酪氨酸激酶抑制剂逆转黏附诱导的骨髓瘤细胞的耐药性

 作者：潘耀柱，陈协群，高广勋，顾宏涛，张永清，董宝侠，白庆咸，朱华峰 

【摘要】  为了探讨酪氨酸激酶抑制剂STI571对RPMI8226细胞黏附、黏附诱导耐药以及靶细胞Rac1 mRNA表达的影响，本研究采用结晶紫染色法分析了RPMI8226细胞与纤连蛋白（fibronectin，FN）的黏附率，用MTT法检测瘤细胞的增殖性，并用半定量RT-PCR研究Rac1 mRNA水平的变化。结果显示：RPMI8226细胞与FN黏附1、6、12小时，其黏附率分别为(43.71±2.18)%、(55.63±1.56)%、(63.42±2.46)%；用20 μmol/L STI571处理1、6、12小时后黏附率分别降为(15.12±1.04)%、(17.58±1.32)%、(17.24±1.59)%；组间差异显著（P＜0.05）；阿霉素作用后，FN黏附组细胞IC50值［（1.46±0.04）μmol/L］显著高于BSA组［（0.78±0.03）μmol/L］(P＜0.05)；FN联合STI571组IC50值［(0.81±0.05)μmol/L］与BSA联合STI571组［（0.74±0.02）μmol/L］相比无显著性差异（P＞0.05），但却低于FN组(P＜0.05)；半定量RT-PCR显示，Rac1 mRNA水平在20 μmol/L STI571处理14小时后明显下降。结论：STI571能降低RPMI8226细胞与FN的黏附率、逆转黏附介导的阿霉素耐药，并且可以下调其Rac1 mRNA水平。

【关键词】  骨髓瘤细胞

　　Tyrosine Kinase Inhibitor Reverses Adriamycin Resistance Mediated by Cell Adhesion in RPMI8226 Cells

　　Abstract    To study the effects of tyrosine-kinase inhibitor STI571 on the adhesion of RPMI8226 cells to fibronectin(FN)，cell adhesion mediated adriamycin-resistance and the Rac1 mRNA expression，the adhesion of RPMI8226 cells to fibronectin and drug resistance mediated by cell adhesion were determined by means of crystal violet staining and MTT assays respectively，Rac1 mRNA levels in RPMI8226 cells were examined by semi-quantitative RT-PCR.The results showed that STI571 could inhibit the adhesion of RPMI8226 cells to fibronectin.When RPMI8226 cells had been adhe-red to FN or BSA-coated wells for 1，6 and 12 hours，the adhesion rates were (43.71±2.18)%，(55.63±1.56)%，and (63.42±2.46)%  respectively.After treatment with  STI571 20 μmol/L，the adhesion rates decreased to (15.12±1.04)%，(17.58±1.32)% and (17.24±1.59)%  respectively (P＜0.05).The experiment revealed that growth of  RPMI8226 cells adhered to FN-coated plates had a significant  advantage over  growth on BSA-coated plates when exposed to adriamycin (Adr) for 1 hour followed by a 24-hour culture period，and the mean IC50 value for FN-adhered cells was (1.46±0.04) μmol/L  while mean IC50 value for  BSA control  was (0.78±0.03)μmol/L (P＜0.05).Following treatment with  20 μmol/L STI571，the mean IC50 values for FN and BSA adhered cells were (0.81±0.05)μmol/L，(0.74±0.02)μmol/L respectively，there was no significant difference between them（P＞0.05）.RT-PCR demonstrated  that the relative Rac1 mRNA level (Rac1/GAPDH) in RPMI8226 cells was downregulated following being treated with 20μmol/L STI571.It is  concluded that  STI571 can inhibit the adhesion of RPMI8226 cells to fibronectin，reverse  cell adhesion mediated adriamycin-resistance，and downregulate Rac1 mRNA level.

　　Key words  tyrosine-kinase inhibitor; STI571; myeloma cell; RPMI8226 cell; adhesion; drug resistance

　　多发性骨髓瘤（MM）至今之所以被认为是一种难以治愈的浆细胞恶性肿瘤，是因为瘤细胞最终会产生多药耐药，其中MM细胞与细胞外基质（extracellular matrix，ECM）间黏附在耐药中占有重要的地位，作为细胞与ECM之间作用的主要介导分子整合蛋白（integrin）与纤连蛋白（fibronectin，FN)结合对凋亡抑制、 细胞存活具有重要作用。 Damiano等［1］发现，经阿霉素（adriamycin，Adr）处理后，与FN黏附的骨髓瘤细胞比不黏附者具有明显的生长优势和较低的凋亡率，他们称这一现象为细胞黏附介导的耐药（cell adhesion  mediated  drug resistance，CAM-DR），采用封闭性抗体阻断整合蛋白与FN间的相互作用后，瘤细胞与FN间的黏附率明显下降，CAM-DR得以完全逆转。

    肌动蛋白（actin）细胞骨架重构与细胞黏附密切相关，Abl酪氨酸激酶与RhoGTP酶Rac1是actin细胞骨架的重要调控分子。因此，Abl与Rac1极可能涉及MM细胞与细胞外基质(entracellular matrix，ECM)间的黏附过程以及靶细胞的CAM-DR形成。那么抑制Abl激酶活性能否逆转黏附诱导的MM细胞耐药呢？针对这一问题，我们以人骨髓瘤细胞株RPMI8226为研究对象，进一步探讨Abl激酶抑制剂STI571对MM细胞CAM-DR的逆转作用及其相关介导机理。

　　材料和方法

　　试剂

　　STI571由诺华制药公司惠赠，分子式为C29H31N7O·CH4SO3，分子量589.7。1粒胶囊含STI571 100 mg，用二甲亚砜（DMSO，Amrosco产品）充分溶解，离心去沉渣，以无菌注射用水稀释至1 mmol/L，分装，-20℃贮存。小牛血清购自杭州四季青生物工程材料有限公司。四氮唑蓝（MTT）、结晶紫（crystal violet）、阿霉素均为Sigma产品。纤连蛋白为Becton Dickinson公司产品，注射用水溶解，分装，-20℃冻存。Taq酶购自大连TaKaRa公司；反转录试剂盒购Promega公司；RNA提取试剂TRIzoL购自Invitrogen公司；Rac1及内参照3-磷酸甘油醛脱氢酶（GAPDH）引物均由上海Sangon公司合成。转贴于 酷文网-论文下载中心 http://www.coolwen.net

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