极限稀释法的原理及其在造血细胞培养中的运用

长期以来,造血细胞体外培养计算克隆的方法一直沿用半固体法。由于支持物琼脂、甲基纤维素粘附于细胞表面,对于进一步采用电镜、染色体、同位素和单克隆抗体等分析带来很大困难,.且克隆计数由于35mm培养皿面积较大,人为误差很多。有无更好的方法?万景华等在此方面做了开创性工作,我们则在万氏的基础上加以发展,首先运用于多向祖细胞(CFU—Mix)的培养和白血病克隆形我的研究. 基本原理和计算公式在同一条件下,重复做几次独立试验,每次有两个对立的结果,事件A发生或不发生。根据这一基本原理设计了极限稀释法,即将细胞数由高到低     

STUDIES ON CFU-MIX OF 47 PATIENTS WITH LEUKEMIA AND MYELODYSPLASTIC SYNDROME

By limiting dilution assay, CFU-Mix of 47 cases of untreated leukemia, myelodysplastic syndrome and 30 cases of normal adults were studied. It is suggested that most cases of acute nonlymphocytic leukemia, like chronic myelogenous leukemia (CML) and myelodysplastic syndrome should have defect in pluripotent hemopoietic stem cell. The CFU-Mix of acute myelogenous leukemia (AML) and CML were obviously lower than that of normal adults (p less than 0.001). By light microscopy, scanning and transmission electron microscopy, it was confirmed that the CFU-Mix colonies, of leukemic patients were chiefly composed of leukemic cells. Their incorporating rate of 3H-TdR was high in the early stage of culture, but that of 55 + 59Fe was low in the whole course. This phenomenon was quite different from that of the normal adults in that the CFU-Mix of the patients showed distinctive proliferation and differentiation into the leukemic progenitor cells.     

正常人骨髓粒单祖细胞的极限稀释微孔培养研究

本文应用先进的极限稀释微孔培养技术,成功地培养出人骨髓粒-单祖细胞(CFU-GM),并在方法学上作了改进,能够获得完整清晰的集落形态。采用线性回归分析、标记性染色体、3_H-TdR参入、扫描电镜等多种手段证实了CFU-GM的克隆性生长和极限稀释微孔培养CFU-GM的优点。     

α一干扰素和前列腺素E_1对多向造血祖细胞和白血病祖细胞的生长影响

本文认为α一干扰素在体外对正常人多向造血祖细胞有抑制作用,10u/4×10~4细胞浓度抑制率为95％,而对白血病祖细胞,1000u/4×10~4抑制率仅为25％;前列腺素E_1对多向造血祖细胞亦有抑制作用,1ug/4×10~4抑制率为77％,而对白血病祖细胞10ug/4×10~4仍无抑制作用,集落产率明显增加(P<0.01)。提示如试用白血病临床治疗时,干扰素需用大剂量,而前列腺素宜进一步探索。     

一种可检测细胞凋亡多项指标的样品处理

目的：建立一种更为简便，可同时用ＤＮＡ电泳、流式的细胞仪，免疫组化分析检测细胞凋亡多项指标的样品处理方法。方法：用７５％乙醇预固定样品后，经柠檬酸缓冲液抽取凋亡细胞内降解的小分子量ＤＮＡ，而保持细胞形态完整，细胞的抗原，大分子ＤＮＡ完好，进而可用于流式细胞仪和免疫组化分析。结果：将柠檬酸抽提液用于琼脂糖电泳，出现典型的“ＤＮＡ梯形”图谱。其敏感性达２×１０６个细胞检出７．６％凋亡率。而标本可贮存５周之久。结论：本方法为对同一标本进行多种指标的同步分析提供了可能性。     

地甘口服液对环磷酰胺损伤小鼠骨髓CFU-F的影响

观察了地甘口服液对环磷酰胺损伤小鼠骨髓基质细胞形成的影响。将小鼠经尾静脉注射环磷酰胺(每次 10 0 mg/ kg· d-1,连续 3天 ) ,后予地甘口服液 号 (浓度 10 0 % ) 、 号 (浓度 2 0 0 % ) ,灌服 ,每日 2次 ,连续 7天。第 8天处死小鼠 ,取股骨 ,制备骨髓细胞悬液 ,并计数之后 ,建立小鼠骨髓长期培养体系 ,第 1 4天计数成纤维细胞集落形成单位 ( CFU- F) 。结果表明 ,正常小鼠 CFU-F为2 0 .6 5± 2 .96 ,模型鼠为 1 1 .3 8± 4 .93 ,两者有显著性差异 ( P<0 .0 1) ;地甘口服液 号和 号 CFU-F分别为 1 8.0 5± 1 .4 1和 1 8.79± 1 .88,均与正常组无差异 ( P>0 .0 5 ) 。故得出地甘口服液有益气养血之功效 ,可促进环磷酰胺损伤小鼠 CFU-F的形成。     

ELECTRON MICROSCOPIC OBSERVATION OF DIFFERENTIATION OF LEUKEMIC CELL CLONE AFTER CFU-MIX CULTURE

By scanning and transmission electron microscopy, leukemlc celb were obwrved after CFU-Mix culture. Even though granulocytlc growth factor, erythropoietin and lymphocytlc growth factor were added at vitor, acute leukemlc celb still showed defects In differentiation and maturation. These were characterized by abnormal colony which consisted of smooth cells, bizarre shape, nuclear-cytoplasmic asynchrony In development, and appearance of nuclear bleb. However, chronic myelogenous leukemlc celb were more nature than the acute ones, manifesting in normal colony with finger- like projections and ruffled membrane. Macrophages and eosinophils could be observed. It b suggested that there b a difference In differentiation between acute and chronic leukemia cells.     

急性髓系白血病Bcl-2/Bax比值与细胞生长类型和耐药关系的研究

为了解白血病耐药与Bcl-2,Bax基因和细胞生长的关系,运用半固体培养,MTT药敏试验和免疫组织化学测定抗原表达的方法研究40例急性髓系白血病(AML)。结果:AML患者Bcl-2/Bax比值显著高于正常对照组,分别为12.21±8.05和0.75±0.05(P<0.001)。细胞集落生长组Bcl-2/Bax比值为(14.84±8.88)较无集落生长组(7.14±3.41)明显增高(P<0.05),耐药组和药物敏感组Bcl-2/Bax比值分别为14.32±8.99和7.50±5.04,有显著性差异(P<0.05)。Bcl-2/Bax高比值患者化疗反应差,缓解率明显低于低比值患者(P<0.05)。结论:Bcl-2、Bax异常表达是AML形成、发展和产生耐药的重要因素,检测Bcl-2/Bax比值对指导治疗,药物选择及判断预后有重要意义。