爪蟾肌细胞的决定

爪蟾肌细胞在什么时期决定尚未有过报道,本文采用体外培养的方法,取原肠晚期到神经褶中期等五个不同时期检测肌细胞的决定。结果表明,这段期间预定肌节在离体培养条件下分化程度有明显差异,直到神经胚中期几乎所有组织块都分化为肌细胞。因此认为爪蟾肌细胞的决定在神经胚中期达到稳定的状态。     

Effects of low-intensity AC and/or DC electromagnetic fields on cell attachment and induction of apoptosis

Rat tendon fibroblast (RTF) and rat bone marrow (RBM) osteoprogenitor cells were cultured and exposed to AC and/or DC magnetic fields in a triaxial Helmholtz coil in an incubator for up to 13 days. The AC fields were at 60 and 1000 Hz and up to 0.25 mT peak to peak, and the DC fields were up to 0.25 mT. At various combinations of field strengths and frequencies, AC and/or DC fields resulted in extensive detachment of preattached cells and prevented the normal attachment of cells not previously attached to substrates. In addition, the fields resulted in altered cell morphologies. When RTF and RBM cells were removed from the fields after several days of exposure, they partially reattached and assumed more normal morphologies. An additional set of experiments described in the Appendix corroborates these findings and also shows that low-frequency EMF also initiates apoptosis, i.e., programmed cell death, at the onset of cell detachment. Taken together, these results suggest that the electromagnetic fields result in significant alterations in cell metabolism and cytoskeleton structure. Further work is required to determine the relative effect of the electric and magnetic fields on these phenomena. The research has implications for understanding the role of fields in affecting bone healing in fracture nonunions, in cell detachment in cancer metastasis, and in the effect of EMF on organisms generally. Bioelectromagnetics 18:264–272, 1997. © Wiley-Liss, Inc.     

FL对脐血造血细胞长期液体培养的影响

用脐血进行干细胞移植有许多优点,但有一个主要的缺点是可获得的细胞数量有限,因此脐血干细胞的体外扩增对于其临床应用具有重要意义。考察了Ｆｌｔ－３配体（ＦＬ）和干细胞因子（ＳＣＦ）,白介素３（ＩＬ－３）,ＩＬ－６,粒细胞集落刺激因子（Ｇ－ＣＳＦ）,粒细胞巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）的组合对脐血细胞扩增和分化的影响,培养４２ｄ,总细胞最多扩增了３８５．３０±１６３．５１倍（ＦＬ＋ＳＣＦ＋Ｇ－ＣＳ     

脐带血造血干/祖细胞体外分化为不同阶段红系祖细胞的动力学观察

目的:探讨体外培养脐带血单个核细胞定向诱导分化为不同阶段红系祖细胞的动力学变化情况。方法:用0.5%甲基纤维素沉降脐带血红细胞及人淋巴细胞分离液密度梯度离心法得到单个核细胞,在含EPO、SCF、IGF-1等细胞因子的无血清培养体系中诱导其定向分化为红系祖细胞,观察细胞增殖、存活率、细胞集落形成情况,并检测不同阶段细胞红系特异性表面标志CD71和CD235a的表达。结果:随着培养时间的延长,细胞数逐渐增多,14 d细胞可扩增140倍左右,收集诱导后的细胞进行瑞氏吉姆萨染色,可见大量红系祖细胞,诱导后的细胞集落形成能力强,形成的克隆大部分为红系集落。诱导过程中,14 d前CD71、CD235a的表达逐渐增高。按细胞表面标志表达的不同可将诱导的细胞分为4群,分别对应红系祖细胞的不同阶段;随着诱导天数的增加,各时间点细胞对应的早期红系祖细胞群(P2、P3)比例逐渐下降,中晚期红系祖细胞群(P4、P5)的比例逐渐上升。结论:无血清培养基添加细胞因子组合的红系诱导培养体系可较好地诱导扩增红系祖细胞,流式分选可获得相对均一而处于不同分化阶段的红系祖细胞群体。获得了红系祖细胞体外分化的动力学数据,为今后进一步优化红系诱导分化体系获得均一的红系祖细胞奠定了基础,并对未来利用干细胞制备均一的红系祖细胞应用于临床治疗有一定的指导作用。     

Initiation of Cartilage Cell Culture from Skate (<Emphasis Type="Italic">Raja porasa</Emphasis> Günther)

Abstract Cartilage tissues from the proboscis of skate (Raja porasa Günther) were used to initiate primary cultures of cartilage cells. Aseptically dissected cartilage tissues were immersed in MEM medium free of fetal bovine serum (FBS), pH 7.6, and minced into small pieces (1 mm³ on average). After hydrolysis with collagenase II, hyaluronidase, and trypsin for 2 hours at room temperature, the acquired cartilage cells were rinsed twice with 20% FBS-supplemented MEM medium and then inoculated into 25-cm³ cell culture flasks, and incubated at 24°C. The primary cultures were initiated successfully, and the cartilage cells grew gradually into a confluent monolayer at day 10. Effects of growth factors were also tested in this study, and it was found that 20 ng/ml of basic fibroblast growth factor and 100 ng/ml of insulin-like growth factor II together had the most prominent stimulating effect on the growth and division of cartilage cells in the series of concentration combinations employed. The induced cartilage cells cultured formed a confluent monolayer at day 7.     

牛睾丸支持细胞分离、纯化及体外生长行为的研究

了解牛睾丸支持细胞体外培养的生物学特性,试验采用组合酶消化和选择贴壁法,将5月龄、6月龄牛胎儿及新生牛睾丸支持细胞分离纯化后进行体外培养。试验结果显示,这一阶段牛睾丸适宜支持细胞分离纯化用;采用0.25 %胰蛋白酶+0.02 %EDTA二次消化法是一种经济有效的牛胎儿睾丸支持细胞分离方案;试验发现,牛胎儿睾丸支持细胞体外研究时间应控制在3天～20天内,处于对数生长期的牛胎儿睾丸支持细胞,对牛体外受精卵体外发育有明显的促进作用。     

Calcium regulation of normal human mammary epithelial cell growth in culture

Summary The concentration of Ca⁺⁺ in culture media profoundly affected the growth and differentiation properties of normal human mammary epithelial cells in short-term culture. In media where Ca⁺⁺ was above 0.06 mM, longevity was limited to an average of three to four cell divisions. The extended growth fraction (those cells able, to divide more than once) was only approximately 50% and diminished to zero quickly with time. Stationary cells inhibited from dividing appeared differentiated in the formation of lipid vacuoles and accumulation of α-lactalbumin. Growth of stationary cultures could be reinstituted in about half the cells, either by disruption and transfer or by a reduction in Ca⁺⁺ to less than 0.08 mM. The reduction of Ca⁺⁺ to levels below 0.08 mM extended the longevity of normal cells to eight to nine divisions. The extended growth fraction was 100%. Under these conditions, cells did not differentiate. The effects of Ca⁺⁺ on growth and differentiation were specific (Mg⁺⁺ and Mn⁺⁺ variations were without effect) and reversible and in many respects resembled Ca⁺⁺ effects on epidermal cells. One major difference is that the dual pathways of growth and differentiation in mammary cells were controlled by glucocorticoid and insulin. Based on the kinetics of the reversible Ca⁺⁺-induced coupling and uncoupling of proliferation and the program of differentiation, we propose that Ca⁺⁺ may be an essential trigger for cell divisions that commit a mammary cell to differentiate progressively in a permissive hormonal milieu. This study was supported by grants NIH-CA18175 and CA36399 and an institutional grant from the United Foundation of Greater Detroit.     

小鼠骨髓源树突状细胞体外诱导培养及初步鉴定

用重组小鼠粒细胞-巨噬细胞集落刺激因子（rmGM-CSF）和重组小鼠白细胞介素4（rmIL-4）体外诱导小鼠骨髓细胞分化为树突状细胞,进行形态学变化观察,分析细胞表面分子,刺激T细胞增殖,探讨小鼠骨髓源树突状细胞（BMDC）体外诱导培养并进行初步鉴定。体外培养9d后BMDC可达80%以上,光镜下可见典型的树突状细胞形态。清楚表达成熟期主要表面标志物,可显著刺激同种异体混合淋巴细胞增殖。获得了较高纯度的BMDC,避免了使用传统磁珠分离方法所带来的成本高,操作复杂,产出率低的弊端,为研究BMDC功能以及运用开展下游实验提供材料。     

人隐静脉血管内皮细胞的分离、培养及鉴定

利用冠脉搭桥术后遗弃的隐静脉段获取内皮细胞,采用消化酶消化收集内皮细胞,扩增、冻存、复苏,在体外建立内皮细胞系。此方法简便易行,能在体外获得大量生物学特性保持良好的内皮细胞,为临床血管内皮化研究提供新的细胞来源。     

体外培养的牛耳成纤维细胞的细胞周期研究

通过细胞体外培养,对牛耳成纤维细胞的周期分布进行了研究。不同代数的细胞在生长至70-85％汇合和血清饥饿72h两种状态的细胞周期分布无显著差异。对于体外培养的第8代细胞,生长至50-60％、70-85％和完全汇合时,细胞周期分布存在显著差异;而培养时间对血清饥饿培养和正常培养至充分汇合时的细胞周期分布无显著影响。结果表明,体外培养代数及培养方法不会明显影响细胞周期的分布,但同代细胞因处于不同生长阶段细胞周期分布会存在显著差异。     

Novel cell culture device enabling three-dimensional cell growth and improved cell function

A better understanding of cell biology and cell-cell interactions requires three-dimensional (3-D) culture systems that more closely represent the natural structure and function of tissues in vivo. Here, we present a novel device that provides an environment for routine 3-D cell growth in vitro. We have developed a thin membrane of polystyrene scaffold with a well defined and uniform porous architecture and have adapted this material for cell culture applications. We have exemplified the application of this technology by growing HepG2 liver cells on 2- and 3-D substrates. The performance of HepG2 cells grown on scaffolds was significantly enhanced compared to functional activity of cells grown on 2-D plastic. The incorporation of thin membranes of porous polystyrene to create a novel device has been successfully demonstrated as a new 3-D cell growth technology for routine use in cell culture.