LIPOPROTEIN INHIBITOR OF BQNE MARROW CELLS IN TUMOR-BEARING RATS

The role of a plasma inhibitor of erythropoiesis is evaluated in rats with Walker-256 carcinoma (W-256). Plasma from tumor-bearing rats was treated by gel filtration chromatography (Sephadex G-150) and fractions were combined into four pools on the basis of mol. wt. Inhibitory activity was assayed by adding an aliquot of the plasma fractions to normal rat marrow cells which were cultured for 24 hr with and without erythropoietin. ⁵⁹Fe-heme synthesis, [³H]thymidine DNA synthesis, and ¹⁴C-leucine protein synthesis were studied. The results indicated that cultures containing the high mol. wt. pool (>400,000 daltons) had significantly decreased heme, DNA and protein synthesis. This inhibitor also diminished the response to erythropoietin in polycythemic mice. The lower mol. wt. pool stimulated heme synthesis in vitro. To identify the inhibitor further, plasma lipo-protein classes were isolated by density gradient ultracentrifugation. The very low density lipoprotein (VLDL) and chylomicron fractions markedly inhibited DNA, protein and heme synthesis. Low density and high density lipoprotein fractions were inactive. A lipoprotein inhibitor of erythropoiesis was also identified in cancerous ascitic fluid, and to a lesser degree, in normal rat plasma. We suggest that this VLDL inhibitor of marrow erythropoiesis is a contributing factor in the anaemia of cancer.     

GROWTH OF MOUSE AND HUMAN BONE MARROW IN DIFFUSION CHAMBERS IN MICE

Both murine and human bone marrow cells were cultured in plasma clots which were formed inside diffusion chambers implanted into cyclophosphamide- and saline-treated mice. After an initial fall, the number of mouse bone marrow cells and numbers of mouse myeloid stem cells (CFU-C) and agar cluster-forming units rose faster in the cyclophosphamide-treated animals. These hosts also favored formation of myeloid (CFU-D-G) and erythroid (CFU-D-E) colonies and myeloid clusters in the plasma clot. The number and growth rate of mouse CFU-D-G were higher than those of CFU-C from the same marrow population. These observations suggest the existence of humoral factors stimulating granulocyte progenitor cell replication and differentiation. At its best the increment of CFU-D-E number was equivalent to that caused by a single 0·1 unit erythropoietin dose. Culture of normal human marrow cells resulted in colonies in the plasma clot containing only granulocytes and macrophages. Cyclophosphamide-treated host animals were essential for human CFU-D-G development. Plating efficiency for human marrow myeloid colonies was better in the conventional in vitro agar cultures than in diffusion chambers.     

射线诱导在体造血细胞凋亡的量时效关系的研究

以4—10Gy射线在体损伤的小鼠为模型,应用DNA电泳和流式细胞术等方法证实细胞凋亡是射线损伤在体骨髓造血细胞的途径之一,发现射线诱导的造血细胞凋亡有明显的量效和时效关系。4、6、8和10Gy照射后,细胞凋亡发生率均表现为升高-降低过程,各剂量诱导的凋亡发生率峰值分别出现在照后12、8、4和4h,6和8Gy诱导的凋亡发生率已达最高水平,约为30％,10Gy诱导的凋亡反而要低。上述结果表明,诱导细胞凋亡是射线损伤骨髓造血细胞的一个重要途径,而且凋亡的发生率受照射剂量和照后时间的影响。因此,深入研究射线诱导的细胞凋亡,将有助于揭示射线损伤造血功能的机理。     

COLONY-FORMING ABILITY OF BONE MARROW CELLS OF W ANAEMIC MICE ON MACROPHAGE LAYER FORMED IN PERITONEAL CAVITY OF MICE

The colony-forming ability of haematopoietic cells of W anaemic mice was examined on the macrophage layer formed in the peritoneal cavity of mice. Bone marrow cells of W anaemic mice formed a considerable number of colonies on the macrophage layer, notwithstanding they did not form any colonies in the spleen of the same recipients. As the colony-forming ability of the bone marrow cells was not reduced by the incubation with ³H-thymidine, most of the cells which formed colonies on the macrophage layer seemed to stay in G₀ state. The interrelationship between the spleen colony-forming cells, the macrophage-layer colony-forming cells, and in vitro colony-forming cells was discussed.     

THE PROLIFERATION OF LYMPHOID CELLS IN GUINEA-PIG BONE MARROW

A double isotope DNA labelling method has been used to determine the duration of DNA synthesis (S) in bone marrow lymphoid cells classified by their nuclear diameters in smears. Incorporation of ³H-thymidine was confined almost entirely to marrow lymphoid cells of 8·0-15·0 μm nuclear diameter (large lymphoid cells). After exposure to ³H-thymidine in vivo and ¹⁴C-thymidine 40-104 min later in vitro , the proportion of cells labelled with ³H alone to those labelled with ¹⁴C(±³H) in radioautographic smears, plotted against time indicated the efflux from S per hour. Collectively, 28·3 ± 1·1% of all large lymphoid cells were in S and the efflux from S was 15·1% per hour. With decreasing cell size (nuclear diameter) the efflux fell progressively from 28·3% per hour (11·0 μm) to 9·2% per hour (8·0-8·9 μm) and the proportion of cells in S declined from 54·9 ± 2·3% to 14·8 ± 1·6%. Influx into S, measured in vitro by reversing the sequence of isotopes, closely resembled the corresponding efflux values in vivo relative to cell size. Most DNA synthesizing marrow large lymphoid cells belonged to a subgroup with deeply basophilic cytoplasm. The results demonstrate that basophilic large lymphoid cells in the marrow are actively proliferating and have a mean S phase duration of 6·6 hr. The largest marrow lymphoid cells (11·0 μm) proliferate most rapidly (S phase, 3·5 hr; maximum cell cycle time, 6·4 hr) while S duration is prolonged progressively to 10·9 hr for the smaller cells (8·0-8·9 μm).     

当归多糖对放射损伤小鼠骨髓单个核细胞黏附分子表达及细胞周期的影响

目的探讨当归多糖(APS)对放射损伤小鼠骨髓单个核细胞(BMNC)黏附分子表达及细胞周期的影响,旨在阐明APS防护辐射性造血损伤的分子机制。方法建立小鼠放射损伤模型后连续给予不同剂量APS 13d,在不同时间点进行外周血白细胞、红细胞、血小板及BMNC计数;流式细胞术检测小鼠Sca-1+BMNC黏附分子CD44和CD49d表达及BMNC细胞周期的变化;Western blot和RT-PCR方法分别检测小鼠BMNC细胞周期蛋白(Cyclin)D2 mRNA和蛋白表达水平的改变。结果与正常组比较,NS组外周血WBC、RBC、PLT及BMNC计数明显减少,Sca-1+BMNC CD44、CD49d表达明显下降,G0/G1期细胞比例显著增加,CyclinD2 mRNA和蛋白表达水平明显降低。2 mg/kgAPS组和8 mg/kgAPS组能增加外周血各指标及BMNC计数,第7d明显提高Sca-1+BMNC黏附分子CD44、CD49d的表达水平,第14d能降低Sca-1+BMNC黏附分子CD44、CD49d的表达水平,降低G0/G1期细胞比例,提高CyclinD2mRNA和蛋白表达水平。结论当归多糖能通过调节放射损伤小鼠Sca-1+BMNC黏附分子的表达水平、上调BMNC的CyclinD2 mRNA和蛋白表达水平来加速BMNC G1期向S期的转换,促进造血恢复。     

THE ROLE OF BONE MARROW OF X-IRRADIATED MICE IN THYMIC RECOVERY

The influence of the bone marrow on the repopulation of the thymus in X-irradiated mice has been investigated.
It was observed that the thymus and a certain population of bone marrow lymphocytic cells were repopulated in parallel in a cyclic fashion. This occurred either after a single exposure of mice to 400 R or after serial weekly X-ray treatments with 170 R. Lethally irradiated recipients which were grafted with bone marrow cells obtained 12-24 days after four weekly irradiations of donor mice with 170 R also exhibited a cyclic repopulation of both the thymus and the bone marrow lymphocytic population. In contrast, mice which were transplanted with bone marrow cells from unirradiated donors, containing an equal number of stem cells (CFU), exhibited a continuous rather than a cyclic recovery of both cell populations. the bone marrow stem cells of mice recovering from X-irradiation were found to have a decreased proliferative activity, since they produced significantly smaller spleen colonies in lethally irradiated recipients than marrow cells from unirradiated mice.
The results were interpreted as indicating that the bone marrow lymphocytic cells may act as thymic precursor cells and that thymic lymphopoiesis is dependent on the presence of such cells. Evidently, the production of lymphocytic cells will decrease when the stimulus for granulocyte production increases due to the limited proliferative activity of the surviving bone marrow stem cells after irradiation. This may result in a cyclic variation of the production of bone marrow lymphocytic cells and it follows that thymic lymphopoiesis will run parallel.     

正常小鼠骨髓血红蛋白含量的测定

小鼠骨髓血红蛋白含量的变化可以间接地反映骨髓微循环系统形态和功能的状况。按Burger and Knyszynski(1969)方法操作繁复,限制了它的推广应用及正常值的问世。最近,我们建立的简易测定方法,为成批标本的测定和正常值的确定创造了条件。 正常小鼠骨髓血红蛋白含量测定的目的:1)在较大量标本的测定中进一步验证该方法的可靠性;2)确定青、成年小鼠骨髓血红蛋白的正常值范围;3)分析其可能的影响因素,以便更好地控制实验条件和判断骨髓微循环障碍的程度。     

小鼠体内脾脏,骨髓及精原细胞姐妹染色单体交换的比较研究

本文对几种化学诱变剂诱发小鼠体内脾脏、骨髓和精原细胞的SCE进行了比较研究,同时分析了几类常见化合物在小鼠脾脏细胞中诱发SCE的活力。结果显示诱变剂在脾脏细胞中诱发SCE比骨髓和精原细胞敏感。几类化合物都能显著地诱发小鼠脾脏SCE的增加,与对照相比差异显著(P<0.05)或极显著(P<0.01),说明利用小鼠脾脏细胞检测环境诱变物是相当灵敏的。     

THE DEVELOPMENT OF FIBROBLAST COLONIES IN MONOLAYER CULTURES OF GUINEA-PIG BONE MARROW AND SPLEEN CELLS

In monolayer cultures of guinea-pig bone marrow and spleen the development of discrete fibroblast colonies takes place on days 9–12. The linear increase in the number of colonies with increasing numbers of explanted cells and the distribution of male and female cells in mixed cultures support the view that fibroblast colonies are clones. The concentration of colony-forming cells in bone marrow and spleen is approximately 10^-5. Bone marrow culture (but not spleen culture) fibroblasts are capable of spontaneous bone formation in diffusion chambers. Fibroblasts from both bone marrow and spleen cultures are inducible to osteogenesis in diffusion chambers in the presence of transitional epithelium.     

β-半乳糖苷酶基因在正常人及白血病人骨髓细胞的表达

为探索组织化学方法检测标志基因在正常骨髓细胞及白血病细胞的表达,本研究利用脂质体介导将ＮｅｏＲ基因和ＬａｃＺ基因共转移正常人及白血病人骨髓细胞。然后,采用Ｘ－ｇａｌ染色的方法,我们观察了ＬａｃＺ基因在转导细胞的表达。结果显示：ＬａｃＺ基因在正常人及白血病人骨髓细胞表达的阳性率分别为１３．３３％±２．６８％和１５．３９±３．６９％。经Ｇ４１８筛选后,转导阳性细胞率达到４６．０６％±３．４７％和４８．２２％±４．４７％。提示：经脂质体介导ＬａｃＺ基因在正常骨髓细胞及白血病细胞可获得高效的转移率。同时表明,利用组织化学方法可有效地检测标志基因的表达。     

HISTOCHEMICAL DEMONSTRATION OF DEHYDROGENASE ACTIVITY IN THE CELLS OF NORMAL HUMAN BLOOD AND BONE MARROW

Endogenous and succinic dehydrogenase activity was demonstrated in the living cells of normal human blood and bone marrow using a buffered nitro BT-succinate incubating solution. With this technique dehydrogenase activity was localized primarily in the granular leukocytes and the sites of enzymatic activity appeared to be non-mitochondrial. The addition of a non-ionic surface active agent to the incubating solution resulted in marked differences in the cellular and intracellular localization of dehydrogenase activity. With this method it was possible to demonstrate dehydrogenase activity in the mitochondria of most of the formed elements of the blood and bone marrow, including developing granulocytes and erythroid cells, agranulocytes, and blood platelets. Mature erythrocytes also exhibited a minimal dehydrogenase reaction with this procedure. This investigation indicated that in order adequately to demonstrate and evaluate dehydrogenase activity in the cells of the blood and bone marrow it was necessary to have increased cellular and mitochondrial permeability, as well as partially viable cells with an intact dehydrogenase system.     

骨髓基质细胞与交联明胶-聚羟基丁酸酯膜的生物相容性研究

目的研究生物材料交联明胶-聚羟基丁酸酯膜与骨髓基质细胞的生物相容性,探讨新型材料在骨组织工程中的应用前景。方法体外培养兔骨髓基质细胞,分别接种于G-PHB(交联明胶-聚羟基丁酸酯)、PHB(聚羟基丁酸酯)和G(交联明胶)材料膜片。采用MTT法检测细胞增殖活性,体视学方法检测细胞粘附能力,荧光双染法检测细胞完整性,扫描电镜观察细胞-材料界面。结果MTT检测发现G-PHB组增殖活性最强,而且表现为最佳的细胞粘附特性,与对照组比较差异有显著性意义。各组细胞完整性分析没有发现显著性差异。扫描电镜观察显示,G-PHB组细胞粘附及铺展良好,优于其他各组。结论交联后的生物降解膜材料G-PHB与BMSCs细胞的体外相容性明显优于单纯膜材料PHB和明胶,在骨组织工程学领域具有良好的研究价值和应用潜力。     

骨髓基质干细胞的诱导分化及其治疗癫痫的实验研究

目的探讨骨髓基质干细胞诱导分化为神经元样细胞的方法及脑内移植治疗大鼠癫痫模型的作用。方法无菌条件下分离纯化BMSCs,用bFGF 10ng/ml、RA0.5μmol/L的DMEM/F12诱导72h后,部分用于免疫荧光检测nestin,其余的继续用bFGF 10ng/ml、RA 0.5μmol/L及神经营养因子NT-3 20ng/ml、BDNF 20ng/ml的DMEM/F12诱导4d,检测GAD67。皮下注射匹罗卡品建立癫痫大鼠模型,采用行为学分析筛选模型。通过立体定位仪,用微量注射器将BMSCs来源的神经干细胞和神经营养因子移植入癫痫鼠海马内,观察大鼠行为变化,存活2、4周后,心脏灌注取脑,冰冻切片免疫组化双标检测移植细胞的存活、迁移、分化情况。结果BMSCs诱导72h后,nestin表达阳性,4d后GAD67检测60%阳性。采用匹罗卡品造模,方法简便,但死亡率较高,仅15%-20%的动物造模成功。BMSCs源神经干细胞移植后可在海马内存活,向周围的脑区内迁移和整合。结论BMSCs源的神经干细胞在体外可诱导为γ-氨基丁酸能神经元并且对慢性癫痫有一定的治疗作用。     

骨髓基质干细胞诱导分化为神经元过程中miR-124和miR-128的表达

目的探讨骨髓基质干细胞诱导分化为神经元过程中miR-124和miR-128的表达变化及作用。方法采用全骨髓培养法体外分离培养获得骨髓基质干细胞,取传代培养至第3代的骨髓基质干细胞,在神经干细胞培养液及细胞因子等条件下诱导其分化为神经元,倒置显微镜下观察其形态变化,应用ABI公司的TaqManMicroRNAAssaysreal-timePCR技术,检测miR-124和miR-128在诱导分化过程中的表达。结果 miR-124分化后神经元的表达是未分化BMSCs的0.051倍(P0.05);miR-128分化后神经元的表达是未分化BMSCs的0.070倍(P0.05)。结论 miR-124和miR-128在骨髓基质干细胞诱导分化为神经元过程中可能起重要作用。     

施万细胞条件培养基对大鼠骨髓间质细胞的诱导分化作用

目的　探讨施万细胞条件培养基对大鼠骨髓间质细胞的诱导分化作用。方法　从大鼠骨髓中分离培养间质细胞并传至第 6代 ,诱导前 2 4h加 1μg·L-1碱性成纤维生长因子 (bFGF)入培养液中以促分裂 ,再以施万细胞条件培养基作诱导剂 ,观察细胞形态的变化 ,并采用免疫组织化学法对诱导后一周的细胞进行Map 2及NSE、GFAP表达的检测。结果　诱导后 48小时间质细胞在形态上表现为神经元样 ,神经元样细胞呈Map 2及NSE阳性 ,而GFAP显阴性。结论　施万细胞的上清液能诱导骨髓间质细胞分化为神经元样细胞。     

DISTRIBUTION AND MULTIPLICATION OF COLONY FORMING UNITS FROM BONE MARROW AND SPLEEN AFTER INJECTION IN IRRADIATED MICE

The distribution and proliferation of CFUs from bone marrow and spleen cell suspensions were followed after injection in lethally irradiated isogeneic mice. It was found that a larger proportion of the injected bone marrow CFUs than of the spleen derived CFUs could be recovered from the recipient's spleen and femur. This consistently higher recovery points to the conclusion that a larger fraction of bone marrow-derived CFUs than of spleen-derived CFUs is capable of producing daughter CFUs, most likely due to a commitment to early differentiation of many spleen CFUs.     

CELL PROLIFERATION IN HAEMATOPOIETIC SPLEEN COLONIES OF MICE: DIFFERENCE BETWEEN COLONIES DERIVED FROM INJECTED ADULT BONE MARROW AND FOETAL LIVER CELLS

Cell proliferation in mouse spleen colonies, derived from injected foetal liver and young adult bone marrow, was studied by measuring incorporation of radio-iodine-labelled 5-iodo-2'-deoxyuridine (IUdR). Foetal liver-derived colonies incorporated significantly more IUdR than marrow-derived colonies on the 8th and 12th days after cell injection. The data are consistent with the view that foetal haematopoietic stem cells are capable, on average, of producing larger descendant populations than are stem cells from young adults.     

THYMIDINE SUICIDE IN VIVO AND IN VITRO OF SPLEEN COLONY FORMING AND AGAR COLONY FORMING CELLS OF MOUSE BONE MARROW

The ‘thymidine suicide’technique for indicating differences in the proliferation rate of early haemopoietic progenitor cells (spleen colony forming and agar colony forming cells) in C57BL mice has been evaluated. Special care was taken to use the same bone marrow cell suspension for the two progenitor cell assays. Both the in vivo and the in vitro techniques were employed. Following ³H-TdR in vivo, about 20% of both types of progenitor cell are killed in normal mice; however, after incubation in vitro with ³H-TdR, 35% of agar colony forming cells but only 4% of spleen colony forming cells are killed. Reasons for the difference between the in vivo and the in vitro results are discussed. With bone marrow from continuously irradiated animals, the thymidine suicide for both agar colony forming and spleen colony forming cells is in the range 42–50%, and there is no difference between in vivo and in vitro suicide. The in vivo results support the conclusion, based on the effect of proliferation dependent cytotoxic agents, that in C57BL mice agar colony forming and spleen colony forming cells are proliferating at the same rate in normal animals, and are speeded up to the same extent by continuous γ-irradiation. It is considered that in normal C57BL mice the in vitro method does not give a correct estimate of the proliferation rate of these progenitor cells. It would seem that the similarity in the proliferation rate of agar colony forming and spleen colony forming cells in C57BL mice is not true for other strains of mice: indeed using normal CBA and in vivo suicide, we have shown a significantly greater thymidine suicide for agar colony forming cells compared to spleen colony forming cells.     

正常人间围血及骨髓单个核白细胞和白血病肿瘤细胞的免疫分型

本文用10种单克隆抗体(McAb)分析了正常人周围血及有髓单个核细胞的免疫表型,以及急性淋巴细胞白血病(ALL)和急性髓细胞白血病(AML)的免疫表型,并以免疫双酶标记法观察了非T-ALL肿瘤细胞的肿瘤相关核仁抗原(HMNA)和细胞表面抗原的表达。结果:不同年龄正常群体周围血CD_4~+细胞数及CD_4/CD_8比值有差异;约3％的正常骨髓单个核细胞CD_(10)~+(ALL的抗原)。应用单克隆抗体对白血病的免疫分型,不仅能确诊肿瘤的谱系,还能了解细胞分化阶段。本文讨论了AML和ALL免疫分型中的鉴别性McAb。HMNA为多种肿瘤细胞的标记,本文观察到幼稚B细胞白血病及毛细胞白血病的肿瘤细胞中HMNA亦为阳性。