小鼠骨髓高增殖潜能内皮祖细胞的培养与纯化（简报）

1997年Asahara等在人外周血中发现内皮祖细胞（endothelial progenitorcell．EPC）,随后有文献报道,从骨髓中分离出EPC．异体骨髓移植研究显示成体外周血EPC来源于骨髓。许多作者报告EPC参与生理性和病理性血管新生．具有潜在的临床应用前景。目前关于EPC的不少基本生物学问题尚不完全清楚,其生理和病理意义也有争议。     

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.     

人骨髓CD34^＋造血细胞体外扩增形成HPP—CFC造血祖细胞的能力

目的和方法：高增殖潜能集落形成细胞（ＨＰＰＣＦＣ）是表达ＣＤ３４＋ＤＲＬｉｎ－的最早期造血祖细胞之一,它在体外的增殖分化能力可反映造血干细胞的某些特征。结果：本文研究了人正常骨髓ＣＤ３４＋造血细胞在体外扩增和形成ＨＰＰＣＦＣ的能力。利用ＣＩＭＳ１００免疫磁性分离术首先获得＞９０％的ＣＤ３４＋造血细胞以富集ＨＰＰＣＦＣ。在含有Ｅｐｏ＋ＧＭＣＳＦ＋ＩＬ３＋ＩＬ６＋ＳＣＦ（简ＥＧＩＩＳ）的无基质液培条件下,ＣＤ３４＋造血细胞在四周内可持续产生单个核细胞和ＨＰＰＣＦＣ,并使其总量最高可达１７７０倍和８倍,以第２和３周为最佳时期,但不同个体ＣＤ３４＋造血细胞的这种能力差别较大。结论：高度纯化的人骨髓ＣＤ３４＋造血细胞能够在含有最佳组合造血生长因子的无基质液培条件下持续扩增,为临床应用提供了重要依据。     

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。     

THE PROLIFERATIVE STATE OF GRANULOCYTIC PROGENITOR CELLS IN HUMAN BLOOD AND MARROW

A double layer agar technique was used to investigate the proliferative state of granulocytic progenitor cells (Colony Forming Units in Culture; CFUc) in human peripheral blood and bone marrow. The sensitivity of the progenitor cells to the S-phase specific agent, hydroxyurea, was used as an index of the proportion of cells engaged in DNA synthesis. In the presence of low concentrations of colony stimulating factor (CSF) the CFUc were found to be virtually insensitive to the drug. However, when cultured in the presence of increasing concentrations of CSF the proportion of CFUc apparently killed by hydroxyurea increased to a maximum of 23% for those cells in the blood and 39% for those in the marrow. The results indicate that CFUc which are slowly proliferating are sensitive to low concentrations of CSF. In contrast, those CFUc which are proliferating more rapidly require high concentrations of CSF before they will form colonies in culture. A model has been devised which suggests that as CFUc mature, their cell cycle time shortens and their sensitivity to CSF decreases.     

骨髓动员对骨髓造血干细胞在心肌梗死后心脏中定植及分化的影响

目的观察小鼠心肌梗死后骨髓造血干细胞在心脏内的分化及细胞因子的影响。方法C57/BL6小鼠60只分为骨髓动员组和对照组,先后行脾切除、骨髓移植（骨髓供体为增强绿色荧光蛋白转基因小鼠）、骨髓动员及建立心肌梗死模型。心肌梗死后3周将小鼠心脏取出并切片行组织学及激光共聚焦显微镜免疫荧光检查。结果骨髓动员可以增加EGFP阳性细胞在心脏中梗死区和边缘区的定植,但绝大多数EGFP阳性细胞都同时表达CD45。仅发现有极少数骨髓来源的心肌细胞、成纤维细胞及血管内皮细胞,且与骨髓动员无相关性。结论骨髓动员能够明显促进骨髓来源细胞定植入小鼠心脏的梗死区;极少数骨髓造血干细胞可以分化为心肌细胞,其数量远不足以修复梗死心肌及改善心功能;骨髓造血干细胞不参与梗死区疤痕形成的病理过程。     

培养骨髓基质细胞的尿嘧啶核苷二磷酸半乳糖-4-表异构酶活性

本实验用液体静置法体外培养小鼠骨髓基质细胞。用细胞化学法观察处于不同分化阶段基质细胞中的尿嘧啶核苷二磷酸半乳糖－－４－表异构酶（Ｕｒｉｄｉｎｅｄｉｐｈｏｓｐｈｏｇａｌａｃｔｏｓｅ－４－ｅｐｉｍｅｒａｓｅ,ＵＤＰＧａｌ,ＥＣ５．１．３．ａ）活性。结果在各类骨髓基质细胞中均可见到很强的酶反应。在由原始网状细胞向成熟星状细胞分化过程中,酶活性逐渐增强。随着培养时间和延长（培养５、７、１０天）,星状细胞和成纤维细胞中酶反应的阳性率和阳性度逐渐增高。这证明在体外培养骨髓基质细胞中由ＵＤＰＧａｌ催化的代谢非常活跃     

骨髓移植后的白血病患者环境微生物控制与感染关系研究

白血病患者骨髓移植后,易引起各类微生物感染而死亡。通过F_1鼠9Gy^(60)Co照射(最大致死量)骨髓移植后,行环境微生物控制,将其数据结合临床特点,设计的白血病患儿骨髓移植,环境微生物监控的层流病房。自1989～1993年12例患儿行骨髓移植无一例感染。     

THE RELATIONSHIP BETWEEN SPLEEN COLONY PRODUCTION AND SPLEEN CELLULARITY

The fraction, f, of injected spleen colony-forming cells which can be recovered from the spleen of a radiated mouse has been determined at various times up to 24 hr following the initial cell injection. the cellularity of the spleen at the time of assay was also measured and compared with the calculated f number. the linear relationship between these two parameters indicated that over the period of 2-24 hr the number of CFU/10⁶ spleen cells was constant, both falling in a parallel fashion. A further experiment using non-irradiated W/W^v mice in which the spleen size did not change showed the same f number at the end of this period as at 2 hr. It is concluded that CFU are expelled from the spleen during its post-irradiation contraction thus leading to the apparent fall in f number. It is also concluded that a more realistic f number is obtained by assaying the splenic CFU content 24 hr after the primary cell transfer rather than 2 hr.     

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。     

多发性骨髓瘤的骨髓细胞长期培养的研究

多发性骨髓瘤的骨髓细胞置于Dexter长期培养体系中,浆细胞可以长期生长。非贴壁层细胞形成CFU-GM的能力达6周以上,但随着培养时间的延长,CFU-GM的形成活性明显下降。浆细胞产生兔疫球蛋白单克隆特异性在Dexter长期培养体系中仍维持不变,但Ki-67阳性的浆细胞随着培养时间的延长而减少,以上结果说明浆细胞虽然可以在Dexter培养体系中生长并保持单克隆特异性,但此种培养体系不是浆细胞最佳的生长条件。本研究还表明Dexter长期培养不宜作为骨髓净化的手段用于多发性骨髓瘤的自体骨髓移植。     

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.     

DNA-SYNTHESIS TIME OF BONE MARROW CELLS IN HEALTHY AND ASCITES TUMOR-BEARING MICE

DNA-synthesis (S) times of myelocytes and nucleated erythroid cells in the bone marrow of healthy mice as well as mice bearing advanced Ehrlich ascites tumors were measured with the aid of a combined in vivo-in vitro double isotope labeling technique. Neither the S-period nor the rate of proliferation of these cells were influenced by the presence of the tumor in these hosts. This finding discounts the possibility that the marked retardation of DNA-synthesis and proliferation rate observed in the tumor cells themselves with advancing tumor-age is a nonspecific effect of the nutritional deterioration in the host.     

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。     

ASSESSMENT OF VIABILITY OF FROZEN BONE MARROW CELLS USING A CELL-CULTURE METHOD

The survival of mouse bone marrow after freezing and thawing was assessed using two methods: the spleen-colony technique; and a cell-culture method. The results of these experiments indicate that both methods yield similar estimates of marrow cell viability. It is suggested that the cell-culture method be used as an assay of marrow-cell viability in situations where the spleen-colony technique is not applicable.     

THE EFFECTS OF COLCEMID ON MOUSE BONE MARROW

Following Colcemid administration, mitoses accumulate preferentially in the subendosteal region of the bone marrow of the mouse. This finding suggests that the most rapidly proliferating cells are localized to the subendosteal region, and complements previous radioautographic studies which have demonstrated a corresponding labelling gradient in the marrow. Quantitative estimates of cell cycle time by the stathmokinetic method were precluded by the presence of significant Colcemid induced interphase cell loss. Colcemid also affected cell differentiation in the marrow. Following Colcemid administration there was a fall in mature granulocytes in the marrow, and a concommitant rise in marrow megakaryocytes.     

P27kip1、EGFR在星形胶质瘤中的表达与其对增殖活性的影响

目的检测P27kip1与EGFR在星形胶质瘤中的表达,探讨其对增殖活性的影响,为治疗提供一定的依据.方法采用免疫组化S-P法检测星形胶质瘤EGFR、P27kip1的表达,每一例肿瘤用ki-67LI表示其增殖活性,应用SAS8.0统计软件进行结果分析.结果 1.P27kip1表达水平与星形胶质瘤病理分级负相关(rs=-0.487,P<0.0001).2.对EGFR、P27kip1及Ki-67LI联合分析显示:P27kip1( )EGFR(-)组的ki-67LI(5.0%)明显低于P27kip1( )EGFR( )、P27kip1(-)EGFR( )、P27kip1(-)EGFR(-)组(10.4%、10.4%、10.8%)(P<0.05).结论 1.P27kip1在星形胶质瘤的发生、发展过程中可能起抑制作用.2.P27kip1、EGFR对星形胶质瘤增殖活性起相互拮抗的作用.     

ESTIMATION OF EFFECTIVE EOSINOPOIESIS AND BONE MARROW EOSINOPHIL RESERVE CAPACITY IN NORMAL MAN

The eosinophil reserve capacity of the post-mitotic granulocyte compartment in the bone marrow and the effective eosinopoiesis in three haematologically normal men have been quantified by means of kinetic parameters of [³H]thymidine flash-labelled peripheral blood eosinophils. From (a) the time of the appearance in the blood of labelled eosinophils after the tracer injection, (b) the inflow characteristics of the labelled eosinophils in the blood and (c) the magnitude of the eosinophil granulocyte pool in the venous blood, the effective eosinopoiesis (i.e. the eosinophil turnover) was calculated to range between 0.014 and 0.031 × 10⁹ cells/kg body weight per day (mean 0.22 × 10⁹ cell/kg per day). The post-mitotic eosinophil reserve capacity of the bone marrow ranged from 0.09 to 0.20 × 10⁹ cells/kg body weight (mean 0.14 × 10⁹ cells/kg). The large reserve pool and the high turnover rate may contribute to sudden rises of the peripheral blood oesinophil counts in some cases of eosinophilia.