造血生长因子联合应用对骨髓粒-单系造血祖细胞体外增殖的影响

目的：研究不同造血生长因子联合应用对骨髓粒－单系造血祖细胞体外增殖的调节作用。方法：应用甲基纤维素体外半固体培养法，检测各种生长因子联合应用组，ＣＦＵ－ＧＭ集落的生长情况。结果：造血生长因子的联合应用不仅可增加ＣＦＵ－ＧＭ集落数，促进ＣＦＵ－ＧＭ集落的体外形成，而且，也能明显地增加ＣＦＵ－ＧＭ集落的体积，大型ＣＦＵ－ＧＭ集落数的比例在各联合应用组增加显著，其中以ＩＬ－３＋ＩＬ－６＋Ｇ－ＣＳＦ＋ＧＭ－ＣＳＦ联合应用组作用最强。同时，结果还显示，造血生长因子的联合应用可加速ＣＦＵ－ＧＭ集落的形成。结论：造血生长因子的联合应用能够协同地促进骨髓粒－单系造血祖细胞的体外增殖，从而为联合应用造血生长因子来扩增少量骨髓，外周血或脐血中造血干／祖细胞进行移植治疗白血病或实体瘤提供了实验依据。     

造血生长因子联合应用对骨髓粒—单系造血祖细胞体匀增殖的影响

目的：研究不同造血生长子联合应用对骨髓粒－单系造血祖细胞体外增殖的调节作用。方法：应用甲基纤维素体外半固体培养法，检测各种生长因子联合应用组，ＣＦＵ－ＧＭ集落的生物情况。结果：造血生长因子的联合应用不仅可增加ＣＦＵ－ＧＭ集落数，促进ＣＦＵ－ＧＭ集落的体外形成，而且，也能明显地增加ＣＦＵ－ＧＭ集落的体积，在型ＣＦＵ－ＧＭ集落数的比例在各联合应用组增加显著，其中以ＩＬ－３＋ＩＬ－６＋Ｇ－ＣＳＦ＋ＧＭ－     

重组集落刺激因子对大鼠正常骨髓及其白血病细胞体外增殖的影响

通过体外半固体培养集落形成和液体培养测定分析了重组的人或小鼠集落刺激因子对大鼠正常骨髓及其白血病细胞生长的影响。实验结果表明，重组的人巨噬细胞集落刺激因子（ｒｈＭ－ＣｓＦ），粒细胞集落刺激因子（ｒｈＧ－ＣＳＦ）和重组的小鼠粒－巨噬细胞集落刺激因子（ｒｍＧＭ－ＣＳＦ）能明显促进褐家（ｂｒｏｗｎＮｏｅｗａｙ）大鼠正常骨髓造血祖细胞的生长，在终浓度１０００ｋＵ·Ｌ－１时，它们的刺激指数分别为２８±ｓ１０，１２±ｓ４，２５±ｓ９。然而，ｒｈＧ－ＣＳＦ和ｒｍＧＭ－ＣＳＦ明显地抑制其白血病细胞集落形成，其ＩＣ５０分别为３５±ｓ８和１３４３±ｓ６３４ｋＵ·Ｌ－１也具有一定的抑制效应。同样，在液体培养中证实ｒｈＧ－ＣＳＦ和ｒｍＧＭ－ＣＳＦ明显地抑制ＬＴ１２白血病细胞ＤＮＡ合成及饱和生长密度（ＩＣ５０分别为１９１３和１８７ｋＵ·Ｌ－１）。重组的人粒－巨噬细胞集落刺激因子和小鼠多系集落刺激因子无论在半固体或液体培养中对大鼠正常骨髓造血祖细胞和白血病细胞的增殖均无明显影响。此外，还观察到大鼠急性白血病ＬＴ１２细胞经ｒｍＧＭ－ＣＳＰ（１０００ｋＵ·Ｌ－１）处理８ｄ后，６±ｓ１％的细胞显示具有还原硝基篮四氮唑的能力。     

脐血造血祖细胞体外培养及脐血清对其影响的观察

应用单层琼脂体外培养法观察了脐血、成人骨髓及成人外周血ＣＦＵ—ＧＭ生长情况，并观察了脐血血清对脐血和成人骨髓ＣＦＵ—ＧＭ形成的影响。结果脐血７天ＣＦＵ—ＧＭ集落产率与成人骨髓相当，而明显高于成人外周血，并有散在成纤维细胞生成，可自发形成红系集落形成单位（ＣＦＵ—Ｅ）。培养到１４天，脐血ＣＦＵ—ＧＭ集落增多、曾大、呈巨大型。表明脐血不仅富含造血细胞，且造血细胞更原始。在培养体系中加１０％脐血血清后，脐血和成人骨髓ＣＦＵ—ＧＭ产率均增多，集落增大，集落中细胞数可高达数千，表明脐血血清中有造血刺激物存在。作者认为胎盘血循环实际上是—个髓外造血场所，脐血造血细胞有望作为—种骨髓造血重建的干／祖细胞来源而用于移植。     

地黄低聚糖对快速老化模型小鼠造血功能的影响

目的：研究地黄低聚糖（ＲＧＯＳ）对快速老化模型小鼠（ＳＡＭＰ８）造血功能的影响。方法：采用造血祖细胞体外克隆培养等实验血液学方法。结果：ＲＧＯＳ１０，２０ｍｇ·ｋｇ－１可使ＳＡＭＰ８小鼠的ＣＦＵ－Ｓ、ＣＦＵ－ＧＭ、ＣＦＵ－Ｅ和ＢＦＵ－Ｅ明显增多，并可使外周血中降低的ＷＢＣ数明显增加。提示ＲＧＯＳ可刺激ＳＡＭＰ８小鼠造血干细胞、祖细胞的增殖分化。集落刺激活性实验显示ＲＧＯＳ可使小鼠体内的集落刺激因子产生明显增多。小鼠骨髓组织学研究结果也进一步证实了ＲＧＯＳ增强ＳＡＭＰ８小鼠造血功能的作用。结论：ＲＧＯＳ可以增强ＳＡＭＰ８小鼠的造血功能。     

脐带血造血祖细胞培养的观察

本组共收集２６份脐带血，１８份骨髓，２２份外周血，用琼脂半固体培养法，培养粒单系造血祖细胞。结果表明，脐带血ＣＦＵ－ＧＭ的平均率接近骨髓，明显高于成人外周血。脐带血富含造血祖细胞，既可作为造血干细胞移植新的来源，又可用于骨髓抑制患者的临床输注。有广阔的应用前景。     

异搏定对化疗药物体外净化白血病细胞的增强效应

用体外克隆形成培养技术研究异搏定（ＶＰＬ）对化疗药物阿霉素（ＡＤＭ）、长春新碱（ＶＣＲ）及足叶乙甙（ＶＰ－１６）体外净化白血病细胞作用的影响，结果显示ＶＰＬ能明显提高不同浓度的３种药物体外对Ｋ５６２、Ｒａｊｉ及Ｌ－ＣＦＵ的杀伤敏感性，而不增加其对ＧＭ－ＣＦＵ的毒性。２．２０μｍｏｌ·Ｌ－１ＶＰＬ联用０．９２μｍｏｌ·Ｌ－１ＡＤＭ、０．０２５μｍｏｌ·Ｌ－１ＶＣＲ或１．７１μｍｏｌ·Ｌ－１ＶＰ－１６对Ｌ－ＣＦＵ的杀伤作用分别为不加ＶＰＬ组的１．９６、１．６５及１．９５倍，而ＧＭＣＦＵ集落存活率则无明显改变，提示ＶＰＬ能选择性提高上述化疗药物的体外净化白血病细胞效果。     

乌贼墨对小鼠造血干细胞、粒-单系祖细胞及外周白细胞的影响

目的探讨乌贼墨对小鼠造血干细胞、粒-单系祖细胞及外周血WBC的影响。方法用不同剂量的乌贼墨灌胃正常小鼠、环磷酰胺(Cy)和辐射骨髓损伤模型小鼠,采用造血祖细胞体外培养方法和实验血液学技术,检测小鼠骨髓造血干细胞生成数(CFU-S)、粒-单系祖细胞集落生成数(CFU-GM)和外周血WBC数量。结果乌贼墨能够显著提高正常小鼠CFU-S,CFU-GM和外周血WBC数量,有效拮抗模型小鼠体内CFU-S,CFU-GM及外周WBC的降低,并显著促进模型小鼠上述各指标的恢复。结论乌贼墨具有显著的促进小鼠骨髓粒系造血作用。其作用机制可能通过调节机体的免疫机能,诱导机体产生粒/单核细胞集落刺激因子和多种细胞因子,促进造血细胞的增殖,并诱导造血细胞向粒单系细胞分化。     

组胺H_2受体激动剂和拮抗剂及抗癌药对正常人造血祖细胞和HL－60白血病细胞生长的作用

采用体外微量克隆培养体系研究了组胺Ｈ＿２受体激动剂４－甲基组胺（４－ＭＨ）和拮抗剂雷尼替叮（ｒａｎｉｔｉｄｉｎｅ）及抗癌药阿糖胞苷分别对正常人外周血粒－巨噬系祖细胞（ＰＢＣＦＵ－ＧＭ）和ＨＬ－６０白血病细胞生长的作用。当４－ＭＨ的浓度为１０￣（－９）～１０￣（－６）ｍｏｌ·Ｌ￣（－１）时，可促进ＰＢＣＦＵ－ＧＭ的增殖，４－ＭＨ的浓度增加至１０￣（－１）ｍｏｌ·Ｌ￣（－１）时则表现为抑制ＰＢＣＦＵ－ＧＭ的增殖。Ｒａｎｉｔｉｄｉｎｅ的浓度为１０￣（－９）～１０￣（－５）ｍｏｌ·Ｌ￣（－１）时，表现出对ＰＢＣＦＵ－ＧＭ增殖的抑制作用，但在１０－６ｍｏｌ·Ｌ￣（－１）剂量时对ＰＢＣＦＵ～ＧＭ的抑制率低于５０％，而在该剂量时对ＨＬ－６０白血病细胞的抑制率已达１００％，具有一定的选择性。抗癌药阿糖胞苷（Ａｒａ－Ｃ）对ＨＬ－６０白血病细胞的抑制作用比对ＰＢＣＦＵ－ＧＭ的抑制作用较强，但两者的ＩＣ＿（５０）值处于同一个数量级。在强化化疗剂量１０￣（－５）ｍｏｌ·Ｌ￣（－１）时，Ａｒａ－Ｃ对ＨＬ－６０白血病细胞和ＰＢＣＦＵ－ＧＭ正常造血祖细胞的抑制率均达１００％。     

粒─巨噬细胞集落刺激因子的临床药理研究进展

粒─巨噬细胞集落刺激因子的临床药理研究进展粒－巨噬细胞集落刺激因子（ｇｒａｎｕｌｏｃｙｔｅ－ｍａｃｒｏｐｈａｇｅｃｏｌｏｎｙ－ｓｔｉｍｕｌａｔｉｎｇｆａｃｔｏｒ，ＧＭ－ＣＳＦ）是一种促进正常造血细胞增殖和分化的多肽类激素，对粒细胞和单核／巨噬细胞的生...     

微量液体克隆形成法体外检测三尖杉酯碱、高三尖杉酯碱对CFU—GM及L—CFU的细胞毒作用

基于琼脂半固体培养法和极限稀释微孔池培养法,建立了一种新的体外药敏检测体系——微量液体克隆形成法。并应用该体系检测了三尖杉酯碱(H)和高三尖杉酯碱(HH)对CFU—GM及L—CFU的细胞毒作用。结果提示:24h及168h作用法时,HH对CFU—GM以及L—CFU的细胞毒作用均大于H,H、HH对L—CFO的细胞毒作用选择性均大于CFU—GM,但HH的细胞毒作用选择性不及H;H、HH的TDI(Time—schedule Depne—dence Index),IC_(90-24h)÷IC_(-90-168h)分别为68、60,说明两药物均为细胞周期时相特异性药物;并对H、HH之间可能存在的同系物间的交叉耐药性作了探讨。     

The effect of cytotoxic drugs on the development of murine granulocyte-macrophage (GM) progenitor cells.

The effect of cytotoxic drugs was tested on the hematopoietic system by assay of the ability of granulocyte-macrophage colony forming cell (GM-CFC) in mice to create GM colonies. The in vivo ability of bone marrow progenitor cells to granulocyte-macrophage colony formation, was tested after long-term peritoneal cytotoxic drug administration. The direct effect of these agents on cells in vitro culture was evaluated also. It was found that cytotoxic drugs inhibit the GM colony formation. The degree of damage to the bone marrow progenitor cells by assaying in vivo colony formation inhibition, depends on the drug dosage and length of therapy (after 25-30 days of treatment the colony growth was below 50%). The in vitro inhibition of granulocyte-macrophage colony formation depends on the concentration of drug (10(-7)-10(-5) M is critical for GM colony growth). The results suggest the possibility of the GM-CFC growth testing as an indicator of the progress or side effects of cytotoxic therapy.     

小鼠干细胞因子基因以脂质体介导转染脐血细胞

目的小鼠干细胞因子 (SCF)以脂质体介导转染脐血细胞并表达。方法采用PCR技术从 pRC/CMV(含SCFcDNA)中钓取SCFcDNA膜外段活性区 ,克隆入真核表达载体 pcDNA3 ,转染传 2代的脐血细胞 ;用RT PCR方法检测mRNA表达水平及通过协同刺激集落形成来检测转染细胞上清的生物活性。结果转染SCFcDNA的脐血细胞SCFmRNA水平表达量高于对照组 ,其细胞培养上清协同GM CSF刺激骨髓细胞形成集落数比GM CSF单独作用高两倍左右。结论脂质体介导质粒载体转染培养富集的脐血细胞并表达 ,且转染上清具有生物学活性 ,为进一步研究转染基因的表达特性及持续时间提供依据。     

Enhanced proliferation of functionally competent bone marrow cells in different strains of mice treated with swainsonine

The immunomodulatory alkaloid swainsonine (8alphabeta-indolizidine-1alpha,2alpha,8beta-triol) has potential for overcoming the bone marrow suppressive effects of cancer chemotherapy and radiotherapy. An earlier study showed that multiple doses of swainsonine enhanced bone marrow cellularity in four different strains (C57BL/6; C3H-HEN; Balb/C and DBA-2 mice) of inbred mice which were not exposed to any chemotherapeutic agents or radiation. In vitro assessment of total colony formation capacity of bone marrow cells (BM CFUs) showed a 2- to 8-fold increase in swainsonine-treated mice compared to control mice that were given sham injections of physiological saline. In the current study, we have evaluated the functional competence of the bone marrow cells produced in response to swainsonine treatment of normal healthy mice. In particular, colony forming units-granulocyte-macrophage (CFU-GM), erythroid-burst forming units (BFUe) and CFU-Mix (or CFU-granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM)) levels, were determined using in vitro assays. The time course of the changes in CFU-GM, BFUe and CFU-Mix (CFU-GEMM) were also followed. Our results demonstrate that swainsonine bolsters the CFU capacity of BM cells without loss of function to levels which are several folds higher than in sham-treated control mice. Swainsonine treatment caused an increase in all lineages of marrow cells without loss of function. This effect was reproduced in all four strains of inbred mice in this investigation. Examination of the peripheral blood did not reveal increase in white blood cells or changes in the hematocrit levels. The long-term effects of swainsonine treatment are not known at present. Nonetheless, swainsonine-induced increase in CFU capacity of bone marrow cells and related cells along the different differentiation paths should find clinical application in cancer treatment with chemotherapeutic agents and/or radiation.     

The Regulation of Ganglioside GM3 Synthesis

Glycosphingolipids (GSLs) exist in the outer leaflet of the plasma membrane, where they form lipid microdomains that function as platforms for the regulation of trans-membrane signal transduction. In mammals, complex GSLs differing in the number and/or type of sugar species are produced in a cell-type specific manner, and the variety of glycan structures in GSLs are believed to determine specific cell functions. The glycan moieties of GSLs are synthesized at the luminal side of the Golgi apparatus by multiple Golgi-resident glycosyltransferases. Since the expression levels of most endogenous glycosyltransferases are relatively low, their detection is generally difficult. Nevertheless, we have succeeded in detecting endogenous mouse GM3 synthase (GM3S), the primary glycosyltransferase responsible for the biosynthesis of ganglio-series gangliosides. Mouse GM3S (mGM3S) has three isoforms (M1-GM3S, M2-GM3S, and M3-GM3S), each with a distinct length in its NH2-terminal cytoplasmic tail. These isoforms are produced by leaky scanning from two mRNA variants, mGM3Sa and mGM3Sb. M1-GM3S is stably localized in the endoplasmic reticulum (ER), as a result of retrograde transport signals (arginine [R]-based motifs); consequently, its in vivo GM3 synthesis activity is very low compared with that of other isoforms. In contrast, both M2-GM3S and M3-GM3S are localized in the Golgi apparatus, yet each exhibits a distinct intracellular fate. M2-GM3S is rapidly degraded in the lysosomes, whereas M3-GM3S is retained in the Golgi apparatus. A system that produces GM3S isoforms having such distinct characteristics is likely to be of critical importance in the regulation of GM3 biosynthesis under various pathological and physiological conditions.     

Acute inflammation effects on in vivo granulopoiesis: comparative studies in healthy and leukaemic mice.

Mice undergoing an inflammatory reaction, induced by subcutaneous implantation of copper rods, elaborate two kinds of humoral stimulatory factors: the diffusible granulopoietic stimulator (DGS) that enhances diffusion chamber (DC) granulopoiesis, and the serum colony stimulating factor (CSF) that stimulates in vitro granulocyte-monocyte colony growth. We demonstrate here that mice suffering from acute myeloid leukaemia (AML) are unable to augment the production of these humoral stimulatory factors when acute inflammation is induced. Moreover, our results show that increased levels of normal humoral stimulatory factors (DGS and CSF) do not influence the proliferation and/or the differentiation of leukaemic cells implanted in DC.     

Swainsonine stimulates bone marrow cell proliferation and differentiation in different strains of inbred mice.

The immunomodulatory alkaloid swainsonine (8alphabeta-indolizidine-1alpha,2alpha,8beta-triol) has potential for overcoming the bone marrow suppressive effects of cancer chemotherapeutic drugs and radiation. The effect of swainsonine on bone marrow cellularity was evaluated in four different strains (C57BL/6; C3H-HEN; Balb/C and DBA-2 mice) of inbred mice subjected to multiple doses of the alkaloid. Swainsonine treatment stimulated bone marrow cell proliferation in all strains of mice. Examination of the peripheral blood did not reveal any increase in total leukocyte count. In vitro assessment of total colony-forming unit (CFU) capacity of bone marrow cells showed a two- to eight-fold increase in swainsonine treated mice of different strains compared to their corresponding controls given sham injections of physiological saline. Swainsonine induced increase in CFU capacity of bone marrow cells should find clinical application in cancer treatment with chemotherapeutic agents and radiation.     

The effect of glucocorticoids on the development of murine granulocyte-macrophage (GM) progenitors.

The effect of glucocorticoids was tested on the hematopoietic system by assay of the ability of granulocyte-macrophage colony forming cells (GM-CFC) in mice to create GM colonies. The in vivo ability of bone marrow progenitor cells to granulocyte-macrophage colony formation was tested after long-term peritoneal glucocorticoids administration. The direct effect of these agents on cells in vitro culture was evaluated also. It was found that glucocorticosteroids inhibit the GM colony formation. The degree of damage to the bone marrow progenitor cells assaying by in vivo colony formation inhibition depends on the drug dosage and the length of therapy. The in vitro inhibition of granulocyte-macrophage colony formation depends on the concentration of the drug. The results suggest the possibility of the GM-CFC growth testing as an indicator of the side effects of prolonged corticoid therapy.