三氧化二砷对急性淋巴细胞白血病原代细胞的作用

探讨三氧化二砷（As2O3）对急性淋巴细胞白血病（ALL）原代白血病细胞的作用及作用机制，为As2O3在急性淋巴细胞白血病中的应用提供实验依据。方法：采用MTT法检测As7O3，对ALL原代白血病细胞生长的抑制作用；采用细胞形态学和DNA琼脂糖凝胶电泳技术，观察As2O3在体外诱导ALL原代白血病细胞的凋亡作用；采用流式细胞术检测原代白血病细胞在As2O3，处理前后的Caspase-3活性水平变化及Bcl-2表达水平。结果：①As2O3浓度为1．0—10．0μmol／L的As20，可以明显抑制ALL原代白血病细胞的生长，并呈时间与浓度依赖性。②As2O3作用于ALL原代白血病细胞出现典型细胞凋亡形态学改变、DNA出现了特征性“阶梯形”条带和细胞凋亡峰。③16例ALL患者BMMNCCaspase-3活性水平为4．11±1．93；经As2O3作用后Caspase-3活性水平为26．39±8．36，明显高于对照组11．54±2．78，P〈0．001。④本组患者治疗有效者9例，As2O3作用前后Caspase-3活性水平为4．83±0．98、29．67±7．93；无效者7例Caspase-3活性水平为3．19±2．50、20．59±4．63；As2O3作用后治疗有效组Caspase-3活性明显升高，P〈0．05。⑤16例ALL患者BMMNCBcl-2表达水平为28．08±6．35，其中治疗有效组为26．32±5．79，无效组为32．63±5．47，P〈0．05。⑦As2O3作用前后Bcl-2高表达组Caspase-3活性水平分别为4．07±1．26、22．10±5．64；而Bcl-2低表达组分别为4．14±2．41、32．63±5．47。As2O3作用后两组cas—pase-3活性水平上升程度明显不同，差异有统计学意义（P〈0．05）。结论：Ass2O3在体外对ALL原代白血病细胞生长具有抑制作用，呈现明显的剂量及时间依赖性关系。As2O3能通过激活Caspase-3途径诱导ALL原代白血病细胞凋亡，而Bcl-2可在一定程度上抑制As2O3激活Caspase-3诱导ALL原代白血病细胞凋亡。

Effect of Arsenic Trioxide On Primary Acute Lymphoblastic Leukemia Cells

Objective： To investigate the effects and the mechanisms of As2O3 on acute lymphoblastic leukemia （ALL） primary cells, and obtain some theoretic evidences for using As2O3 to treat ALL in clinic.Methods： The effect of As2O3 on the proliferation of the primary ALL cells was evaluated by b y MTT colorimetric . The apoptosis of the primary ALL cells was detected by morphological assay, flow cytometry （FCM） and DNA electrophoresis. The levels of Caspase-3 and Bd-2 in the primary ALL cells before and after treatment with As2O3 were detected by FCM.Results： ① 1.0-10.0μmol/L As2O3 could significantly inhibit the proliferation of the primary ALL cells in concentration-dependent and time-dependent manners. ②After treatment with As2O3 ,the primary ALL cells underwent chromatin aggregation, nucleus condensation, cytoplasm vacuous degradation and membrane shrinkage ,typical DNA ladder appeared in DNA electrophoresis, sub-diploid region in FCM analysis. ③The level of Caspase-3 of the primary ALL cells was 4.11 ± 1.93, the＇level of Caspase-3 was markedly increased in ALL primary cells treated with 2.0 μ mol/L As2O3 for 48 hours than that in control group （26.39 ± 8.36vs11.54 ± 2.78, p〈0.001）. ①In the 16 cases of ALL ,9 cases had clinical curative effect and 7 cases had not. The caspase-3 levels of the two groups were 4.83 ± 0.98 and 3.19 ±2.50 respectively, there was no significant difference between them. （P〉0.05） ⑤The caspase-3 levels of the group who had clinical curative effect and that who had no clinical curative effect were 29.67 ±7.93 and 20.59 ± 4.63 respectively, there was significant difference between them. （P〈0.05）⑥The Bcl-2 level of the primary ALL cells was 28.08±6.35,the levels of the group who had clinical curative effect and that who had no clinical curative effect were 26.32 ± 5.79 and 32.63± 5.47 respectively ,there was significant difference between them. （P〈0.05） ⑦Before and after treatment with As2O3 ,the levels ofcaspase-3 of the group who had high expression level of Bcl-2 were 4.07 ± 1.26,22.10 ± 5.64 respectively, and the levels of caspase-3 of the group who had low expression level of Bcl-2 were 4.14 ±2.41,32.63 ± 5.47 respectively, there was significant difference between the caspase-3 changing level of the two group after treatment with As2O3. （P〈0.05） ⑧The levels of leukocyte,hemoglobin,platelets count of peripheral blood were not correlated with the levels ofcaspase-3 in the primary cells of ALL. （P〉0.05） Conclusions： ①1.0-10.0 μmol/L As2O3 can inhibit the primary ALL cells proliferation and in concentration-dependent and time-dependent manners. ②As2O3 can induce the primary ALL cells apoptosis .The appoptosis of the primary ALL cells induced by As2O3was via activeting caspase-3. The caspase-3 level of ALL primary cells treatment with As2O3 was correlated with the prognostic of ALL ,and might become a altemafive prognostic marker for judging the clinical curative effectiveness of patients with acute lymphoblastic leukemia. ③The Bcl-2 level of the primary ALL ceils was correlated with the prognostic of ALL ,and Bcl-2 could partly inhibit the activating ofcaspase-3 induced byAs203 in the primary ALL cells.④The levels of hemoglobin, leukocyte and platelet count of peripheral blood cells were not correlated with the levels of caspase-3 in the ALL primary ceils.