breakdown not being important. Two mechanisms of fasting secretion exist. The first, starting at a slow rate after 20 min, is inferred to result from fortuitous contacts of young secretory granules with the apical cell membrane. The rate of secretion is the same in vivo as in vitro, at least during the first 4 hr after pulse labeling. Within 7 hr about 20% of the total amount of newly synthesized protein has left the cell. The second mechanism consists of an orderly movement of the mass of secretory granules towards the apical cell membrane as caused by the continuous assembly of new granules. The granules that come into contact with the cell membrane are discharged. It takes about 7–12 hr for secretory protein transported in this way to reach the cell membrane. The addition of new secretory granules to those present is essential for the second mechanism, for the blockade of protein synthesis by cycloheximide decreases the rate of this phase of secretion without interfering with the secretory process proper. Atropin does not inhibit the fasting secretion in vitro, nor does extensive washing of the tissue slices, excluding possible secretagogues as important factors in fasting secretion.     

DEMONSTRATION AND PRELIMINARY CHARACTERIZATION OF REAGGREGATION-PROMOTING SUBSTANCES FROM EMBRYONIC SEA URCHIN CELLS

Morulae or early blastulae of sea urchins dissociate readily on treatment with isotonic urea containing EDTA. During dissociation a certain substance was extracted. This substance was found to accelerate reaggregation of dissociated cells. The manifestation of the reaggregation-accelerating activity of the substance requires rather complicated handling of divalent cations. On the basis of these findings, a working hypothesis on the mode of function of the substance is presented.
Some properties of the substance were investigated and were found to be very similar to those of hyalin, the calcium-insoluble protein of the hyaline layer surrounding the fertilized egg.     

KINETICS OF DEVELOPMENT OF EMBRYONIC ERYTHROID CELLS

Embryonic erythropoiesis is an intrinsically non-steady-state process. A method of non-steady-state analysis is employed to approximately determine the kinetics of maturation of embryonic erythroid cells during the hepatic phase of erythropoiesis in the mouse. It appears from this analysis that embryonic erythroid cells have significantly shorter maturation times than their adult counterparts. In the embryo, there is insufficient time for more than three divisions between the proerythroblast and the orthochromatic erythroblast.