影响人骨髓CFU-C产率某些因素的分析

在CFU-C(粒系或单核-巨噬细胞集落生成单位)体外琼脂培养中,只有在CSF(细胞集落生成刺激因子)的作用下粒系祖细胞才能增殖、分化,生长成为细胞集落。CSF来源广泛,其刺激活力文献报告不一。此外,CFU-C产率波动较大,影响因素甚多。本文在相同的实验条件下,比较了不同来源的CSF刺激活力,并对影响CFU-C产率的某些因素进行了分析。     

骨髓基质支持造血功能观察

正常人和小鼠骨髓细胞体外平皿液体培养中形成的贴壁细胞层,具有骨髓基质成分和功能,在此贴壁细胞层或基质层上,培养粒系祖细胞 CFU-C(Colony Forming Unit-Culture),以不带基质层的 CFU-C 为对照(100%)。结果第一周基质层上 CFU-C 为对照51.07±3.54%(人)和27.30±4.68%(鼠);第二周者则与对照相近;笫三周之 CFU-C 则上升达150.70±17.63%(人)与146.89±10.01%(鼠)。示基质层对 CFU-C 的增殖分化,在1～3周呈先抑制后刺激的效应。并观察了10例用山莨菪碱治疗的再生障碍性贫血患者之骨髓三周基质层,对正常骨髓CFU-C 影响。治疗前4例,基质层上 CFU-C 较对照<80%,示有抑制作用。此4例均治疗有效。其中2例复查,抑制作用消失。其余6例,其基质层上 CFU-C,均较对照>80%,仅1例用山莨菪碱治疗有效。这种基质层上培养 CFU-C 的方法,可用于实验或临床,研究骨髓基质支持造血的功能。     

增加Gi和β2AR表达对异丙肾上腺素损伤的大鼠心肌细胞存活的影响

目的：观察增加β2肾上腺素受体（β2AR）和抑制性G蛋白（Gi）基因的表达是否可改善长期异丙肾上腺素（ISO）刺激引起的心肌细胞死亡。方法：用腺病毒作载体,在培养的心肌细胞增加β2AR和Gi基因的表达;然后在培养基中加入5μmol/L的异丙肾上腺素以损伤心肌细胞,24h后记数各组细胞的存活率。结果：β2AR和Gi表达增加,对正常培养的大鼠心肌细胞的死亡率均没有影响,但能够减少异丙肾上腺素损伤引起的细胞死亡。这种作用可分别被β2AR选择性阻断剂ICI118,551和Gi阻断剂百日咳毒素（PTX）所阻断。结论：增加β2AR或Gi基因的表达,对异丙肾上腺素损伤的心肌细胞有保护作用。     

儿茶酚胺和凝血酶对人胎肝细胞富组氨酸糖蛋白分泌的影响

采用人胎肝细胞原代培养方法,观察儿茶酚胺和凝血酶对肝细胞富组氨酸糖蛋白（ＨＲＧ）分泌的影响。结果表明：抗原测定和生物活性检测,均证明细胞能分泌ＨＲＧ;而肾上腺素、去甲肾上腺素、异丙肾上腺素以及凝血酶则对肝细胞白蛋白分泌无明显影响,但它们均能刺激肝细胞分泌ＨＲＧ。肾上腺素与心得安联用,虽仍然刺激肝细胞分泌ＨＲＧ,但其作用远较单独使用肾上腺素弱。故推测肾上腺素刺激肝细胞分泌ＨＲＧ主要是通过β肾上腺素能受体介导的,但部分由非β肾上腺素能受体介导,其中α－肾上腺素能受体亦可能具有一定作用     

豚鼠骨髓细胞在体内扩散盒培养条件下的增殖和分化

我们研究了豚鼠骨髓细胞在体内扩散盒培养条件下的生长特性。骨髓细胞悬液在体内扩散盒培养中,细胞处于增殖活动,培养后第5天,收集的有核细胞量或增殖性粒系细胞量与最初种入的骨髓有核细胞量之间呈比例关系。骨髓细胞在体内扩散盒血浆凝块培养中,逐渐生成由红系细胞组成的细胞团(CFU-E、BFU-E)和由粒系细胞组成的细胞团(CFU-C)。培养后第7天,CFU-E 和 CFU-C 的生成量分别与种入的骨髓有核细胞量之间呈正比关系。应用体内扩散盒血浆凝块培养技术测定了整体照射条件下豚鼠骨髓CFU-C 和 CFU-E 的辐射敏感性,它们的 D_0值分别为84.2和67.6拉德。     

异丙肾上腺素和氨甲酰胆碱对心肌延迟后除极的影响

用乙酰毒毛旋花子甙元2.0×10~(-7)mol/L,在绵羊心室浦肯野纤维产生延迟后除极(DAD)为实验模型,观察异丙肾上腺素与氨甲酰胆碱对DAD的影响。异丙肾上腺素1.0—3.0×10~(-8)mol/L能增加DAD幅值,为剂量依赖性,并可诱发出触发型心律失常。氨甲酰胆碱2.0×10~(-6)mol/L单独作用对DAD幅值无影响,然而在异丙肾上腺素增强DAD幅值或产生触发型心律失常后,同样浓度的氨甲酰胆碱能显著地减小DAD幅值以及消除触发型心律失常。但氨甲酰胆碱对由高钙、氨茶碱和组胺等所增加的DAD幅值却无抑制作用。以上结果表明,氨甲酰胆碱能拮抗异丙肾上腺素引起的DAD增强作用,可能与M受体的激活,并继而降低膜上β受体激活时所提高的腺苷酸环化酶活性有关。     

雌激素受体在人舌鳞癌中的表达及β-雌二醇对其增殖的影响

目的：观察雌激素受体（ER）在人舌鳞癌Tca8113细胞系细胞上的表达,研究β－雌二醇（estrabiol,E2)对培养的舌癌细胞增殖的影响。方法：用免疫细胞化学方法和RT－PCR方法检测雌激素受体在人舌鳞癌细胞上的表达,将β－雌二醇（β－E2）作用于体外培养的人舌鳞癌细胞,测定^3H-TdR掺入量,并用流式细胞仪测定细胞周期。结果：经免疫细胞化学和RT－PCR方法可检测到ER－α和ER－β在人舌鳞癌细胞上均有表达,其中ER－β表哒相对较弱。不同浓度的β－E2可增加^3H-TdR掺入量,并存在着剂量效应。106-6mol/L的β－E2能使人舌鳞癌细胞处于G1期的细胞比例从对照组的76．5％下降至62．3％,而处于S期和G2期的细胞比例从23．5％上升至37．7％,细胞的DNA合成增加,促进细胞的增殖。β－E2对舌癌细胞的促增殖作用可被Tamoxifen阻断。结论：在人舌鳞癌细胞上有雌激素受体的表达,且β－E2在体外能明显促进培养的人舌鳞癌细胞的增殖。     

海马内注射肾上腺素能受体激动剂对细胞免疫功能的影响

目的 研究肾上腺素能受体激动剂对机体细胞免疫功能的作用及下丘脑-垂体-肾上腺轴(HPA轴)在此作用中的地位.方法以刀豆蛋白A(ConA)刺激脾淋巴细胞的增殖活性为细胞免疫功能指标,检测在正常及去肾上腺大鼠海马内注射去甲肾上腺素(noradrenaline,A)对机体细胞免疫功能的影响.结果①在正常大鼠,A(4 μl,.0×10-3mol/L)、β1受体激动剂杜丁胺 (dobutamine,ob,μl,.0×10-3mol/L) 和β2受体激动剂异丙喘宁(metaproterenol,et, μl,.0×10-3mol/L)均可抑制Con A刺激的脾淋巴细胞增殖反应、降低NK细胞的活性,其中NA的作用最强,et 次之,ob的作用最弱.α及β受体阻断剂酚妥拉明(Phen, μl,.6×10-2mol/L) 和心得安(Prop,μl,.6×10-2mol/L)均可部分阻断NA的免疫抑制作用,且Prop的作用较强.②在去肾上腺组,A的免疫抑制作用不明显.结论海马内NA对机体的细胞免疫功能具有明显的抑制作用,此作用由α及β受体共同介导,其中β受体的作用大于α受体,且β2受体的作用大于β1受体.此外,保持肾上腺结构和功能完整在NA调节机体细胞免疫功能作用中具有重要意义.     

人骨髓造血干细胞体外培养中的动态变化

本文报告100多批人的骨髓细胞长期体外培养的实验结果。在首次接种骨髓细胞建立起贴壁层细胞基础上,再将另一个体的骨髓细胞接种在贴壁细胞层上面,这样就可使骨髓中的造血干细胞在贴壁细胞层上增殖和分化,维持较长时间。人类多向性造血干纽胞(CFU-S)目前尚无测定的方法,但由CFU-S分化出来的粒系祖细胞(CFU-C)和红系祖细胞(CFU-E)是有方法可以测定的,从此获得的结果表明,在体外培养条件下,CFU-C可存活9周,CFU-E可存活4.5周。在培养悬液细胞中,形态上可以识别的主要有粒细胞,其存在时间与CFU-C大致相同,而红系细胞仅在3周内可以见到,成熟淋巴细胞最多能存活5周,以后就不复存在了。在培养7周内,每个培养瓶中平均悬液细胞数是增加的。从干细胞自杀实验证明,CFU-C至少在6周内不断地有一部分细胞进入DNA合成期。在整个培养期间,各系统的造血祖细胞及其后代细胞,它们的存活时间虽有差别,但均保持着正常的生物活性。7周以后,培养体系中的细胞总数就逐渐减少,到了9周后因为造血干细胞业已消失,所以各种成熟的或未成熟的血细胞也就逐渐不见了。在骨髓细胞培养液中还存在着CSF,其浓度随着CFU-C总数的下降而平行地降低,提示在体外培养体系中可能存在对造血干细胞活动的调节因子。此外,也观察了培养瓶中悬液细胞与贴壁细胞间的相互关系及其某些造血特征。     

17β—雌二醇抑制内皮素诱导的血管平滑肌细胞增殖作用

目的和方法：利用组织块贴壁法进行大鼠VSMC培养,胰蛋白酶分散细胞法传代。实验采用第4-6代细胞。采用氚-胸腺嘧啶核苷（[^3H]-TdR)掺入和细胞计数来作为VSMC增殖的指标,以RT-PCR的方法检测ETAR的表达,观察17β-雌二醇（E2）对内皮素-I（endothelin-l,ET-1)介导的血管平滑肌细胞（VSMC）增殖反应以及对内皮素A型受体（ETAR）表达的影响。结果：ETAR特异性拮抗剂BQ123能完全阻断ET-1介导的VSMC增殖反应;E2可明显抑制ET-1促进VSMC增殖的作用,RT-PCR结果显示E2能抑制ETAR的表达,12h时抑制作用最为明显;E2受体阻断剂Tamoxifen亦能部分抑制ET-1对VSMC的增殖及ETAR的mRNA的表达。结论：ET-1促进VSMC增殖作用主要通过ETAR介导的,雌激素可通过抑制ETARmRNA表达来发挥对ET-1促进VSMC增殖的抑制作用。     

KINETICS OF GROWTH OF HAEMOPOIETIC COLONY CELLS IN AGAR

Cell kinetic parameters of mouse granulocytic and mononuclear cells growing in colonies in agar cultures have been measured. Analysis of flash and continuous labelling studies with ³H-thymidine together with determinations of colony size, growth fraction and mitotic indices, gave the following values for the phases of the cell cycle: G₁= 6·3 1·6 hr, S = 5·8 ± 1·4 hr, G₂= 1·7 ± 0·1 hr and M = 0·7 ± 0·1 hr (42 ± 8 min). No difference in the cell cycle parameters of granulocytic and mononuclear cells were found in this study.
Colonies of different size from cultures of the same age group had similar labelling indices, indicating that the size of a colony is not a function of the rate of proliferation of cells in the colony. Rather, variation in colony size is probably representative of an initial delay in the onset of colony development.     

COLONY FORMATION IN AGAR: IN VITRO ASSAY FOR HAEMOPOIETIC STEM CELLS

Using a method in which embryo fibroblasts were used as feeder layers, the colony forming capacity in agar of a variety of mouse haemopoietic suspensions was compared with their CFU s content. A striking parallelism between the results of the two assays was found. In addition, under certain conditions higher numbers of CFU s could be retrieved from 5-day-old agar colonies than were originally plated, indicating that the CFC a (Colony Forming Cell agar) may fulfil the requirements of pluripotency as well as of self-renewal, both prerequisites for any haemopoietic stem cell candidate. Although our data by no means provide direct proof that the CFC s and the CFC a are identical, they certainly support such a concept. the contradictory findings by others that CFU s and CFU c (Colony Forming Unit culture) can be separated on a velocity gradient is attributed to different culture conditions, in other words, that their CFU cè are not identical with our CFU a .
Our findings also indicate that for mouse cells our soft agar colony assay meets the criteria of a quantitative assay for haemopoietic stem cells and that extension of this technique to bone marrow of primates including humans seems to be justified.     

DISSECTION OF TWO DISTINCT DEFENSE‐RELATED RESPONSES TO AGAR OLIGOSACCHARIDES IN GRACILARIA CHILENSIS (RHODOPHYTA) AND GRACILARIA CONFERTA (RHODOPHYTA)1

The two agar‐producing red algae, Gracilaria chilensis C. J. Bird, McLachlan & E. C. Oliveira and Gracilaria conferta (Schousboe ex Montagne) Montagne, responded with hydrogen peroxide (H₂O₂) release when agar oligosaccharides were added to the medium. In G. conferta, a transient release was observed, followed by a refractory state of 6 h. This response was sensitive to chemical inhibitors of NADPH oxidase, protein kinases, protein phosphatases, and calcium translocation in the cell, whereas it was insensitive to inhibitors of metalloenzymes. Transmission electron microscopic observations of the H₂O₂‐dependent formation of cerium peroxide from cerium chloride indicated oxygen activation at the plasma membrane of G. conferta. A putative system, consisting of a receptor specific to agar oligosaccharides and a plasma membrane‐located NADPH oxidase, appears to be responsible for the release of H₂O₂ in G. conferta. Subcellular examination of G. chilensis showed that the H₂O₂ release was located in the cell wall. It was sensitive to inhibitors of metalloenzymes and flavoenzymes, and no refractory state was observed. The release was correlated with accumulation of an aldehyde in the algal medium, suggesting that an agar oligosaccharide oxidase is present in the apoplast of G. chilensis. The presence of this enzyme could also be demonstrated by polyacrylamide electrophoresis under nondenaturating conditions and proven to be variable. Cultivation of G. chilensis at 16 to 17°C resulted in significantly stronger expression of agar oligosaccharide oxidase than cultivation at 12°C, which indicates that the enzyme is used under conditions that generally favor microbial agar macerating activity.     

COLONY-FORMING UNITS IN DIFFUSION CHAMBERS (CFU-d) AND COLONY-FORMING UNITS IN AGAR CULTURE (CFU-c) OBTAINED FROM NORMAL HUMAN BONE MARROW: A POSSIBLE PARENT–PROGENY RELATIONSHIP

A series of experiments was performed to elucidate the relationship between cells that form granulocytic colonies in fibrin clot diffusion chambers implanted into the peritoneum (i.p.) of irradiated mice (CFU-d) and day 7 and day 14 CFU-c which give rise to colonies after 7 and 14 days in agar cultures in vitro, respectively. Normal human bone marrow cells were cultured in suspension in vitro or in diffusion chambers implanted into irradiated or non-irradiated mice. During these culture conditions there was an initial decrease in the number of CFU-c per culture. This was followed by an increase between day 2 and day 7 of culture. No similar increase of neutrophilic CFU-d was observed. When CFU-d, day 14 and day 7 CFU-c in normal marrow were separated by velocity sedimentation and cultured in suspension culture or in diffusion chambers for 7 days, the maximum increase of day 7 and day 14 CFU-c was observed in slowly sedimenting cell fractions which contained the majority of CFU-d. After 3 days in suspension culture, the maximum increase of day 14 CFU-c was found in fractions which also gave rise to maximum numbers of CFU-c after 7 days. However, day 7 CFU-c were found in fractions which initially contained the majority of day 14 CFU-c. No increase in CFU-d was found in fractions initially containing peak numbers of CFU-c. Between 53 and 71% of CFU-c harvested from diffusion chambers in irradiated mice or from suspension cultures were sensitive to pulse incubation with tritiated thymidine, suggesting that the cells were proliferating during these culture conditions. In diffusion chambers implanted into non-irradiated mice, however, CFU-c were found to be relatively resistant to this treatment (3–11% sensitive to tritiated thymidine). Thus marked increases in CFU-c were also observed during experimental conditions, where no significant DNA synthesis was detected. A reproducible time sequence of increase in CFU-c populations in culture was observed. Day 14 CFU-c and cells that gave rise to clusters on day 7 in agar increased between day 2 and day 4, whereas day 7 CFU-c increased between day 4 and day 7. The results suggested that CFU-d gave rise to CFU-c in culture and that day 14 CFU-c were precursors of day 7 CFU-c.     

ARRESTED GROWTH OF CHINESE HAMSTER OVARY (CHO) CELL COLONIES ON AGAR

Chinese hamster ovary (CHO) cells plated on agar form two classes of colonies; those which increase continuously in diameter and those which become arrested in outward growth. All colonies continue to increase in mass and thickness as demonstrated by computer-assisted analysis of time sequence photographs of several thousand colonies and by examination of histological sections. Colonies which shift from a monolayer to a mounded morphology at fairly large colony diameter (?1 mm) continue to increase in diameter. Colonies in which mounding occurs at smaller colony diameter (?1 mm) cease to increase in diameter but continue to increase in thickness, as demonstrated by histological examination and by computer-assisted analysis. Rapid cell division occurs at the edge of all colony classes, as shown by the distribution of mitotic figures. In arrested colonies these dividing cells must move toward the colony core to compensate for dying cells. Necrotic cells are found as a discrete zone at the air-colony interface in all cases for CHO cell colonies growing on agar. As such, necrosis is probably due to limitations in nutrient diffusion upward from the agar rather than oxygen diffusion downward.