体外培养骨髓巨核细胞脱氢酶活性的细胞化学观察

本文采用液体培养体系结合酶细胞化学方法,对体外培养不同发育阶段的小鼠肾髓巨核细胞乳酸脱氢酶、苹果酸脱氢酶、谷氨酸脱氢酶的活性变化进行了动态观察。在9天培养期间,巨核细胞的增殖数在5—7天达到高峰,并随时间有不同程度分化。对培养3、5、7、9天的巨核细胞进行酶细胞化学研究,结果表明,巨核细胞在发育成熟前,三种酶活性均有增高。提示巨核细胞在分化过程中,糖酵解及三羧酸循环代谢均有增强。     

原红细胞在体外EPO诱导下的增殖分化特征

目的 系统了解小鼠红细胞终末分化过程中细胞增殖和分化的动力学。方法选用FVA细胞模型,利用密度梯度离心的方法从感染Friend致贫血病毒(anemia-inducing strain of Ffiend virus,FVA)的小鼠脾脏中分离出同步的原始阶段红细胞(FVA细胞)。通过研究EPO诱导下细胞的生长曲线,^3H-TdR掺入率以及细胞分化过程中β-珠蛋白(β-globin)基因的表达和血红蛋白合成的时空关系,分析FVA细胞在体外EPO诱导分化过程中增殖及分化的特性。结果 FVA细胞在EN3诱导早期生长旺盛,诱导24小时细胞数量达高峰,为0时的4倍;^3H-TdR掺入在诱导后12小时达高峰;从诱导后12小时开始有β-globin基因的转录,在24小时达高峰,随后减低;从18小时开始出现血红蛋白,至48小时,几乎全部细胞都表达大量血红蛋白。结论 本研究结果阐明了FVA细胞在体外EPO诱导下增殖及分化的动力学,为利用FVA细胞模型研究红细胞终末分化的机理提供了有用的基础信息。     

小鼠胎肝和骨髓造血基质细胞贴壁层对红系祖细胞生长的影响

本实验以Dexter培养体系作小鼠胎肝和骨髓造血基质细胞贴壁培养。在所获的基质细胞贴壁层上作红系造血祖细胞集落培养,观察两种来源造血基质细胞对红系集落生长的影响。实验结果表明,胎肝造血基质细胞贴壁层能明显促进早期红系造血祖细胞(BFU-E)形成集落,却不明显影响晚期红系造血祖细胞(CFU-E)的生长。成年小鼠骨髓造血基质细胞贴壁层对BFU-E和CFU-E均有刺激生长的作用;但对前者生长的刺激性影响较胎肝造血基质细胞贴壁层为弱。造血基质细胞贴壁层对红系集落生长的促进作用主要是通过体液因子实现的,细胞间短距离调节的影响亦不能除外。     

狗外周血造血干细胞体外培养条件的探讨

近年来,利用骨髓细胞、胎肝细胞、外周血单个核细胞治疗造血障碍性疾病是引人注目的研究动向,也是治疗极重度放射病包括肠型放射病的可能有效措施。它们有效成分是造血干细胞。就造血干细胞的来源来看,在外周血中的造血干细胞数量虽少,但分离浓集比较方便,也容易为人们所接受。关于外周血干细胞的测试技术,对各种动物都已有报道,但对狗     

体外化学诱导人骨髓间充质干细胞分化为心肌样细胞

为了探讨人骨髓间充质干细胞(MSCs)的体外培养及化学诱导向心肌细胞分化的过程及条件,我们用1.073g/mL密度梯度离心法分离健康人骨髓单个核细胞,经骨髓间充质干细胞培养基传代培养后用流式细胞仪检测细胞表面抗原,在完全培养基中分别加入3、5、10μmol/L的5氮胞苷(每组n=5)进行化学诱导分化,阴性对照组采用完全培养基培养,诱导后21天细胞爬片免疫荧光法鉴定,透射电镜观察细胞超微结构。结果显示人MSCs为形态均一的梭形细胞,生长旺盛时呈旋涡样分布,流式细胞仪检测细胞表面CD44阳性,CD34、CD45阴性;5、10μmol/L的5氮胞苷进行化学诱导后细胞形态变长,诱导后14天时20%-30%细胞融合形成多核肌管样结构,3μmol/L组MSCs未出现肌管结构,诱导后21天5、10μmol/L组MSCs中desmin、心肌早期转录因子GATA4、心肌特异性cTnI及闰盘蛋白connexin43的表达阳性,10μmol/L组cTnI阳性染色细胞数目(65.3±4.7%)高于5μmol/L诱导组(48.2±5.4%)(p<0.05);3μmol/L组及阴性对照组无心肌特异性蛋白的表达。细胞诱导后28天透射电镜下可见肌丝形成。本实验说明,人MSCs在体外经化学诱导可分化为心肌样细胞,而且5-氮胞苷对于心肌相关蛋白的表达呈浓度依赖性正相关。     

血小板生成刺激因子对巨核血小板系统体外生成的影响

本 文应用血小板生成液体培养体系及纯化的血小板生成刺激因子(TSF)研究了 TSF对巨核细胞成熟及血小板生成的作用。TSF 在0.5—2U/ml 浓度范围内能够刺激巨核细胞DNA 合成,胞浆成熟,胞体直径增加以及血小板直径增加,但对巨核细胞与血小板计数没有影响。实验表明 TSF 作为一种血小板生成素,通过促进巨核细胞分化成熟,以增加血小板体积的方式,促进血板小生成。     

体外定向诱导大鼠骨髓基质干细胞向成骨细胞分化的实验研究

目的研究大鼠骨髓基质干细胞的生长特点和诱导条件下的成骨能力。方法通过密度梯度离心和贴壁培养法分离成年大鼠骨髓基质干细胞,应用含地塞米松、p甘油磷酸纳和维生素c的诱导分化培养液定向诱导传代细胞向成骨细胞分化并检测碱性磷酸酶活性和细胞矿化作用。结果原代培养基质干细胞首先形成细胞集落,14d时集落间接近融合;传代细胞体积变大,约5～7d传代一次。诱导条件下,细胞碱性磷酸酶活性明显增高,并出现了矿化结节。结论骨髓基质干细胞易于分离培养及体外扩增,成骨能力肯定,可作为骨组织工程的种子细胞。     

体外证据不支持5-氮胞苷诱导骨髓基质细胞向心肌细胞分化

目的探讨胞嘧啶类化学物质5-氮胞苷是否可诱导体外培养的骨髓基质细胞 (marrow stromal cells, MSCs)向心肌细胞分化.方法用不同浓度(3, 5, 10 μmol/L)的5-氮胞苷以各种方法(一次处理与反复处理)诱导原代及传代MSCs.采用免疫荧光及Western Blot技术检测心肌特异性肌球蛋白重链 (myosin heavy chain α/β, MHCα/β)及肌钙蛋白I(troponin I, Tn I)的表达;并用心室肌特异性肌球蛋白轻链(ventricular myosin light chain 2, MLC2v)启动子(250 bp)控制的增强型蓝绿色荧光蛋白(enhanced cyan fluorescent protein, ECFP)报告基因表达技术检测被诱导MSCs是否发生MLC2v的转录启动.上述检测技术的特异性和可靠性用培养的心肌细胞进行验证.结果在本试验最长观察时间内(30 d),各种浓度及各种方法5-氮胞苷诱导的MSCs培养物中未见细胞自发搏动、肌管形成、心肌特异性MHC和Tn I表达;亦无MLC2v转录启动阳性细胞出现.结论从转录启动到翻译后水平的证据均不支持体外5-氮胞苷处理可诱导MSCs表达心肌特异性蛋白.     

骨髓高增殖潜能集落形成细胞（HPP—CFC）分化产生巨核细胞的研究

本研究用正常和５－ＦＵ处理的小鼠骨髓细胞,作血浆凝块培养,对一种称作ＨＰＰ－ｍＣＦＵ－ＭＫ的ＨＰＰ－ＣＦＣ亚型细胞集落进行体外追踪。发现ＨＰＰ－ｍＣＦＵ－ＭＫ不但能形成符合ＨＰＰ－ＣＦＣ标准的巨大集落,而且能产生大量的巨核细胞。该巨大集落的生长,依赖添加再生障碍性贫血猪血清（ＡＡＳ）或合用三种以上的基因重组造血生长因子而增强,在体外不被ＴＧＦ－β１和ＰＦ４抑制。原代培养１２天所得到的ＨＰＰ－ｍＣＦ     

CONTROL OF IN VITRO SEPAL DEVELOPMENT OF EXCISED FLORAL BUDS OF AQUILEGIA (RANUNCULACEAE)

Excised floral buds of Aquilegia formosa Fisch. were grown on a coconut-milk medium containing the minerals and vitamins of Murashige and Skoog, sucrose, and kinetin. The plant growth regulators indoleacetic acid (IAA, 0.5 mg/liter) and gibberellic acid (GA, 2.0 mg/liter) were added singly and in combinations; both were omitted from the control medium. The addition of GA to the basal medium was required to support sepal development on flowers at all phases of development. The formation of stomatal complexes in the epidermis of the sepals occurred only in the presence of GA. Sepals grown in the presence of GA also contained trichoblasts and developing trichomes, while none were formed in the absence of GA. The role of IAA in the development of these idioblasts was not clear but it appeared to have no effect. The hormones GA and IAA had different effects on the growth of the sepals. In the presence of GA the sepals increase in length until comparable with sepals grown in vivo. However, the sepals remained small when GA was omitted from the medium. Upon closer examination of this effect, it was determined that there was a direct proportionality between an increase in cell number in the epidermis and an increase in sepal length. The role of the two hormones in increasing epidermal cell length in sepals was distinct and separate. Exogenous IAA had no effect upon cell division but was required for cell elongation, while GA was required for cell division and had no effect on cell elongation. The GA effect in promoting cell division in the sepals was substantiated by use of autoradiography. If the buds were grown on media with GA, twice as many epidermal cells along the central file incorporated significant amounts of tritiated thymidine. The cell cycle of the epidermal cells of the GA-treated sepals was ca. 8.7 hr in duration and ca. 13.0 hr if GA was deleted from the medium.     

INDUCTION OF WOUND-VESSEL DIFFERENTIATION IN ISOLATED COLEUS STEM SEGMENTS IN VITRO

Excised internodes and 2-mm-thick transverse stem segments of Coleus blumei were incubated 7 days on media containing 2% sucrose, 1% agar, and various growth substances. Wound-vessel members differentiated in the 2-mm-thick tissue slices incubated on medium containing no exogenous auxin (control). Compared to control slices, the addition to the medium of either IAA (50 or 5 ppm), 2, 4-D (10, 1, or 0.1 ppm), TIBA (50, 5, or 0.5 ppm), or kinetin (50, 5, 0.5 or 0.05 ppm) inhibited wound-vessel differentiation. Simultaneous treatment of tissue slices with IAA and kinetin inhibited wound-vessel differentiation, as did the incubation of tissue slices on medium containing no sucrose. Low concentrations of IAA (0.05 ppm) or 2, 4-D (0.01 ppm) resulted in over a 100% increase in the numbers of wound-vessel members differentiated. These results are interpreted as indicating auxin synthesis by the tissue slices and the participation of auxin as a limiting factor in xylogenesis. The inhibition of wound-vessel differentiation by relatively high concentrations of 2,4-D, TIBA, or kinetin is interpreted as a reflection of the inhibition of polar auxin transport by these substances, and an indication that polar auxin transport enhances xylogenesis.     

人畸胎瘤细胞株PA-1的体外诱导分化

PA-1细胞是一株从人卵巢性畸胎瘤衍生的细胞株,经10~(-5)mol/L视黄酸诱导后,部分细胞的形态和排列方式发生一定的改变。通过细胞免疫荧光染色,发现诱导后细胞的结蛋白和胞外基质(纤粘连蛋白、层粘连蛋白和腱粘连蛋白)表达与分布模式有较大的变化,并且这些变化与细胞形态改变相关。但大部分PA-1细胞诱导后的生长状况并没有较大的改变,仍分泌与表皮生长因子、类胰岛素生长因子-Ⅰ相关的活性物质。以上结果提示PA-1细胞经视黄酸诱导后一部分细胞可能向肌细胞方向分化。     

GROWTH AND DIFFERENTIATION OF JUICE VESICLES OF ORANGE GROWN IN VITRO

Juice vesicles of Valencia orange were grown on agar bases containing different concentrations of kinetin plus mineral and organic constituents, or in comparable liquid solution (shake cultures), maintained at approximately 26 C. Shake cultures enlarge most rapidly, but both maintain similar patterns of anatomical development. Typically, marginal parenchymatous cells of the sac become meristematic and develop, largely by periclinal division, generalized cambial meristems that enlarge the callus by the addition of linear rows of cells. Chloroplasts and wound tracheids with bordered pits mature within 30 days, but to date no further differentiation has been noted. Surface cells frequently enlarge, detach, and grow into branched hyphal aggregations of cells. Media containing 1.0 mg/liter kinetin causes greater enlargement of callus than media with 0.02 mg/liter kinetin.     

MITOSIS AND THE PROCESSES OF DIFFERENTIATION OF MYOGENIC CELLS IN VITRO

The relation between the mitotic cycle and myoblast fusion has been studied in chick skeletal muscle in vitro. The duration of the cell cycle phases was the same in both early and late cultures. By tracing a cohort of pulse-labeled cells, it was found that myoblast fusion does not occur in S, G₂, or M. Cell surface alterations required for fusion are dependent upon the position of the cell in the division cycle. In early cultures, fusion takes place only after a minimum delay of 5 hr from the time the cell has entered G₁. The mitosis preceding fusion may condition the cell for the abrupt shift in synthetic activity that occurs in the subsequent G₁. In older cultures fusion of labeled cells is diminished. Two factors account for the cessation of fusion in older cultures. First, the number of myogenic stem cells declines, but these cells do not disappear as the cultures mature. Their persistence was demonstrated by labeling dividing mononucleated cells in older cultures and challenging them with nascent myotubes. Some of these labeled cells were incorporated into the forming myotubes. Second, a block to fusion develops during myotube maturation. Well developed myotubes challenged with labeled competent myogenic cells failed to incorporate the labeled nuclei.     

INDUCED DIFFERENTIATION OF GERANIUM PLANTS FROM UNDIFFERENTIATED CALLUS IN VITRO

Geranium callus produced from explants of stem tip, internode pith with vascular tissue on synthetic media with or without coconut milk and 2,4-dichlorophenoxyacetic acid grew well for many generations, but only tracheids were induced in them. If callus produced on these media was subcultured immediately on Murashige and Skoog medium with 0.1 mg/liter/α-naphthalene acetic acid (NAA) and 10.0 mg/liter kinetin (K) and incubated at 16/8-hr light/dark cycle, shoots were induced in 8-10 weeks and roots in another 8-10 weeks. Callus produced on the MS medium with the same supplements of NAA and K, subcultured on the same medium and incubated in 16/8-hr light/dark cycle, produced shoots in 6-8 weeks. However, on callus subcultured more than three times, shoots differentiated with greater difficulty and none differentiated after six subcultures. Some abnormal shoot-like structures were also produced, the cells of which showed virus-like inclusion bodies. Requirements of the different varieties for differentiating organs differed. Among 12/12-, 15/9-, 16/8-, and 20/4-hr light/dark photoperiods that induced differentiation, 15/9- and 16/8-hr were more effective than the others. Continuous illumination did not induce differentiation. Differentiated shoots formed roots more readily on a medium with reversed proportions of auxin and kinetin. On agar roots were devoid of root hairs. Root hairs were formed when the shoots and plantlets were cultured on filter-paper bridges. Many “mother” stock plants were produced. These are being studied for their growth qualities and for possible viruses and other pathogens.     

IN VITRO EFFECT OF SUCROSE FEEDING IN THE DIFFERENTIATION OF WOOD ELEMENTS OF PLUMERIA

Wood with cambium of Plumeria rubra Linn. was cultured in modified Schenk and Hildebrandt medium for 75 days. Change in dimension and frequency of cells was compared with that grown in situ for 75 days. Feeding of 41% sucrose in vitro brought about the same type of differentiation as occurred in situ , where non-reducing sugar percentage increased (from 0.0004 before culture to 0.0032 after 75 days' growth in situ ). Sucrose favoured enlargement of axial elements and numerical increase of all lignified elements.     

LABELING OF MURINE MASTOCYTOMA CELLS IN VITRO WITH PLASMA TRITIATED THYMIDINE-LABELED ANIMALS

40 min after injecting tritiated thymidine into an animal, 20–30% of the total plasma radioactivity is nonvolatile. This fraction decreases to about 6% 10 hr after the injection and 3% 24 hr after the injection. There appears to be material in this nonvolatile fraction that can label mastocytoma cells in culture. The labeling indices decrease with time after injection in the same way as the nonvolatile fraction. The 40 min plasma sample contains sufficient material to allow accurate assessment of the fraction of cells in S in culture after a 6 wk exposure. The circulating material is not apparently available for incorporation into those cells in cycle in the donor animal. The material appears to be related to the G₀ cell-specific pool that has been described elsewhere. The trichloroacetic acid-soluble or ethanol-soluble nonvolatile activity appears to contain thymine, and some thymidine-phosphorylated compounds.     

蛇床子素对大鼠成骨细胞增殖分化的影响

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