川楝子醇提物通过线粒体途径诱导白血病CEM细胞的凋亡  

Ethanol extract of toosendan induces apoptosis of leukemia CEM cells through mitochondrial pathway

作　　者：吴慧婷 朱大诚[1] 徐笑明 常娜 Wu Huiting;Zhu Dacheng;Xu Xiaoming;Chang Na(Jiangxi University of Chinese Medicine,Nanchang 330004,Jiangxi Province,China)

机构地区：[1]江西中医药大学,江西省南昌市330004

出　　处：《中国组织工程研究》2022年第30期4873-4878,共6页Chinese Journal of Tissue Engineering Research

基　　金：江西省研究生创新专项资金项目(YC2020-B146),项目负责人:吴慧婷。

摘　　要：背景:苦寒中药川楝子具有小毒,对生长环境要求不高,在实体瘤中能显著抑制肿瘤细胞的增殖,但在血液肿瘤中报道较少,基于前期研究结果,并结合中医“以毒攻毒”理论探讨其主要成分川楝素抗急性淋巴细胞白血病的作用机制。目的:探究川楝子醇提物抑制白血病CEM细胞增殖的作用机制。方法:人急性T淋巴细胞白血病CEM细胞培养至对数生长期,经5种浓度梯度川楝子醇提物作用24,48,72 h后,通过MTT实验求得各组细胞抑制率。考虑到受渗透压影响并保证后续实验的开展,筛选出川楝子醇提物低、中、高质量浓度(16,80,400 mg/L),作用大鼠外周血淋巴细胞24,48,72 h,采用MTT实验和吉姆萨-瑞氏染色探究其毒性作用,通过Hoechst 33258染色观察CEM细胞凋亡小体,RT-qPCR实验检测川楝子醇提物作用CEM细胞24,48 h后p53、Bcl-2、Bax、Cyt-C、Caspase-9和Caspase-3基因转录情况,Western blot实验检测川楝子醇提物作用CEM细胞24,48 h后p53、Bcl-2、Bax蛋白的表达。结果与结论:①16,80,400 mg/L川楝子醇提物对正常淋巴细胞无明显抑制作用;②16,80,400 mg/L川楝子醇提物作用CEM细胞经Hoechst 33258染色能看到蓝色亮光的凋亡小体;③与对照组比较,80,400 mg/L川楝子醇提物作用CEM细胞后能提高p53、Bax、Cyt-C、Caspase-9和Caspase-3基因转录水平(P<0.05或P<0.01),降低Bcl-2基因转录水平(P<0.05或P<0.01),尤其以48 h较为突出;④给药24 h后,相较于对照组,16,80,400 mg/L川楝子醇提物组Bcl-2蛋白表达下调,Bax蛋白表达升高(P<0.05或P<0.01),400 mg/L川楝子醇提物组p53蛋白表达升高(P<0.05),给药48 h后,相较于对照组,80,400 mg/L川楝子醇提物组p53蛋白表达明显上调(P<0.01),Bcl-2蛋白表达下调(P<0.05或P<0.01),Bax蛋白表达则显著升高(P<0.05或P<0.01);⑤结果表明,川楝子醇提物能显著抑制白血病CEM细胞的增殖,在一定范围内呈现浓度和时间依赖性,其机制可能是通过诱导线粒�BACKGROUND:Toosendan,a bitter and cold traditional Chinese medicine,has little toxicity and does not require high growth environment.It can significantly inhibit the proliferation of tumor cells in solid tumors,but it is rarely reported in blood tumors.Based on the previous research results,combined with the theory of“combating poison with poison”of traditional Chinese medicine,this paper discusses the mechanism of its main component toosendanin against acute lymphocyte leukemia.OBJECTIVE:To explore the mechanism of ethanol extract of toosendan inhibiting proliferation of leukemia CEM cells.METHODS:Human acute T lymphoblastic leukemia cell line(CEM cells)was cultured to logarithmic growth stage.After being treated with ethanol extracts of toosendan at five concentration gradients for 24,48,and 72 hours,their inhibition rates were obtained by MTT assay.Considering the influence of osmotic pressure of cells and ensuring the development of subsequent experiments,the suitable ethanol extracts of toosendan with low,medium,and high concentrations(16,80,and 400 mg/L)were screened out.The ethanol extract of toosendan had been exposed to peripheral blood lymphocytes of rats for 24,48,and 72 hours.MTT assay and Giemsa-Reich staining were used to explore its toxic effects.Hoechst 33258 staining was used to observe the apoptotic bodies of CEM cells.RT-qPCR assay was used to detect the transcription of p53,Bcl-2,Bax,Cyt-C,Caspase-9,and Caspase-3 genes in CEM cells after treatment for 24 and 48 hours.Western blot assay was used to detect the expression of p53,Bcl-2,and Bax protein in CEM cells after treatment for 24 and 48 hours.RESULTS AND CONCLUSION:(1)Low,medium and high doses(16,80,and 400 mg/L)of ethanol extract of toosendan had no significant inhibitory effect on normal lymphocytes.(2)After treatment with 16,80,and 400 mg/L of ethanol extract of toosendan in CEM cells,the apoptotic bodies with blue light could be seen by Hoechst 33258 staining.(3)Compared with the control group,the 80 and 400 mg/L ethanol extract of

关 键 词：川楝子醇提物 中药 以毒攻毒 白血病 CEM细胞 线粒体途径 凋亡 分子机制 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学]