[Effect of Huaier Aqueous Extract Combined with Routine Chemo-therapeutic Drugs on Human Acute Lymphoblastic Leukemia Cells Nalm-6 and Sup-B15].

To investigate the proliferation inhibition and pro-apoptotic effect of Huaier aqueous extract combined with routine chemotherapeutic drugs including Vincristine (VCR), Daunorubicin (DNR), L-aspartase (L-Asp) on human acute lymphoblastic leukemia cell lines Nalm-6 and Sup-B15.Nalm-6 and Sup-B15 cell lines were treated with different concentrations of Huaier aqueous extract and chemotherapeutics including VCR, DNR, L-Asp alone or in combination for 48 h, and the growth inhibitory effect and IC50 values (the half maximal inhibitory concentration) were detected by CCK-8. Jin's formula was used to estimated the synergistic effect of these combinations. Apoptosis rates of Nalm-6 and Sup-B15 cells and expression of apoptosis-related proteins BAX, BCL-2, cleaved Caspase-3 were determined by flow cytometry and Western blot respectivcly.Huaier aqueous extract, VCR, DNR and L-Asp had inhibition effect on Nalm-6 and Sup-B15 cell lines. The inhibition rate of Huaier aqueous extract combined with VCR, DNR and L-Asp were all higher than those of each dug alone (P＜0.05) and the combination index (q) was between 0.85 and 1.15 or greater than 1.15. The two kinds of drugs showed had additive or synergistic effects. The results of flow cytometry showed that the cell apoptosis rates in combined treatment group were higher than those of each drug alone (P＜0.05). The results of Western blot revealed that Huaier aqueous extract and VCR all decreased protein expression of BCL-2 (P＜0.05) and increase protein expression of BAX (P＜0.05) and cleaved Caspase-3 (P＜0.05) in Nalm-6 and Sup-B15 cells. Compared with Huaier aqueous extract or VCR alone, the effect of two drug combination were more significant. DNR down-regulated protein expression of BCL-2 (P＜0.05) and up-regulated cleaved Caspase-3 (P＜0.05). However, it had no effect on the expression of BAX in Nalm-6 and Sup-B15 cells. When it was combined with Huaier aqueous extract, the expression of cleaved Caspase-3 and BCL-2 showed more significant changes. The expression of BAX in combined treated group did not show significant difference, compared with group treated with Huaier aqueous extract in Nalm-6 and Sup-B15 cells. L-Asp did not show significant effect on the three apoptosis-related proteins and there was no significant difference between the combination group and the Huaier aqueous extract group.the combination of Huaier aqueous extract and VCR, DNR, L-Asp shows additive or synergistic effects on human acute lymphoblastic leukemia cell lines Nalm-6 and Sup-B15.槐耳清膏联合常用化疗药物对人急性淋巴细胞白血病细胞Nalm-6和Sup-B15的影响.探讨槐耳清膏联合临床常用化疗药物长春新碱（VCR）、柔红霉素（DNR）及左旋门冬氨酸酶（L-Asp）对急性淋巴细胞白血病细胞株Nalm-6和Sup-B15的抗增殖和促凋亡作用.以Nalm-6和Sup-B15细胞为研究模型, 采用CCK-8法检测不同浓度槐耳清膏和3种化疗药物（长春新碱, 柔红霉素, 左旋门冬氨酸酶）单独及联合处理Nalm-6和Sup-B15细胞48 h后的增殖抑制作用, 确定药物的半数抑制浓度（IC50）, 金氏公式判断2药物的联合作用效果；采用流式细胞术和Western blot检测槐耳清膏与化疗药物单独及联合作用对Nalm-6和Sup-B15细胞凋亡率和凋亡相关蛋白BAX, BCL-2, cleaved Caspase-3的影响.槐耳清膏、长春新碱、 柔红霉素、左旋门冬氨酸酶均能抑制Nalm-6和Sup-B15细胞的增殖, 槐耳清膏联合化疗药物处理组比相应的单药处理组抑制率更高（P＜0.05）, q值在0.85-1.15之间或大于1.15, 2药物具有相加或协同作用。流式细胞术检测结果显示, 联合组的凋亡率高于相应单药组的凋亡率（P＜0.05）。Western blot结果显示, 长春新碱和槐耳清膏均能上调Nalm-6和Sup-B15细胞蛋白BAX和cleaved Caspase-3的表达（P＜0.05）, 同时下调蛋白BCL-2表达（P＜0.05）, 2者联合作用更明显；柔红霉素单独作用于Nalm-6和Sup-B15细胞时, BCL-2蛋白下调（P＜0.05）, 而cleaved Caspase-3蛋白上调（P＜0.05）, 但蛋白BAX无明显改变, 当与槐耳清膏联合后, 蛋白BCL-2和cleaved Caspase-3变化更明显, 蛋白BAX与槐耳清膏单药处理组无明显差异。而左旋门冬氨酸酶对3种凋亡蛋白无明显影响, 2者联合与单用槐耳清膏无显著差异.槐耳清膏与常用化疗药物长春新碱、柔红霉素、左旋门冬氨酸酶联合对急性淋巴细胞白血病细胞株Nalm-6和Sup-B15具有协同或相加作用.