人参皂苷及其三醇对白血病细胞增殖抑制、凋亡和药敏的研究

关键词 人参皂苷 人参三醇 白血病细胞 凋亡 药敏试验

Effects of ginsenosides and panaxatriol extracted

from ginseng on inhibition of proliferation，inducing apoptosis and

cytotoxic drug sensitivity in leukemic cells

Gao Ruilan，Lin Xiaojie，Qian Xudai，etal.

Affiliated Hospital of Zhejiang College of Traditional Chinese Medicine，Hangzhou310006.

【Abstract】 Objective To investigate if growth suppression and apoptosis ofleukemic progenitor cells can be induced by ginsenosides（GS）or panaxatriol（GPT）for exploring the effective components on leukemia within GS.Methods The components of GPT were purified from GS that were extracted from ginseng herb.Both primary leukemia progenitor cells from patients with acute myeloid leukemia and cell lines of K562/VCR，K562/Adr（resisted to cytotoxic drugs），HL-60，Meg-01，K562were incubated in semi-solid or liquid culture system.After being treated by GS or GPTwith various concentrations，the leukemia cells were observed for their proliferation，apoptosis and sensitivity of chemotherapy drugs by usingMTT assay，flow cytometry，Annexin V analysis，DNA ladder and drug sensi_ˉtive test.Results（1）GS displayed two activities of suppression and stimulation in proliferation of leukemic cells de_ˉpending on dose applied.Both normal and leukemic progenitor cells were susceptible to low concentration of GS to pro_ˉliferate and increase the colony numbers.The leukemic progenitor cells were obviously suppressed by medium concen_ˉtration of GS to decrease their colony numbers although growth of normal progenitor cells was still prompted.（2）In combination of GS with any one of chemotherapeutic drugs-homoharringtonin，cytarabine，adriamycin and etoposidi，the leukemic progenitor cells became more sensitive to cytotoxic drugs，Theinhibition rates of colony number were at1.84～2.23fold as more as those of non-GS control by four kinds of cytotoxic drugs respectively.Most interestingly，partial of drug sensitive tests also could be reversed by GS from resistance to sensitivity.（3）In combination of GS50mg/L with cytotoxic drugs，K562/VCR and K562/Adr cells became more sensitive to low concentration of cytotoxic drugs.The suppression effects of GS were obviously in dose-dependence manner.（4）The apoptosis of HL-60cells could be specifically induced by GS50mg/L for3days displaying Annexin V positive cells with flow-cytomery and DNA fragments ladder on gel electrophoresis.（5）The proliferation of K562cells could be enhanced obviously at low dosage of GPT20mg/L，while medium concentration of50mg/L GPT exerted inhibitory effects on cells significantly.The synergetic inhibition effects of GPT with cytotoxic drugs-homoharingtonin，cytarabine and etoposide on leukemic cells were obviously observed with high inhibition rates.Conclusion GS or GPT displayed both suppression and stim_ˉulation activities on leukemic cells depending on dosage applied.Both GS and GPT at low dose could drive non-cy_ˉcling leukemic progenitors into cell cycle，thereby potentiate their susceptibility to cytotoxic drugs.Meanwhile，GS could specifically induce apoptosis of leukemic cells at its medium concentrations.It suggested that GPT is an efficient component for anti-leukemia within ginsenosids.Our results might provide reliable evidence for clinicaltrial as GPT-assistant chemotherapy drugs.

Key words ginsenosides panaxatriol leukemic progenitor cells apoptosis cytotoxic drug sensitivity

近年来有关人参及其皂苷抗肿瘤作用的研究表明对肿瘤细胞具有抑制生长、诱导分化等作用 ^［1～3］ ，我们以前的研究表明，人参总皂苷（GS）对白血病祖细胞具有双向作用，低浓度时通过刺激白血病祖细胞增殖而增加对化疗药物的敏感性，中等浓度仍促进正常祖细胞生长，但抑制白血病祖细胞增殖，其原因未明 ^［4］ 。由于总皂苷的成分还较复杂，为了解GS中抗白血病的有效成分，本文从人参中提取GS，再从GS中分离出有效成分人参三醇（GPT）用作研究。应用造血祖细胞半固体集落培养、流式细胞术、药敏试验、细胞凋亡检测等多种实验方法，观察GS及GPT对白血病细胞抑制增殖、诱导凋亡和增加药敏的作用，以寻找GS中抗白血病的有效成分。

1 材料和方法

1.1 研究用药 GS干粉剂由本院中药研究室从人参中提取，纯度为81.6%。又从GS分离出有效成分GPT，经测定含量为75.8%。将GS或GPT配制成1g/L的工作液，无菌过滤，4℃保存备用。

1.2 液相—半固体相二步法培养白血病祖细胞集落（CFU-AML） 急性髓细胞白血病（AML）患者的骨髓细胞先用20%的植物血凝素—白细胞条件培养液（PHA-LCM）作刺激因子孵育20h，然后加入琼脂进行半固体集落培养，含20%小牛血清、10%PHA-LCM、0.3%琼脂和2×10 ⁵/ml骨髓有核细胞。培养体系分别加入不同浓度的GS或GPT。置37℃，5%CO ₂ 中培养7d后，计数白血病集落数（>40个细胞）。

1.3 白血病细胞药敏试验 选择临床上常用治疗AML的四种化疗药物高三尖杉酯碱（HHr）1mg/L、阿糖胞苷（Ara）55mg/L，阿霉素（Adr）5.5mg/L和足叶乙甙（Vp-16）50mg/L，分别与白血病细胞作用1h，洗去化疗药后作CFU-AML集落培养。以药物对CFU-AML集落的抑制率>30%作为对该药物敏感的标准。

1.4 DNA梯形条带电泳分析 细胞经冷PBS洗涤后，用裂解液（1%NP-40、0.5M EDTA、1M Tris.HCl，pH7.5）破碎细胞，离心沉淀后，取上清100μl。加入RNase A（3mg/L）和1%SDS，56℃作用2h，再用蛋白酶K（2mg/L）37℃作用3h。经乙酸钠和无水乙醇沉淀DNA，用1.2%琼脂糖凝胶电泳2～3h，紫外光下观察并摄影。