摘要
目的探讨扁蒴藤素(PM)对骨肉瘤细胞阿霉素(ADM)耐药性的影响及其作用机制。方法采用ADM浓度梯度递增长期培养法在体外建立ADM耐药细胞MG-63/ADR,CCK-8检测不同浓度ADM对骨肉瘤细胞MG-63和MG-63/ADR细胞活力的影响,验证ADM耐药性;随后将MG-63/ADR细胞随机分为MG-63/ADR组(正常培养)、PM低剂量组(加入0.5µmol/L PM)、PM中剂量组(加入1.0µmol/L PM)、PM高剂量组(加入2.0µmol/L PM),CCK-8检测不同浓度ADM处理下各组细胞活力;平板克隆形成实验检测各组细胞对ADM的敏感性;显微镜下观察细胞形态;流式细胞术检测细胞凋亡水平;Western blot检测磷酸化哺乳动物STE20样蛋白激酶1(p-MST1)、MST1、磷酸化大肿瘤抑制因子1(p-LATS1)、LATS1、磷酸化Yes相关蛋白(p-YAP)、YAP蛋白表达。结果25、125、625、3125、15625 ng/mL ADM干预下,MG-63/ADR细胞活力较MG-63细胞显著升高(P<0.05),说明ADM耐药细胞模型建立成功。与MG-63/ADR组相比,PM低、中、高剂量组细胞数量减少且细胞逐渐皱缩、间距变大,细胞活力、细胞克隆形成率以及p-YAP/YAP水平下降(P<0.05),细胞凋亡率和p-MST1/MST1、p-LATS1/LATS1水平显著提高(P<0.05)。结论PM对骨肉瘤细胞ADM耐药性具有抑制作用,其可能与激活Hippo通路、下调YAP信号有关。
Objective To investigate the effect of pristimerin(PM)on adriamycin(ADM)resistance in osteosarcoma cells and its mechanism.Methods The ADM resistant cells MG-63/ADR was established in vitro by using the ADM increasing concentration gradient long-term culture method,CCK-8 was used to detect the effects of different concentrations of ADM on the viability of osteosarcoma cells MG-63 and MG-63/ADR cells to verify the drug resistance of ADM;subsequently,MG-63/ADR cells were randomly divided into the MG-63/ADR group(normal culture),low-dose PM group(adding 0.5µmol/L PM),middle-dose PM group(adding 1.0µmol/L PM)and high-dose PM group(adding 2.0µmol/L PM).The cell viability of each group under different concentrations of ADM treatment was detected by CCK-8;the sensitivity of cells to ADM in each group was detected by plate clone formation assay;the cell morphology of each group was observed under microscope;the cell apoptosis level was detected by flow cytometry;Western blot was used to detect the expression of phosphorylated mammalian STE20-like protein kinase 1(p-MST1),MST1,phosphorylated large tumor suppressor 1(p-LATS1),LATS1,phosphorylated Yes associated protein(p-YAP)and YAP proteins.Results With the intervention of 25,125,625,3125 and 15625 ng/mL ADM,the activity of MG-63/ADR cells was significantly higher than that of MG-63 cells(P<0.05),which indicated that the ADM resistant cell model was successfully established.Compared with the MG-63/ADR group,the number of cells in the low,middle and high-dose PM groups decreased,and the cells gradually shrank,the spacing between cells became larger,and the cell viability,cell clonal formation rate and p-YAP/YAP level decreased(P<0.05),the apoptosis rate and the levels of p-MST1/MST1 and p-LATS1/LATS1 significantly increased(P<0.05).Conclusion The inhibitory effect of PM on ADM resistance in osteosarcoma cells may be related to the activation of Hippo pathway and the downregulation of YAP signal.
作者
李浩
戎帅
刘连涛
LI Hao;RONG Shuai;LIU Lian-tao(Department of Pediatric Orthopedics,Shijiazhuang Third Hospital,Shijiazhuang Hebei050000,China)
出处
《局解手术学杂志》
2024年第9期796-800,共5页
Journal of Regional Anatomy and Operative Surgery
基金
河北省医学科学研究重点课题计划项目(20191425)。
