造血干细胞辐射敏感性的比较

本文研究了在体内外照射条件下小鼠骨髓多向性造血干细胞(CFU-S)和粒系定向千细胞(CFU-C)的辐射敏感性。它们的 D_0值分别为103.4拉德和173.7拉德,反映了CFU-S 和 CFU-C 是两类在性质上不全相同的干细胞群。在目前尚未建立起除啮齿类动物以外的多向性造血干细胞测定技术的情况下,我们应用体外琼脂培养技术,比较了在体外照射条件下狗与小鼠骨髓 CFU-C 的辐射敏感性,它们的 D_0值分别为58.6拉德和190.2拉德,反映了两者在辐射敏感性上存在着明显的差异,为阐明不同种属动物的不同辐射耐受性提供了一个根据。联系到 AET 具有降低造血干细胞辐射敏感性以及提高动物的辐射耐受性的事实,进一步论证了在造血型放射病的发生和发展中,造血干细胞的损伤和修复是起着重要作用的。     

豚鼠骨髓巨核细胞集落(CFU-M)和粒细胞集落(CFU-C)的特性

小鼠胸腺细胞条件培养基可促进豚鼠骨髓造血祖细胞在体外琼脂和血浆凝块培养基中形成 CFU-M 和 CFU-C。培养基中种入1×10~4—2×10~5个骨髓有核细胞,在培养7天后 CFU-M 和 CFU-C 形成的数量与种入的骨髓细胞数之间呈线性关系。CFU-M 和 CFU-C 的祖细胞具有很高的辐射敏感性,骨髓细胞体外接受~(60)钴γ线照射后 CFU-M 的计数呈指数下降,其D_0值为92拉德与 CFU-C(D_0=88拉德)相近似。     

IL-33转基因小鼠骨髓造血干／祖细胞向髓系细胞分化的能力增强

目的：利用IL-33转基因小鼠研究IL-33对造血干/祖细胞的增殖和分化影响。方法利用流式细胞仪分析IL-33转基因小鼠及同窝野生对照小鼠的外周血、脾脏、骨髓细胞的免疫表型及造血干细胞分化不同阶段细胞的数量变化;利用体外成克隆实验和细胞周期分析研究IL-33对于造血干细胞增殖能力的影响。结果与野生型小鼠相比,IL-33转基因小鼠B细胞和T细胞在外周血中都明显降低,粒细胞在外周血和骨髓中都有明显增加;IL-33转基因小鼠的骨髓造血干细胞和多能祖细胞数量减少,共同淋系祖细胞数量减少,共同髓系祖细胞和粒单系祖细胞数量增加;IL-33转基因小鼠的造血干细胞处于S-G2-M的细胞增多;体外单克隆实验发现IL-33转基因小鼠造血干细胞形成的集落数增加。结论 IL-33转基因小鼠造血干细胞增殖能力增强,更易向髓系细胞分化。     

大剂量~(60)Co照射后存活小鼠造血干细胞阈值的测定

本文应用CFU_s的剂量-存活曲线和干细胞移植后受体存活率测定法,摸清了F_1小鼠在临界死亡剂量—8.5 Gy γ-线照射后,存活的造血干细胞数量只有照前的1×10~(-4),约为20个左右的CFU_s或200个左右的HSC。因此,用造血干细胞计数可以作为估计辐射剂量和放射病早期诊断的敏感检测指标。这一细胞阈值的测定,为一些疾病的骨髓输注或骨髓移植治疗时的细胞数量提供了重要的依据。     

循环血造血干细胞的性能

造血干细胞是一类能够自我更新和多向分化的原始细胞群,主要分布于骨髓,也有少部分分布于循环血。在造血干细胞研究这一领域,过去对循环血中造血干细胞的研究较少,直至七十年代才渐渐活跃起来。为了对造血干细胞群体有一个较全面的了解,本文就循环血造血干细胞的性能作简要的综述。     

粒单系造血祖细胞(CFU-GM)琼脂半固体培养的原位固定及鉴定染色

用粒单系造血祖细胞（CFU－GM）体外琼脂半固体培养法,琼脂块原位固定,Wright染色与细胞化学和免疫细胞化学技术,对培养人的骨髓CFU－GM进行集落性质鉴定,结果表明：Wright染色能区别集落的性质及细胞的分化程度：CFU－GM的细胞中PAS、POX呈阳性反应;CD14、CD15分别在单核细胞和粒细胞的胞浆或胞膜上表达。该方法对鉴定细胞集落类型、集落中细胞的分化与成熟程度是简便可靠的手段。     

四物汤对再生障碍性贫血小鼠骨髓细胞增殖的影响研究

目的研究四物汤对再生障碍性贫血（AA）小鼠骨髓细胞体外增殖的影响,探讨其治疗AA的机制。方法采用流式细胞仪、骨髓造血祖细胞培养等技术,检测四物汤对AA小鼠骨髓细胞的增殖变化。结果 四物汤能促进骨髓有核细胞进入G2／S期、增加CFU—GM、CFU-E、BFU-E集落数,且与自然恢复组有明显增强的差异。结论 四物汤在体内有促进AA小鼠骨髓细胞增殖的作用,为补血药治疗AA提供了实验与理论依据。     

低能量激光对造血细胞的增殖作用

激光能刺激造血细胞增殖,促使粒-巨噬细胞集落形成单位(GM—CFUc)增多。从人脐带血中分离制备的有核细胞,加入集落刺激因子(CSF)后,形成的GM—CFUc为18.6±12.6/10~4;如果加入的CSF是经激光照射后的细胞制成的,其GM—CFUc为40.5±20.2/10~4;若先用激光照射脐带血的有核细胞,在培养时加入的是未经激光处理细胞制成的CSF,也有增殖效应,GM—CFUc为36.5±18.5/10~4;如果脐带血有核细胞和制备CSF的细胞都用低能激光照射,就可以见到GM—CFUc明显地增多,其值为57.4±41.2/10~4是未经激光处理者的三倍。表明激光照射脐带血有核细胞及制备CSF的细胞均可使GM—CFUc增殖。     

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。     

造血干细胞相关microRNAs的筛选及其促分化研究

为了获得人脐血造血干细胞(HSCs)的microRNAs(miRNAs)表达谱,并对相关miRNAs功能进行初步鉴定.利用免疫磁珠(MACS)和流式细胞仪(FACS)细胞分选技术分离人脐血造血干细胞(HSCs),分别提取细胞总RNA并分离小分子RNA,经荧光标记后与miRNAs基因芯片杂交,获得HSCs的miRNAs表达谱,集落形成实验(CFC)研究在HSC中高表达miR-520h对HSC的促分化作用.成功分离人脐血CD34 细胞和HSC,经基因芯片杂交获得31个造血干细胞相关miRNAs,其中22个为低表达,9个为高表达;经实时定量RT-PCR验证miR-520h显著升高,CFC实验表明其可增加多种集落形成,具有促进HSC向祖细胞分化的作用.上述结果表明,人脐血HSC具有自身特征性miRNAs,参与并调控HSC生物学功能,为深入探讨miRNAs在造血系统发育中的作用打下基础.     

小鼠巨核系祖细胞增殖,分化特性的研究

小鼠骨髓细胞经7d培养后进行细胞形态学观察,可见不同发育阶段的巨核细胞及不同大小的巨核细胞集落。通过计数每个集落中的细胞数,可确定相应祖细胞的有丝分裂能力。结果表明,具有不同有丝分裂能力的祖细胞的体外增殖动力学有所不同。祖细胞的数量与其有丝分裂次数呈负相关(r=-0.986)。进行0、1、2和3次有丝分裂的祖细胞的阿糖胞苷自杀率分别为48.9,58.7,48.0和41.2％;放射敏感性的D_O值(Gy)分别为1.71,1.24,1.03和0.77,D_O值的大小与有丝分裂次数呈负相关(r=-0.958)。经3Gy全身照射后CFU-Meg与CFU-GM的恢复动态过程具有不同特点。     

Comparative radiosensitivity of CFUs of the mouse bone marrow and embryonal liver forming 7- and 11-day colonies

The authors studied the radiosensitivity of CFU-s, forming 7- and 11-day colonies from fetal liver (FL) of 14 and 17 day gestation and bone marrow (BM) of adult mice. The index of D0 for 7-day colonies, formed by CFU-s from 14-day, 17-day FL and BM was 2.02; 1.57 and 0.78 Gy, accordingly. 11-day CFU-s both from FL, and from BM did not distinguish statistically at their radiosensitivity (their D0 was 1.25 Gy).     

Ex vivo expansion and clonality of CD34+ selected cells from bone marrow and cord blood in a serum-free media

Although umbilical cord blood is increasingly being used in allogeneic marrow transplantation, delayed platelet engraftment is often a concern for cord blood transplant recipients. We evaluated the potential of ex vivo expansion and clonality in CD34+ cells separated from a bone marrow source, and cord blood, in a serum-free Media. The CD34+ cells, selected from bone marrow (BM) and umbilical cord blood (CB), were expanded with hematopoietic growth factors. They were then cultured for burst-forming units of erythrocytes (BFU-E), colony-forming units of granulocytes and monocytes (CFU-GM) and colony-forming units of megakaryocytes (CFU-Mk) at days 0, 4, 7, and 14 under the combination of growth factors, with cell counts. The cytokines included the recombinant human megakaryocyte growth and development (100 ng/ml), interleukin-3 (10 ng/ml), stem cell factor (100 ng/ml), flt-3 ligand (50 ng/ml) and interleukin-11 (200 ng/ml). The CB-selected CD34+ cells showed significantly higher total cell expansion than those from the BM at day 7 (3.0 fold increase than BM), day 14 (2.4 fold), and day 17 (2.6 fold). The colony count of the BFU-E/CFU-E per CD34+ cell at day 0 was 0.14 +/- 0.023 in the CB, which was significantly higher than 0.071 +/- 0.015 in the BM. The CB-selected CD34+ cells produced more BFU-E colonies than the BM on culture days 4, 7, and 14. The BFU-E colonies from the CB cells increased markedly on culture days 4 and 7, with a 4-fold increase at day 14. The colony count of the CFU-Mk per CD34+ cell at day 0 was 0.047 +/- 0.011 in the CB-selected CD34+ cells cultures, which was higher than the 0.026 +/- 0.014 in the BM. The CB-selected CD34+ cells produced more CFU-Mk colonies than the BM on culture days 4, 7 and 14. In conclusion, the ex vivo expansion of the CB cells may be very promising in producing total cellular expansion, CFU-Mk and BFU-E compared with BM, especially at day 7. The ex vivo expansion of the CB may have rationale in making an ex vivo culture for 7 to 14 d.     

Radiosensitivity of human bone marrow granulocyte-macrophage progenitor cells and stromal colony-forming cells: effect of dose rate

Study of the radiation biology of human bone marrow hematopoietic cells has been difficult since unseparated bone marrow cell preparations also contain other nonhematopoietic stromal cells. We tested the clonogenic survival after 0.05 or 2 Gy/min X irradiation using as target cells either fresh human bone marrow or nonadherent hematopoietic cells separated from stromal cells by the method of long-term bone marrow culture (LTBMC). Sequential nonadherent cell populations removed from LTBMC were enriched for hematopoietic progenitors forming granulocyte-macrophage colony-forming unit culture (GM-CFUc) that form colonies at Day 7, termed GM-CFUc7, or Day 14 termed GM-CFUc14. The results demonstrated no effect of dose rate on the D0 or n of fresh marrow GM-CFUc (colonies greater than or equal to 50 cells) after plating in a source of their obligatory growth factor, colony-stimulating factor (CSF) (GM-CFUc7 irradiated at 2 Gy/min, D0 = 1.02 +/- 0.05, n = 1.59 +/- 0.21; at 0.05 Gy/min, D0 = 1.07 +/- 0.03, n = 1.50 +/- 0.04; GM-CFUc14 at 2 Gy/min, D0 = 1.13 +/- 0.03, n = 1.43 +/- 0.03; at 0.05 Gy/min, D0 = 1.16 +/- 0.04, n = 1.34 +/- 0.05). There was a decrease in the radiosensitivity of GM-CFUc7 and GM-CFUc14 derived from nonadherent cells of long-term bone marrow cultures compared to fresh marrow that was observed at both dose rates. In contrast, adherent stromal cells irradiated at low compared to high dose rate showed a significantly greater radioresistance (Day 19 colonies of greater than or equal to 50 cells; at 2 Gy/min, D0 = 0.99 Gy, n = 1.03; at 0.05 Gy/min D0 = 1.46 Gy, n = 2.00). These data provide strong evidence for a difference in the radiosensitivity of human marrow hematopoietic progenitor compared to adherent stromal cells.     

Differentiation from precursors in normal bone marrow of spontaneously cytotoxic cells showing anti-self-MHC specificity

Colonies containing spontaneously cytotoxic effector cells with specificity for target cells carrying self-MHC can be grown from normal mouse bone marrow (BM). BM was first depleted of nylon wool-adherent cells and was then cultured at low cell number (1 to 300 cells/culture) in multiple replicate microcultures in liquid culture medium containing supernatant from EL4 thymoma cells stimulated with PMA. Frequency of colony growth followed one-hit limiting dilution kinetics. Colonies contained lymphoid, myeloid, or both kinds of cells. About 5% of colonies contained self-specific cytotoxic effector cells. Analysis using the X chromosome-linked isoenzyme PGK-1 confirmed that colonies containing autoreactivity could be clonal. A factor other than IL 2, IL 3, or PMA appears to be required for the growth of autoreactive colonies. Similar colonies, both with and without autoreactive effector cells, could also be grown from the BM of athymic nude mice with frequencies and cytotoxic activities directly comparable to those found for normal BM. C.B-17 scid mice lack both B and T cells, apparently due to a block in the development of lymphoid stem cells. Colonies could be grown with comparable frequency from their BM, but these colonies lacked both lymphoid cells and spontaneous cytotoxic activity. Evidence is presented against the self-reactive effector cells being NK cells, macrophages, or mature T cells. It is speculated that they represent an early stage of the T cell differentiation pathway.     

Stimulation of retroviral vector infection of murine hematopoietic progenitors

Conditioned media (CM) from a cloned murine marrow-derived stromal cell line, AC6.21 (ALC), was shown to stimulate retroviral vector infection of hematopoietic progenitors in culture. Inclusion of ALC CM during cocultivation of normal murine bone marrow (BM) with vector-producing fibroblasts improved infection efficiency of day 13 spleen colony-forming cells (CFU-s) from 63% (15 provirus-positive spleen colonies/24 total), without added growth factor, to 90% (36 provirus-positive colonies/40 total). In addition, stimulation of BM cells with ALC CM during cocultivation improved retroviral infection of stem cells capable of repopulating the hematopoietic system of irradiated recipient animals. Because ALC CM was found to have 50 to 100 U/ml of IL-6 activity, purified recombinant human IL-6 was tested for an effect in this system. Stimulation with IL-6 alone increased retroviral infection efficiency of CFU-s from 15% (17 colonies provirus-positive/111 total analyzed) without added growth factor to 66% (97 provirus-positive colonies/148 total analyzed). These experiments support and extend previous studies which have demonstrated the necessity for growth factor stimulation in optimizing retroviral vector transduction of hematopoietic precursors.     

球形棕囊藻的生长特性及生活史研究

对球形棕囊藻不同的藻株(香港株HK和汕头株ST)在实验室条件下分别进行了形态学,生活史及生长曲线的研究,结果表明:球形棕囊藻具有一个复杂的异型生活史,具有两种不同的生活形态:群体和游离的单细胞,香港株和汕头株二者单细胞呈球形或近球形,直径大多为3-9μm;群体呈中空的球形囊泡,细胞包埋在胶质囊中较均匀分布,但二者群体大小有较大差异。当培养达到一定阶段,群体衰亡破裂释放大量单细胞。批次培养中球形棕囊藻的生长周期约为20-30d,香港株最适生长温度接近25℃,最大比生长率为038;汕头株的最适生长温度接近30℃,最大比生长率为042,这说明温度是重要的生长限制因子之一。       

FETAL HEMOPOIESIS IN DIFFUSION CHAMBER CULTURES

The growth pattern of fetal liver (FL), normal adult bone marrow (NABM) and regenerating (post Velban treatment) adult bone marrow (RABM) colony forming units (CFU) cultured in diffusion chambers (DC) was studied. When twenty CFU were implanted into DC the recovery of CFU after 4 days with FL, NABM or RABM was 133 ± 7, 19 + 2 and 34 ± 2 CFU, respectively. The transplantation fraction of CFU from NABM decreased from 10-4% on day 0 to 6–9 % on day 4; that of FL did not change from the initial 6-2%. The growth rate of CFU derived from FL was substantially greater than that from NABM. The relative growth of FL and RABM CFU was clearly inhibited when the concentration of cells cultured was increased. Spleen colonies from FL cells before culture were larger (P < 0–005) than colonies from NABM but after 7 days of culture there was no difference between the two groups. Histological examination of spleen colonies showed that after DC culture FL and NABM CFU were differentiating along the three normal pathways. These data suggest that intrinsic differences exist between fetal and adult stem cells in the in vivo diffusion chamber culture system.     

云南红豆杉细胞发酵培养的研究

利用云南红豆杉(Taxus yunnanensis)细胞悬浮培养的最佳培养条件,进行了细胞10L规模的发酵培养研究。对细胞发酵培养过程中的pH值变化、糖利用率以及细胞生长周期等实验参数进行了测定。已进行的3次发酵培养的实验结果表明:培养细胞的生长率已达0.4g/L.d(即12.0g/L),培养细胞中紫杉酵的含量为0.119％(其中培养液中的紫杉醇含量约占42％)。初步建立了优化的云南红豆杉细胞大量培养系统。     

The sensitivity of mesenchymal stromal cells subpopulations with different adhesion properties and derived from hemopoietic organs to growth factors EGF,bFGF, and PDGF

The action of three growth factors (EGF, bFGF, and PDGF) on mesenchymal stromal cell (MSC) subpopulations from rat adult bone marrow (BM) and embryo liver (EL) was investigated. These cells are plastic-adhesive and have different rates of adhesion (AS1–AS4 subpopulations). The efficiency of colony-formation, the size of colonies, and the number of early osteogenic progenitors with alkaline phosphatase activity in colonies and induced osteogenesis were analyzed. It was shown that EGF increased the number of bone marrow (BM) MSC colonies, but it had no influence on osteogenic differentiation. bFGF suppressed colony formation, but it stimulated both early and late stages of osteogenesis. PDGF increased the size and the number of colonies in AS2 and AS3 subpopulations, but it stimulated only the terminal stage of ostegenesis. The distinction between MSC subpopulations from two organs was found: MSC from EL had small osteogenic capacities and low sensitivity to grow factors; MSC from BM had no such characteristics. MSC subpopulations with different adhesion properties and from different tissues had compatible sensitivity to growth factors.