预激态补骨脂素对白血病移植瘤裸鼠的治疗作用

 目的　观察预激态补骨脂素在动物体内的抗白血病作用。方法　人类白血病细胞系HL-60细胞移植于裸鼠背部皮下,实验组于不同时间、不同部位注射预激态补骨脂素数天,对照组于相应时间、部位注射生理盐水,观察两组在瘤重、成瘤率和存活期方面的差异。结果　移植HL-60白血病细胞后第8天,成瘤率为100 %。瘤内注射实验表明,用药组瘤体平均重量为（3.21±1.32）g,对照组为（5.0±1.0）g,两组瘤体重量差异有统计学意义（t = 3.42,P = 0.0031）;病理检查瘤体内坏死面积实验组明显多于对照组;用药组裸鼠的平均存活为（35±9.92）d,对照组为（28.3±5.14）d,两组存活天数差异无统计学意义（Z = 1.52,P = 0.1294）。肌肉注射实验表明,用药组第8天的成瘤率为80 %,对照组为100 %,两组成瘤率差异无统计学意义（χ2 = 2.22,P = 0.1360）;用药组裸鼠平均存活（31.5±6.39）d,对照组为（17.8±12.07）d,两组存活天数差异无统计学意义（Z=1.37,P = 0.1713）。移植局部注射实验表明,用药组第8天的成瘤率为60 %,对照组为100%,两组成瘤率差异有统计学意义（χ2 = 5.0,P = 0.0250）;用药组裸鼠平均存活（37.9±6.21）d,对照组为（20.8±4.10）d,两组存活天数差异有统计学意义（Z = 4.30,P = 0.0000）。结论　预激态补骨脂素在动物体内也能发挥抗白血病作用。     

阿克拉霉素诱导原代白血病细胞凋亡的实验研究

目的　研究阿克拉霉素 (ACM)体外抑制原代急性白血病 (AL)细胞的生长及其机理。方法　MTT法研究 37例初发AL细胞的生长抑制。DNA片段原位末端标记 (TUNEL)法检测 2 0例AL细胞的凋亡率。结果　ACM体外能明显抑制原代AL细胞的生长 ;细胞形态学、DNA琼脂糖凝胶电泳均证实ACM能诱导原代AL细胞凋亡 ;与空白对照相比 ,ACM在体外与原代AL细胞共同培养 15小时后 ,TUNEL阳性细胞率明显增高 ,差异有统计学意义 (30 89± 15 90 %对 14 85± 15 90 %;P <0 0 1) ;且TUNEL阳性细胞率与MTT抑制率呈正相关 (r =0 32 6 ,P =0 0 4)。结论　诱导细胞凋亡是ACM抑制原代AL细胞增殖的重要机制之一。     

高三尖杉酯碱诱导急性白血病原代细胞凋亡和杀伤作用的研究

目的　探讨高三尖杉酯碱 (HHar)对AML的疗效明显优于ALL的可能机制。方法　以成人AL原代细胞为研究对象 ,采用高、中、低三种浓度梯度对HHar进行研究。采用光镜、扫描电镜下观察细胞凋亡的形态变化 ,透射电镜下观察细胞超微结构改变 ,DNA凝胶电泳、二苯胺法、流式细胞术等方法 ,证实HHar确可诱导AL原代细胞凋亡。尔后采用光镜计数细胞凋亡率、二苯胺定量测定凋亡细胞的DNA片段化率及台盼蓝染色光镜下计数坏死率三种方法来观察HHar对ALL及AML原代细胞的诱导凋亡和杀伤 ,得出相应的时间、剂量、效应关系 ,并进行对比分析。结果　HHar对AML原代细胞诱导凋亡的敏感性高于ALL原代细胞。结论　Hhar对AML原代细胞诱导凋亡的敏感性高是其临床疗效优于ALL的可能机制     

高三尖杉酯碱诱导急性白血病原代细胞凋亡和杀伤作用的研究

目的：探讨高三尖杉酯碱（HHar）对AML的疗效明显优于ALL的可能机制。方法：以成人AL原代细胞为研究对象，采用高、中、低三种浓度梯度对HHar进行研究。采用光镜、扫描电镜下观察细胞凋亡的形态变化，透射电镜下观察细胞超微结构改变，DNA凝胶电泳、二苯胺法、流式细胞术等方法，证实HHar确可诱导AL原代细胞凋亡。尔后采用光镜计数细胞凋亡率、二苯胺定量测定凋亡细胞的DNA片段化率及台盼蓝当然光镜下计数坏死率三种方法来观察HHar对ALL及AML原代细胞的诱导凋亡和杀伤，得出相应的时间、剂量、效应关系，并进行对比分析。结果：HHar对AML原代细胞诱导凋亡的敏感性高于ALL原代细胞。结论：Hhar对AML原代细胞诱导凋亡的敏感性高是其临床疗效优于ALL的可能机制。     

DC-CIK对急性髓细胞白血病干细胞的杀伤作用

目的:研究树突状细胞(dendritic cell,DC)联合同源细胞因子诱导的杀伤细胞(cytokine-induced killer cell,CIK)对急性髓细胞白血病细胞株KG-1a中白血病干细胞(leukemic stem cell,LSC)的体外杀伤和诱导凋亡作用。方法:分离健康人外周血单个核细胞,贴壁细胞用GM-CSF和IL-4诱导培养DC,悬浮细胞用IL-2、IL-1、IFN-γ和CD3 mAb诱导培养CIK。将KG-1a细胞冻融物作为抗原负载DC(即Ag-DC),与CIK共培养作为实验组(Ag-DC-CIK),无抗原负载的DC与CIK共培养作为对照组(DC-CIK),单独CIK作为空白对照组,与KG-1a共育后流式细胞术检测各组细胞中CD34+CD38-CD123+白血病干细胞的比例。DC-CIK与KG-1a细胞共培养,流式细胞术检测各组细胞中KG-1a细胞与CD34+CD38-CD123+细胞的凋亡率。结果:外周血单个核细胞成功诱导DC。CIK组、DC-CIK组及Ag-DC-CIK组中CD3+CD56+细胞比例为(17.36±4.44)%、(28.22±3.66)%和(36.16±5.88)%,依次升高(P<0.05)。与对照组相比,Ag-DC-CIK组与DC-CIK组细胞中CD34+CD38-CD123+细胞比例显著降低[(8.78±0.62)%vs(3.95±0.53)%、(3.03±0.62)%,P<0.01〗。DC-CIK可诱导KG-1a细胞凋亡,凋亡率由(2.34±0.74)%上升至(12.27±1.01)%,但对其中CD34+CD38-CD123+细胞无明显的诱导凋亡作用。结论:DC联合CIK能杀伤急性髓细胞白血病干细胞,但无明显的诱导凋亡作用。     

克隆化人LAK细胞杀伤白血病细胞的作用机制研究

探讨了克隆化的人外周血LAK细胞对人白血病细胞的杀伤作用及其机制。克隆化人LAK细胞由半固体液体二步培养法获得，其抗原标志属T细胞系境。^51Cr4小时释放试验和6天半固体集落形成测定结果表明：克隆化LAK细胞对白血病细胞系K562、     

Cytotoxic effects on viable human leukemic cells by combinations of lymphokine activated killer cells and monoclonal antibodies

Earlier studies with individually phenotyped monoclonal antibody combinations and complement or lymphokine activated killer (LAK) cells showed that many acute myeloid leukemic cells were resistant to these cytotoxic agents when used singly. Therefore, a combination of both agents was studied. When the leukemic target cells were submitted to killer cells activated with 100 or 800 IU of recombinant interleukin-2 (rIL-2), only averages of 6.0 and 16.7% of the targets were killed respectively. When the remaining, refractory cells were confronted with a cocktail of individually phenotyped monoclonal antibodies and complement, an additional significant cell kill was obtained, but it amounted to only between 7.4 and 5.5% (for LAK-100 and LAK-800, respectively). In contrast, of the target cells initially refractory to the same cocktail of monoclonal antibodies, all were cross-resistant both to LAK-cells activated with 100 and to those activated with 800 IU of rIL-2. This cross-resistance was caused neither by sub-optimal LAK-cell activation, nor by antibody blocking of hypothetical LAK-cell receptors, since pre-incubation with monoclonal antibodies without complement did not inhibit LAK-cell cytotoxicity. Although only partial cross-resistance was found in the present study, it still remains that only a minority of the tumor cells could be killed. A higher in-vitro cell kill should be attempted prior to clinical trials in order to avoid clinical effects resembling those of a partial surgical tumor resection.     

RNA干扰对白血病细胞系HL-60增生的影响

 目的 研究针对c-myc基因的小分子干扰RNA片断（small interfering RNA, siRNA）对白血病细胞系HL-60细胞增生的影响,探讨应用siRNA进行白血病基因治疗的可能性。方法 制备特异性对应c-myc基因的siRNA转染HL-60细胞,倒置显微镜观察细胞形态学改变,MTT法检测细胞增生状态,流式细胞仪进行细胞周期分析。结果 siRNA转染HL-60细胞后,细胞生长受抑,增生能力减弱,其增生抑制作用于转染后48 h、72 h最明显,增生抑制率分别为53.2 %和51.5 %;S期细胞比值由（56.1±4.7）%降至（17.8±3.2）%,细胞阻滞在G0／G1期。结论 针对c-myc基因的siRNA对HL-60细胞增生有明显抑制作用,有望为白血病提供新的治疗途径。     

CD3AK细胞对耐药白血病细胞的体外净化作用

目的 :观察CD3 AK细胞对耐药的白血病细胞系及慢性髓细胞白血病 (chronicmyelogenousleukemia ,CML)急变患者原代肿瘤细胞的体外净化作用。方法 :采用固化的抗CD3单克隆抗体联合小剂量IL 2诱导CD3 AK细胞 ;MTT法观察CD3 AK细胞对K5 6 2、HL6 0及其耐药株的细胞毒活性 ;肿瘤细胞集落培养 (tumorcolonyassay ,TCA)观察CD3 AK细胞对K5 6 2、HL6 0及其耐药株集落形成的抑制作用 ;流式细胞仪 (flowcytometry ,FCM)检测耐药的CML急变患者原代细胞经CD3 AK细胞净化后Pgp阳性细胞的比例变化。结果 :MT法显示CD3 AK细胞在体外对K5 6 2细胞、HL6 0细胞及其耐药株有相似的杀伤作用 ;集落培养观察CD3 AK细胞对HL6 0细胞株及其耐药株的集落形成均有较强的抑制作用 ;FCM结果显示耐药CML原代细胞经CD3 AK细胞净化后Pgp阳性细胞比例下降 2 3 2 0 %。结论 :CD3 AK细胞在体外对耐药白血病细胞株及耐药白血病原代细胞均有较强的净化作用。     

Resistance of acute myeloid leukemic cells to the triterpenoid CDDO-Imidazolide is associated with low caspase-8 and FADD levels

The synthetic triterpenoid CDDO-Im-induced apoptosis of patient-derived AML blasts: 11/25 AMLs were highly sensitive, while the remaining were moderately sensitive to CDDO-Im. The addition of TRAIL significantly potentiated the cytotoxic effect of CDDO-Im, through mechanisms involving the induction of TRAIL-R1/TRAIL-R2 and downmodulation of TRAIL-R3/TRAIL-R4. Biochemical studies showed that CDDO-Im: induced a rapid and marked GSH depletion and antioxidants (GSH or NAC) completely inhibited its pro-apoptotic effect; sequentially activated caspase-8, -9 and -3; caspase inhibitors partially protected AML blasts from CDDO-Im-induced apoptosis; resistance of AML blasts to CDDO-Im-induced apoptosis correlated with low caspase-8/FADD and high Bcl-X_L expression in leukemic blasts.     

Cytotoxic effects of treosulfan and busulfan against leukemic cells of pediatric patients

PURPOSE: The alkylating agent treosulfan exerts a high cytotoxic activity against various malignant cells. Due to limited non-hematological toxicity, treosulfan might be a promising compound in myeloablative therapy for hematopoietic transplantation in children. Since in vitro data regarding the activity of treosulfan against childhood leukemic cells are limited, we compared the effect of treosulfan and busulfan against pediatric leukemic and non-malignant cells. EXPERIMENTAL DESIGN: Both agents were tested alone and in combination with fludarabine by means of the MTT and/or a five color-flow cytometric assay. Moreover, the induction of apoptosis by treosulfan was investigated via regulation of the proteinase caspase 3. RESULTS: Treosulfan was more active against leukemic cells of 20 children as well as against 3 leukemia-derived cell lines than busulfan, with increasing IC(50) values from initial diagnosis to relapse. Overall purified stem cells were most sensitive, followed by CD56(+)CD3(-) NK and CD3(+) T cells. The combination of treosulfan with fludarabine resulted in a synergistic effect against leukemic cells. In malignant cells, treosulfan induced rapid cell apoptosis measured by the activation of the centrally proteinase caspase 3. CONCLUSION: Our results indicate that treosulfan has activity against pediatric leukemic cells, myeloablative potential and immunosuppressive properties suitable for conditioning regimen in childhood malignancies.     

In vitro chemosensitivity testing of leukemic cells: development of a semiautomated colorimetric assay

A rapid chemosensitivity assay was developed, employing the human continuous leukemic cell lines HL 60, K 562, FLG 29.1. This automated colorimetric assay is based on the characteristic of viable, metabolically active cells to cleave p-iodonitrotetrazolium violet (INT) into a red formazan derivative, whose optical density is readable at 492 nm by an automated microtiter-plate reader photometer. A linear relationship was found between the viable cell number and the optical density of INT cleaved by the cellular samples. Dead cells did not reduce INT and did not interfere with the formazan derivative generation and the photometric reading. Leukemic cell lines were also tested for INT formazan derivative generation after exposure to antileukemic drugs at various concentrations, representative of plasma levels obtainable in vivo. A dose-dependent inhibition was detected, with different sensitivity patterns, related both to the drugs and to the different cell lines. A significant correlation between the viable cell number and the amount of tetrazolium salt cleaved was also demonstrated after drug exposure. INT assay allows the processing of a great number of samples and gives the opportunity to screen several drugs, saving time and yielding fully reliable results.     

HPLCASSAY FOR INTRACELLULAR ACCUMULATION OF VERAPAMIL IN VER-RESISTANT HUMAN LEUKEMIC CELL SUSLINES AND THEIR PARENTAL CELL LINES

Ｖｅｒａｐａｍｉｌ（ＶＥＲ），ａｃａｌｃｉｕｍｃｈａｎｎｅｌｂｌｏｃｋｅｒ，ｈａｓｂｅｎｆｏｕｎｄｔｏｅｆｅｃｔｉｖｅｌｙｒｅｖｅｒｓｅｍｕｌｔｉｄｒｕｇｒｅｓｉｓｔａｎｃｅｉｎｔｕｍｏｒｃｅｌｓｔｈｒｏｕｇｈｃｏｍｐｅｔｉｔｉｖｅｃｏｍｂｉｎａｔｉ...     

Human interleukin-2 activated cytotoxic cells kill autologous glioma cells in vitro

Summary We have cultured peripheral blood lymphocytes (PBL) from glioblastoma patients in recombinant interleukin-2 (IL-2) containing medium for a period of 5 days. The cytotoxicity of these cells was tested on ⁵¹Cr-labelled autologous dissociated glioblastoma cells which had not been cultured. Significant cytotoxicity against glioma cells was observed in seven out of nine cases. IL-2 activated PBL from normal donors were equally cytotoxic against these glioma cells. Autologous lymphocytes activated by phytohaemagglutinin were also lysed in most cases, and the erythroleukemia cell line K562 was highly susceptible to the cytotoxic capability of the IL-2 activated PBL. In cold target inhibition experiments, K562 inhibited the cytotoxicity against both autologous and allogenic glioma cells, and glioma cells inhibited the cytotoxicity against K562. Following immunomagnetic separation, the IL2 activated cells demonstrated cytotoxicity against glioma cells, K562 cells, and PHA blasts in both the CD8⁺ and the CD8^– subsets.