蟾酥逆转白血病多药耐药细胞CEM/VCR的作用及其机制研究

At present most leukemia drug-resistant reversal agents are western medicine, but the effect is not ideal and it has significant toxicity. Based on the traditional Chinese medicine theory which called "combating poison with poison", we devoted to using traditional Chinese medicine with toxic effects to the leukemia cell research, proving toxic Chinese traditional medicine that called venenum bufonis has strong inhibition on lymphocyte leukemia CEM cell growth, western medicine vincristine (VCR) takes second place. It was found tbat CEM cells have antibiotic resistance to VCR by preliminary experiments, and venenum bufonis still possess the inhibition on CEM cells which have the VCR drug resistance. The project centred on whether venenum bufonis can reverse MDR cells CEM/VCR and its mechanism. We established the leukemia MDR cell lines CEM/VCR. Using the method of MTT to test each venenum bufonis concentration on the CEM/VCR inhibition rate; With light and electron microscopic to observe changes of each group cells in morphology; From molecular level to measure the apoptosis rate and the express of agrp Bcl-2, Bcl-X, bax, bak and P53, P-gp in signal transduction in apoptosis, resistance genes mdr1 mRNA positive express, the topological isomerase Ⅱ activity determination to discuss the inhibition mechanism.Using experiments to prove that venenum bufonis can reverse CEM/VCR through multi-target and multi-channel. Enriching the " combating poison with poison " theory to provide theoretical basis of the venenum bufonis clinical treatment of the lymphocyte leukemia.

目前白血病多药耐药（MDR）逆转剂多为西药，但效果不理想且毒副作用大。基于中医"以毒攻毒"理论，我们长期致力于用有毒中药作用于白血病细胞的研究，证明有毒中药蟾酥对淋巴细胞白血病CEM细胞生长抑制能力强，西药长春新碱（VCR）次之；预实验发现CEM细胞对VCR具有耐药性，而蟾酥对VCR耐药的CEM细胞仍有抑制作用。本项目围绕蟾酥是否能逆转MDR细胞CEM/VCR及机制问题。首先建立白血病MDR细胞株CEM/VCR。用MTT法测蟾酥对CEM/VCR的抑制率；光镜、电镜观察细胞形态变化；从分子水平测凋亡率及凋亡信号通路中的相关蛋白Bcl-2、Bcl-X、bax、bak和P53、P-gp的表达，耐药基因mdr1 mRNA的阳性表达，拓扑异构酶Ⅱ的活性测定等来探讨其抑制机制。以实验证明，蟾酥能通过多靶点多途径逆转CEM/VCR。从而丰富"以毒攻毒"理论，为蟾酥走向临床治疗淋巴细胞白血病提供理论依据。

针对白血病治疗易出现MDR，西药逆转剂效果不理想且毒副作用大。基于中医“以毒攻毒”理论，在前期证明有毒中药蟾酥、西药长春新碱（VCR）对白血病CEM细胞具有抑制作用，且长期应用后出现对VCR耐药现象的基础上，以VCR作诱导剂，历时14个月建立了白血病MDR CEM/VCR细胞株。该细胞株被化疗药阿霉素、阿糖胞苷、表柔比星、VCR、柔红霉素及门冬酰胺酶作用72h后耐药倍数（RI）分别为563.71、10.79、75.55、905.34、158.08和436.85倍，差异显著（P<0.01）；该耐药细胞与亲本CEM细胞相比体积较小；倍增时间为24.27h，其在细胞周期中G0/G1、S和G2/M期的分布分别为50.96%、47.92%和1.12%；CEM/VCR细胞的MDR1 、P-gp和topoⅡ的表达显著高于亲本细胞（p﹤0.01）。MTT实验说明蟾酥对CEM、 CEM/VCR细胞具有很强的抑制作用，随浓度增加、时间的延长，抑制率增强；24、48、72h的IC50 CEM细胞为1.5915、0.2774、0.0123µg/ml；CEM/VCR细胞为2.1992、0.7027、0.0433µg/ml；RI分别为1.38、2.53及3.53倍，两者的抑制作用没有差异（ P﹥0.05）；镜下见细胞体积缩小，细胞膜皱缩、破损，染色质浓缩、聚集，边聚于核膜，形成凋亡小体；流式术也证实蟾酥能诱导CEM/VCR细胞凋亡。蟾酥逆转实验：选用浓度0.0008µg/ml蟾酥为逆转剂量与CEM/VCR细胞共同培养72h脱药后，用不同浓度VCR分别作用于蟾酥处理和未加蟾酥处理的CEM/VCR细胞，显示VCR对蟾酥处理的CEM/VCR细胞的抑制率明显高于未加蟾酥的抑制率（p〈0.01）；蟾酥逆转剂量可将VCR对CEM/VCR细胞作用24、48、72h的IC50值由27.14、16.34、10.01μg/ml降至16.89、6.357、2.224μg/ml，其逆转倍数为1.61、2.57、4.50倍；免疫印迹显示，实验组与对照组比较能下调p-gp、Bcl-2、Bcl-XL和上调bax、p53、bak的表达（p〈0.01），凋亡相关蛋白的这种变化蟾酥处理组更为显著（p〈0.01）。本研究建立了可靠的模型—白血病多药耐药CEM/VCR细胞株；证明蟾酥能逆转白血病多药耐药CEM/VCR细胞的耐药性。