补肾养骨汤对人多发性骨髓瘤耐药细胞KM3/BTZ多药耐药性的影响
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摘要
目的:探讨补肾养骨汤对人多发性骨髓瘤耐药细胞KM3/BTZ耐药性及增殖的影响,为补肾养骨汤在骨髓瘤治疗方面的前景和应用提供参考。方法:采用硼替佐米(bortezomib,BTZ)浓度梯度诱导法建立人多发性骨髓瘤耐药细胞KM3/BTZ,通过MTT法检测常用化疗药物以及含补肾养骨汤药血清对KM3细胞和KM3/BTZ细胞增殖的影响,采用RT-qPCR及Western blot法检测Par-4、HSP27和P-gp基因表达变化,采用流式细胞术检测细胞凋亡情况。结果:建立的KM3/BTZ细胞对常用化疗药物均可产生不同程度的耐药性,其中BTZ的耐药指数(RI)最高,为20.269。MTT法检测结果显示,经含补肾养骨汤药血清处理过的KM3/BTZ细胞增殖受抑制,且随含补肾养骨汤药血清浓度升高抑制作用呈增强趋势。含补肾养骨汤药血清可抑制KM3/BTZ细胞增殖,诱导其凋亡,明显降低KM3/BTZ细胞耐药性,上调Par-4表达,下调HSP27和P-gp表达。结论:补肾养骨汤能有效抑制KM3/BTZ细胞增殖,诱导其凋亡,逆转KM3/BTZ细胞多药耐药性,其机制可能与HSP27和P-gp表达下调,Par-4表达升高有关。
Objective: To explore the effect of Bushen Yanggu Decoction(BYD) on drug resistance and proliferation of human multiple myeloma-resistant KM3/BTZ cells. Methods: Human multidrug-resistant KM3/BTZ cells were established by Bortezomib(BTZ) gradient induction. The effects of commonly used chemotherapeutic drugs and serum containing Bushen Yanggu Decoction(BYD) on the proliferation of KM3 cells and KM3/BTZ cells were detected by MTT assay. RT-qPCR and Western blot were used to detect the expression of Par-4, HSP27 and P-gp genes. Flow cytometry was used to detect cell apoptosis. Results: The established KM3/BTZ cells could produce varying degree of resistance to commonly used chemotherapeutic drugs. Among them, the highest resistance index(RI) to BTZ was 20.269.MTT assay showed that the proliferation of KM3/BTZ cells treated with serum containing Bushen Yanggu Decoction was inhibited, and the inhibitory effect increased with the serum concentration incranse of Bushen Yanggu Decoction.The serum containing Bushen Yanggu Decoction could inhibit the proliferation of KM3/BTZ cells, and induce apoptosis,significantly reduce the drug-resistance of KM3/BTZ cells, up-regulate the expression of Par-4, down-regulate the expression of HSP27 and P-gp. Conclusion: Bushen Yanggu Decoction can effectively inhibit the proliferation of KM3/BTZ cells and induce apoptosis. Bushen Yanggu Decoction can effectively reverse the multidrug-resistance of KM3/BTZ cells. The mechanism may be related with the decrease of expression of HSP27 and P-gp and the increase of expression of Par-4.
Objective: To explore the effect of Bushen Yanggu Decoction(BYD) on drug resistance and proliferation of human multiple myeloma-resistant KM3/BTZ cells. Methods: Human multidrug-resistant KM3/BTZ cells were established by Bortezomib(BTZ) gradient induction. The effects of commonly used chemotherapeutic drugs and serum containing Bushen Yanggu Decoction(BYD) on the proliferation of KM3 cells and KM3/BTZ cells were detected by MTT assay. RT-qPCR and Western blot were used to detect the expression of Par-4, HSP27 and P-gp genes. Flow cytometry was used to detect cell apoptosis. Results: The established KM3/BTZ cells could produce varying degree of resistance to commonly used chemotherapeutic drugs. Among them, the highest resistance index(RI) to BTZ was 20.269.MTT assay showed that the proliferation of KM3/BTZ cells treated with serum containing Bushen Yanggu Decoction was inhibited, and the inhibitory effect increased with the serum concentration incranse of Bushen Yanggu Decoction.The serum containing Bushen Yanggu Decoction could inhibit the proliferation of KM3/BTZ cells, and induce apoptosis,significantly reduce the drug-resistance of KM3/BTZ cells, up-regulate the expression of Par-4, down-regulate the expression of HSP27 and P-gp. Conclusion: Bushen Yanggu Decoction can effectively inhibit the proliferation of KM3/BTZ cells and induce apoptosis. Bushen Yanggu Decoction can effectively reverse the multidrug-resistance of KM3/BTZ cells. The mechanism may be related with the decrease of expression of HSP27 and P-gp and the increase of expression of Par-4.
引文
1中国医师协会血液科医师分会.中国多发性骨髓瘤诊治指南(2017年修订).中华内科杂志, 2017; 56(11):866-870.
2 Michels TC, Petersen KE. Multiple myeloma:diagnosis and treatment. Am Fam Physician, 2017; 95(6):373-383.
3 Mateos MV, Ocio EM, Paiva B, et al. Treatment for patients with newly diagnosed multiple myeloma in 2015. Blood Rev, 2015;29(6):387-403.
4 魏占杰.补肾养骨汤联合VAD方案对老年多发性骨髓瘤血清IL-6和VEGF水平的影响.西部中医药, 2017; 30(5):88-91.
5 董静,吴勃岩,车艳新,等.熟地黄多糖诱导H_(22)荷瘤小鼠细胞凋亡作用的研究.中医药信息, 2015; 32(4):32-34.
6 胡吉梦,姜昊文.淫羊藿素及相关衍生物的抗肿瘤机制研究进展.上海中医药杂志,.2018; 52(4):107-112.
7 李志远,李振杰,陈旭,等.淫羊藿素对人多发性骨髓瘤耐药细胞株KM3/BTZ多药耐药性的逆转作用.中国实验血液学杂志, 2017; 25(6):1690-1695.
8 吕艳芬,苏子成.丹参抗肿瘤转移的作用及其机制.肿瘤研究与临床, 2009; 21(2):138-139.
9 沈莹,胡晔,蒋树龙.丹参抗肿瘤作用的临床研究进展.天津中医药, 2015; 32(9):574-576.
10 王爱云,陶丽,陆茵,等.丹参干预肿瘤和缺血性疾病血管生成研究进展.中草药, 2015; 46(9):1399-1404.
11 薛均来.覆盆子抑制基质金属蛋白酶活性与抗肿瘤机理的实验研究:吉林大学; 2010.
12 Wu QN, Gan XG, Lei XT, et al. Par-4/NF-κB mediates the apoptosis of is letβcells induced by glucolipotoxicity. J Diabetes Res, 2016;2016:4692478.
13 Saegusa M, Hashimura M, Kuwata T, et al. Transcriptional regulation of pro-apoptotic Par-4 by NF-κB/p65 and its function in controlling cell kinetics during early events in endometrial tumourigenesis. J Pathol, 2010; 221(1):26–36.
14 Han JY, Lim YJ, Choi JA, et al. The role of prostate apoptosis response-4(Par-4)in mycobacterium tuberculosis infected macrophages.Sci Rep, 2016; 6:32079.
15 Sookoian S, Casta?o GO, Scian R, et al. Heat shock protein 27is down-regulated in ballooned hepatocytes of patients with nonalcoholic steatohepatitis(NASH). Sci Rep, 2016; 6:22528.
2 Michels TC, Petersen KE. Multiple myeloma:diagnosis and treatment. Am Fam Physician, 2017; 95(6):373-383.
3 Mateos MV, Ocio EM, Paiva B, et al. Treatment for patients with newly diagnosed multiple myeloma in 2015. Blood Rev, 2015;29(6):387-403.
4 魏占杰.补肾养骨汤联合VAD方案对老年多发性骨髓瘤血清IL-6和VEGF水平的影响.西部中医药, 2017; 30(5):88-91.
5 董静,吴勃岩,车艳新,等.熟地黄多糖诱导H_(22)荷瘤小鼠细胞凋亡作用的研究.中医药信息, 2015; 32(4):32-34.
6 胡吉梦,姜昊文.淫羊藿素及相关衍生物的抗肿瘤机制研究进展.上海中医药杂志,.2018; 52(4):107-112.
7 李志远,李振杰,陈旭,等.淫羊藿素对人多发性骨髓瘤耐药细胞株KM3/BTZ多药耐药性的逆转作用.中国实验血液学杂志, 2017; 25(6):1690-1695.
8 吕艳芬,苏子成.丹参抗肿瘤转移的作用及其机制.肿瘤研究与临床, 2009; 21(2):138-139.
9 沈莹,胡晔,蒋树龙.丹参抗肿瘤作用的临床研究进展.天津中医药, 2015; 32(9):574-576.
10 王爱云,陶丽,陆茵,等.丹参干预肿瘤和缺血性疾病血管生成研究进展.中草药, 2015; 46(9):1399-1404.
11 薛均来.覆盆子抑制基质金属蛋白酶活性与抗肿瘤机理的实验研究:吉林大学; 2010.
12 Wu QN, Gan XG, Lei XT, et al. Par-4/NF-κB mediates the apoptosis of is letβcells induced by glucolipotoxicity. J Diabetes Res, 2016;2016:4692478.
13 Saegusa M, Hashimura M, Kuwata T, et al. Transcriptional regulation of pro-apoptotic Par-4 by NF-κB/p65 and its function in controlling cell kinetics during early events in endometrial tumourigenesis. J Pathol, 2010; 221(1):26–36.
14 Han JY, Lim YJ, Choi JA, et al. The role of prostate apoptosis response-4(Par-4)in mycobacterium tuberculosis infected macrophages.Sci Rep, 2016; 6:32079.
15 Sookoian S, Casta?o GO, Scian R, et al. Heat shock protein 27is down-regulated in ballooned hepatocytes of patients with nonalcoholic steatohepatitis(NASH). Sci Rep, 2016; 6:22528.