抗胸腺细胞球蛋白治疗再生障碍性贫血25例临床研究

本文报道抗胸腺细胞球蛋白（ＡＴＧ）治疗２５例再生障碍性贫血（再障ＡＡ）的疗效。总有效率７２．０％，基本治愈＋缓解４４．０％，病死率１６．０％。死亡的４例均为重型再障。８０．０％ＡＡ病人治疗前Ｔ＿４／Ｔ＿８倒置，治疗后半数转为正常或好转。血清病反应轻者疗效明显优于反应重者（Ｐ＜０．０１）。     

抗胸腺细胞球蛋白治疗重症再障获缓解1例

抗胸腺细胞球蛋白治疗重症再障获缓解１例北京第５１４医院内四科夏鹄，胡升华，熊锡娥，周晓南关键词抗胸腺细胞球蛋白，贫血．重症再生障碍性重症再生障碍性贫血（ＳＡＡ）死亡率高。我院１９９５年２月首次应用抗胸腺细胞球蛋白（ＡＴＧ）治疗１例ＳＡＡ－Ⅱ获得满意疗...     

二次抗淋巴细胞球蛋白/抗胸腺细胞球蛋白治疗重型再生障碍性贫血4例临床分析

目的:观察二次抗淋巴细胞球蛋白(ALG)/抗胸腺细胞球蛋白(ATG)治疗重型再生障碍性贫血(SAA)的疗效和安全性。方法:结合文献报道,回顾性分析4例应用二次ALG/ATG治疗SAA的临床资料。结果:4例均获得明显疗效,其中3例获得完全缓解,1例部分缓解,脱离输血时间为4～17个月;随访2～13年,总体生存率100%,且无一例复发,仅1例用药前即并发PNH克隆的患者在恢复后发作过一次血红蛋白尿症,尚未发生骨髓增生异常综合征、急性白血病及其他肿瘤等病变。结论:二次ALG/ATG的疗效肯定,且具有良好安全性,对于不适合allo-BMT以及无法找到合适供者不能进行allo-BMT或难治复发的SAA患者应首选重复疗程的ALG/ATG。     

槲皮素对白血病细胞增殖、凋亡的影响

目的 探讨槲皮素对白血病细胞增殖、凋亡的影响。方法 人原髓细胞白血病细胞(HL-60)加入10、20、40μmol/L浓度的槲皮素100μl,分别记为槲皮素10、20、40μmol/L组,另加入RPMI1640培养液100μl为空白组。比较各组细胞增殖、凋亡、黏附能力、迁移能力、侵袭情况;检测各组HL-60细胞中半胱氨酸天冬氨酸特异性蛋白酶(caspase)家族蛋白8、9、3的表达情况。结果 槲皮素抑制白血病细胞的增殖活力,其活力随槲皮素浓度和作用时间的增加而加强。槲皮素各组凋亡指数较空白组增加,黏附率、迁移率、侵袭数较空白组降低,差异有统计学意义(P<0.05),且槲皮素各组呈显著浓度依赖性(P<0.05)。40μmol/L槲皮素能够显著上调caspase9、3蛋白表达(P<0.05)。结论 槲皮素能够抑制白血病HL-60细胞的增殖、迁移和侵袭,促进凋亡,对其具有较强的杀伤作用,凋亡机制与细胞中caspase9、3蛋白有关。     

哇巴因诱导白血病细胞凋亡的作用机制

目的：研究哇巴因对白血病细胞株Jurkat是否具有凋亡诱导作用，探索其可能的信号传导通路，阐明哇巴因诱导细胞凋亡的作用机制。方法：采用四甲基偶氮唑盐(MTT)法观察哇巴因对白血病细胞生长的抑制程度；应用电镜及流式细胞术观测细胞凋亡；应用Western-blot检测细胞外信号调节蛋白激酶(extracelluar sig-nal-regulated kinase1/2简称 ERK1/2)的表达。结果：哇巴因能诱导白血病细胞凋亡，细胞凋亡的数量与哇巴因的浓度呈正相关，Western-blot检测结果显示哇巴因作用后，磷酸化：ERK1/2的表达减低。结论：细胞外信号调节蛋白激酶ERK通路可能参与了哇巴因引起的白血病细胞的凋亡过程。     

苦参碱对白血病KG1a细胞增殖及凋亡的影响

目的 观察苦参碱对人急性髓白血病细胞株KG1a体外增殖抑制和凋亡的影响.方法 CCK-8法和台盼蓝拒染色法检测苦参碱对KG1a细胞增殖抑制作用和细胞存活率;甲基纤维素培养体系分析细胞克隆形成能力;流式细胞仪法检测作用前后KG1a细胞的凋亡率及细胞周期变化.结果 苦参碱能够抑制KG1a细胞增殖,随着药物浓度增大和作用时间延长,抑制率逐渐升高、存活率逐渐下降;作用后细胞克隆形成率明显低于对照组(P＜0.05);各浓度药物组均能诱导KG1a细胞凋亡;苦参碱作用后细胞大部分被阻于Go/G1期.结论 苦参碱能抑制KG1a细胞的增殖,改变其周期,诱导其凋亡.     

肾移植术后应用抗胸腺细胞球蛋白的价值与前景(64例次随访分析)

目的：探讨抗胸腺细胞球蛋白（ＡＴＧ），在肾移植术后的应用价值与前景。方法：对６４例次使用ＡＴＧ的肾移植受者进行回顾性分析，比较其①对急性排异反应（ＡＲ）、急性加速性排异反应（ＡＡＲ）、超急性排异反应（ＨＡＲ）的预防与治疗情况；②对部分肾移植术后因各种原因暂不能使用常规免疫抑制剂者的替代与过渡治疗情况；③获得以上各种情况的治疗总剂量与总时限。结果：①ＡＴＧ对肾移植术后ＡＲ的逆转率为８２．３５％（１４／１７），对耐激素性ＡＲ的逆转率为７８．５７％（１１／１４），对ＡＡＲ的逆转率为１００％（１０／１０）；②ＡＴＧ制品适用于肾移植术后因各种原因暂不能服用常规免疫抑制剂者的替代与过渡治疗；③ＡＴＧ对ＨＡＲ不能起到预防与治疗的作用；④抗ＡＲ的ＡＴＧ治疗总剂量与时限以８００ｍｇ、５天为宜，抗ＡＡＲ的ＡＴＧ治疗总剂量与时限以７００ｍｇ、６天为宜，预防与替代治疗的总剂量与时限以７００ｍｇ、６天为宜。结论：ＡＴＧ适用于肾移植术后ＡＲ与ＡＡＲ的预防与治疗，特别对耐激素性排异反应以及暂时的替代与过渡治疗尤为合适，如果经济状况允许，应提倡使用。     

普萘洛尔对急性髓系细胞白血病U937细胞增殖和凋亡的影响

目的:探究普萘洛尔对急性髓系细胞白血病U937细胞的增殖和凋亡的影响及可能的机制。方法:采用CCK8法检测不同药物对U937细胞增殖率的影响,分别为普萘洛尔(0、200、400、800、1000、2 000μmol/L)作用24、48、72 h,阿糖胞苷单用(0、10、50、100、200、400、800 nmol/L)24 h,普萘洛尔(400μmol/L)联合阿糖胞苷(10、50、100、200、400、800 nmol/L)作用24 h。普萘洛尔(0、200、400、800μmol/L)作用U937细胞24 h后,用流式细胞术检测细胞的凋亡和周期、线粒体膜电位的变化,用碱性彗星实验检测DNA的损伤,Caspase-Glo试剂盒检测Caspase-3、8、9相对活性的变化,Western blot法检测Bcl-2、Bax蛋白表达量的变化。结果:普萘洛尔可以有效抑制U937细胞的增殖,其效应具有一定的时间(200、400μmol/L,P0.05)和剂量相关性(0、200、400、800μmol/L,P0.01),普萘洛尔还可协同阿糖胞苷的抗肿瘤效应。流式、彗星、Caspase、Western blot结果分别表明普萘洛尔可增加细胞凋亡率,增加G_0/G_1期和减少G_2/M期细胞比例,降低线粒体膜电位水平,诱导DNA损伤,增强Caspase3、8、9活性,上调Bax的表达、下调Bcl-2的表达,且在实验浓度范围内与剂量呈相关性(P0.05)。结论:普萘洛尔可抑制急性髓系细胞白血病U937的增殖并诱导其凋亡,这一机制可能与其诱导G_0/G_1期细胞阻滞、损伤DNA,激活内、外源性凋亡途径有关。     

三氧化二砷对非霍奇金淋巴瘤细胞的增殖抑制及凋亡作用

目的:探讨三氧化二砷(ATO)对非霍奇金淋巴瘤(NHL)细胞株Raji和Jurkat增殖和凋亡的影响及其相关机制。方法:运用CCK-8法、Annexin V FITC/PI双染流式检测法、蛋白免疫印迹法分别检测ATO对Raji和Jurkat的增殖、凋亡以及凋亡相关蛋白caspase-3、PARP、caspase-8和caspase-9表达的变化。结果:ATO能明显抑制Raji细胞和Jurkat细胞生长增殖,且呈时间依赖性和浓度依赖性; ATO可诱导Raji细胞和Jurkat细胞凋亡,且呈现一定的时间依赖性; ATO可引起Raji细胞cleaved-caspase-3、cleaved-PARP、cleaved-caspase-9表达增加(均P 0. 05),而对caspase-8的影响无明显差别; ATO可引起Jurkat细胞cleaved-caspase-3、cleaved-PARP、cleaved-caspase-8、cleaved-caspase-9表达增加(均P 0. 05)。结论:ATO明显抑制Raji细胞和Jurkat细胞的增殖,并诱导细胞凋亡,Raji细胞凋亡效果较弱,主要由线粒体途径介导,而Jurkat细胞凋亡明显,由线粒体途径和死亡受体途径共同介导。     

Comparable TNF-alpha,IFN-gamma and GM–CSF production by purified normal marrow CD3 cells in response to horse anti-lymphocyte and rabbit antithymocyte globulin

Abstract: In vitro priming of T cell with horse antilymphocyte globulin (HALG) results in cytokine release, and this has been associated with its clinical efficacy in patients with severe aplastic anaemia (SAA). Rabbit antithymocyte globulin (RATG) has been studied less extensively. In this study we compare the in vitro priming effect of HALG and RATG on purified normal marrow T cells: end-points of the study were 1) levels of TNF-alpha (TNF-α), IFN-gamma (IFN-γ) GM–CSF in T cell supernatants, and 2) effect of T cell supernatants on colony formation with or without exogenous GM–CSF. TNF-α, IFN-γ and GM–CSF levels were comparable for HALG, RATG and phytohaemagglutinin (PHA). T cell supernatants showed comparable enhancement of colony formation in the presence of recombinant human GM–CSF (rhGM–CSF) and supported colony forming unit granulomacrophage (CFU–GM) growth in the absence of growth factor. This study shows that horse and rabbit derived ALG/ATG and PHA have a comparable in vitro priming effect on T cells: both agents should probably be tested for their clinical efficacy in SAA patients.     

Efficacy of rabbit anti-thymocyte globulin in severe aplastic anemia

Background

A combination of horse anti-thymocyte globulin and cyclosporine produces responses in 60–70% of patients with severe aplastic anemia. We performed a phase II study of rabbit anti-thymocyte globulin and cyclosporine as first-line therapy for severe aplastic anemia.

Design and Methods

Twenty patients with severe aplastic anemia treated with rabbit anti-thymocyte globulin were compared to 67 historical control cases with matched clinical characteristics treated with horse anti-thymocyte globulin.

Results

Response rates at 3, 6 and 12 months were similar for patients treated with rabbit anti-thymocyte globulin or horse anti-thymocyte globulin: 40% versus 55% (P=0.43), 45% versus 58% (P=0.44) and 50% versus 58% (P=0.61), respectively. No differences in early mortality rates or overall survival were observed. We then performed multivariable analyses of response at 6 months and overall survival and identified the presence of a paroxysmal nocturnal hemoglobinuria clone (P=0.01) and a pretreatment absolute reticulocyte count greater than 30×10⁹/L (P=0.007) as independent predictors of response and younger age (P=0.003), higher pretreatment absolute neutrophil (P=0.02) and absolute lymphocyte counts (P=0.03) as independent predictors of overall survival. None of the immunogenetic polymorphisms studied was predictive of response to immunosupressive therapy.

Conclusions

Despite reports suggesting differences in biological activity of different anti-thymocyte globulin preparations, rabbit and horse anti-thymocyte globulin appear to have a similar efficacy for up-front treatment of severe aplastic anemia. Clinicaltrial.gov: {"type":"clinical-trial","attrs":{"text":"NCT01231841","term_id":"NCT01231841"}}NCT01231841)     

Effect of androgens on the response to antithymocyte globulin in patients with aplastic anaemia

30 patients with aplastic anaemia (18/30 with severe aplastic anaemia) were prospectively randomized to be treated with 100 mg/kg ATG with or without the oral androgen Methenolone (3 mg/kg). 15 of 30 patients responded. Among the 15 patients receiving ATG plus androgen, 11 patients (73%) responded, including 8 complete and 3 partial responses. 4 of the 15 patients (31%) receiving ATG only responded, including 2 complete and 2 partial responses. The difference in response rate was statistically significant (p = 0.01). The survival rate in the total population of 30 patients was 64%. The survival rate in the group receiving ATG plus androgen was 87%; in the group receiving ATG only it was 43%. The difference in survival rates between both groups did not reach statistical significance (p = 0.15). Toxicity of ATG and androgens was considerable but manageable. These data support the result of the recent European reevaluation of a large pool of patients by the EBMT (39), that androgens in addition to ATG increase survival in patients with aplastic anaemia. They are, however, in contradiction to a controlled American study showing no benefit of a combined treatment with androgens as compared to ATG only. Further controlled studies on a larger number of patients are indicated to determine the therapeutic efficacy of androgens in addition to immunosuppression in aplastic anaemia.     

Cytotoxic and stimulatory effects of antilymphocyte globulin (ALG) on hematopoiesis

Summary Four different preparations of antilymphocyte/antithymocyte globulin were tested in vitro for their toxicity to lymphocytes and to hematopoietic precursor cells, depending on concentration and time. Complete lymphocytotoxicity was observed at concentrations from 6.3 to 25µg/ml, and suppression of colony formation by hematopoietic precursors was seen at concentrations from 12.5 to 250µg/ml. Prolonged incubation time did not increase lymphocytotoxicity but augmented precursor cell damage. Lymphocytotoxicity was comparable among the four preparations tested whereas precursor cell toxicity varied widely. Antilymphocyte globulin is mitogenic and stimulates the release of hematopoietic growth factor activity by peripheral blood cells. Absorption of ALG with human T-cells eliminated precursor cell toxicity and mitogenicity but not the capacity to release hematopoietic growth factors. These results show that dose/time schedules for ALG administration may be relevant and ALG acts by virtue of inhibitory and stimulatory antibody effects.Former Clinical Fellow of Haematology     

二甲双胍及甘精胰岛素对子宫内膜癌细胞增殖和凋亡的影响

目的 探讨二甲双胍（Met）、甘精胰岛素（Gla）或二者联合对入子宫内膜癌Ishikawa细胞增殖和凋亡的影响. 方法 不同浓度Met、Gla或二者联合干预人子宫内膜癌Ishikawa细胞株不同时间.CCK-8检测细胞增殖,流式细胞仪检测细胞凋亡. 结果 与对照1组比较,Met各浓度均可抑制Ishikawa细胞增殖,小剂量（5 mmol/L）可使增殖率下降18.4％.Met可诱导Ishikawa细胞凋亡,高浓度（20mmol/L）晚期凋亡率升高最显著（26.11％）.Gla可促进人子宫内膜癌Ishikawa细胞增殖,细胞凋亡率降低.Gla联合不同浓度Met,与同浓度同时间Gla干预对比,可抑制增殖,增加凋亡率. 结论 Met可抑制人子宫内膜癌Ishikawa细胞增殖,促进其凋亡,并可阻断Gla对人子宫内膜癌细胞的促增殖作用.     

家兔颈总动脉内皮剥脱术后细胞凋亡与增殖的动态研究

目的　探讨细胞凋亡在血管损伤后内膜增厚过程中的作用。方法　采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法和免疫组化技术对内皮剥脱术后 3、9、1 4、2 8天家兔颈总动脉壁的凋亡及增生细胞分别进行检测 ,并采用计算机图像分析系统进行定量分析。结果　术后各损伤组凋亡及增生指数示 9天时均显著高于 3天 ,1 4天达峰值 ,2 8天呈下降趋势 ,其中凋亡指数仍较高 ;阳性表达面积率示高峰及之前的凋亡 /增殖比 <1 ,而之后 >1。对照组染色呈阴性。结论　细胞凋亡调节着损伤动脉壁的细胞数目 ;它与细胞增生伴存 ,在增生后期占主导地位 ,调控细胞不再增多。     

Expression of the Fas antigen on primary human leukemia cells

Summary The antigen defined by the monoclonal antibody anti-Fas can mediate apoptosis, is associated with the receptor for tumor necrosis factor, and is expressed on a limited number of human tissues. In this study we analyzed the expression of Fas on primary human leukemic cells and on mononuclear cells from other hematologic disorders. A total of 95 samples of blood or bone marrow were studied by indirect immunofluorescence. These samples included the normal controls, 47 cases of acute myelogenous leukemia (AML), 11 cases of acute lymphoblastic leukemia (ALL), 21 cases of leukemic lymphoma, seven cases of chronic myelogenous leukemia (CML), five cases of plasma cell leukemia or multiple myeloma, and five cases of myelodysplastic or myeloproliferative syndromes. Normal controls were negative without exception. Among AML, 13/47 cases (28%) were positive; among ALL, 1/11 cases (9%) was positive; among leukemic lymphomas, 3/21 cases (14%) were positive. In a case of plasma cell leukemia which strongly expressed the Fas antigen, we demonstrated that the antibody mediates cell lysis, which was synergistically enhanced by the addition of rabbit complement. In patients with AML, Fas positivity had no obvious clinical relevance. Taken together, our results show that approximately 30% of cases of AML and occasionally other leukemias express the Fas antigens, whereas normal controls are negative in our test system. These findings may be useful in the treatment of refractory leukemias or may permit the purging of autologous transplants.Presented in part at the 34th annual meeting of the American Society of Hematology, Anaheim, CA, December 1992     

二甲双胍对人胰腺癌细胞株CFPAC-1增殖与凋亡的影响

目的观察二甲双胍对人胰腺癌细胞株CFPAC-1增殖、凋亡的影响,探讨其相关分子机制。方法应用1、2．5、5、10、20、40、60mmol／L二甲双胍处理人胰腺癌CFPAC-1细胞24、48、72h,以未处理的细胞作为对照组。采用CCK-8法检测细胞的增殖抑制率,流式细胞仪分析细胞周期,AnnexinV／PI双染法检测细胞凋亡,蛋白质印迹法检测磷酸化AMPK（p-AMPK）、FAS、cyclinD1、Bcl-xl、Bax、caspase-3、survivin蛋白的表达。结果二甲双胍呈浓度及时间依赖性抑制CFPAC-1细胞的增殖。40mmol／L二甲双胍处理细胞48h后,G0／G1细胞比例显著增多[（65．93±0.27）％比（42．89±1．02）％],G2／M期、s期细胞比例显著减少[（22．01±2．95）％比（38．28±4．93）％,（13．58±0．43）％比（20．12±3．38）％],差异均有统计学意义（P值均〈0．05）;细胞凋亡率从对照组的（3．01±0．49）％增加到（32．97±3．19）％（P〈0．05）;p-AMPK、Bax、caspase-3表达增强,FAS、cyclinD1、Bcl-xl、survivin表达减弱。结论二甲双胍可以显著抑制CFPAC-1细胞增殖,促进细胞凋亡,其机制可能通过激活AMPK信号通路,下调FAS、cyclinD1、survivin表达及Bcl-xl／Bax比值,上调caspase-3表达所致。     

Direct binding of antithymoctye globulin to haemopoietic progenitor cells in aplastic anaemia

Antithymocyte globulin (ATG) is widely used in the treatment of aplastic anaemia (AA) and a response occurs in 60-80% of patients. However, its exact mechanism of action in the treatment of AA has yet to be determined. Previously, we have shown that ATG increases colony growth from purified bone marrow CD34+ cells of AA patients in vitro, and decreases stem cell apoptosis and the expression of soluble Fas receptor after ATG therapy in vivo. The aim of this study was to further examine the association of ATG with AA haemopoietic progenitor cells. We describe here that ATG bound directly to CD34+ cells. Forty-six patients and 20 normal control subjects were studied. ATG bound to CD34+ cells in normal control subjects (mean 90.38%) as determined by flow cytometry. The mean percentage of CD34+ cells binding to ATG was 59.90% in untreated aplastic patients, 83.24% in partial responders, 58.3% in non-responders and 62.73% in relapsed patients. In completely recovered patients, ATG binding was indistinguishable from control subjects. The functionality of AA patients' haemopoietic progenitor cells was assessed using colony assays. These results demonstrate the direct binding of ATG to CD34+ cells and suggest that differences in its binding to AA CD34+ cells could reflect functional differences in the haemopoietic stem cell compartment throughout the disease process.