hUMSCs外分泌上清联合替莫唑胺在不同胶质瘤细胞系中的协同增敏作用  

Synergistic sensitization of hUMSCs-derived supernatant combined with temozolomide in different glioma cell lines

作　　者：刘雨思 王明明 张玉富 靳小燕 贺晶[2] 史海燕 陈美霓[1] 张静[1,3,4] LIU Yusi;WANG Mingming;ZHANG Yufu;JIN Xiaoyan;HE Jing;SHI Haiyan;CHEN Meini;ZHANG Jing(Medical College of Yan'an University,Yan'an 716000,China;The Affiliated Hospital of Yan'an University,Yan'an 716000,China;The Second Affiliated Hospital of Xi'an Medical University,Xi'an 710038,China;Shaanxi Stem Cell Engineering Co.,Ltd,Xi'an 710075,China)

机构地区：[1]延安大学基础医学院,陕西延安716000 [2]延安大学附属医院,陕西延安716000 [3]西安医学院第二附属医院,陕西西安710038 [4]陕西干细胞工程有限公司,陕西西安710075

出　　处：《中国病理生理杂志》2024年第3期385-394,共10页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.82260489);陕西省科技厅自然科学基础研究计划项目(No.2022JQ-931)。

摘　　要：目的:探讨人脐带间充质干细胞外分泌上清(hUMSC-CM)联合替莫唑胺(TMZ)在不同胶质瘤细胞系中的协同增敏作用及潜在机制。方法:采用2种血清剥夺法(24和48 h分批次撤血清法)收集hUMSC-CM并制备成冻干粉,设置5种浓度(0、1、3、6和9 g/L)处理大鼠恶性胶质瘤细胞系RG-2、人星形细胞瘤细胞系U251和人胶质母细胞瘤细胞系LN-428。通过CCK-8实验检测hUMSC-CM作用于胶质瘤细胞24、48和72 h后的肿瘤抑制可行性及敏感度。HE染色结合CCK-8法确定6种浓度(0、25、50、100、200和400µmol/L)的TMZ作用于胶质瘤细胞48 h后化疗敏感性的差异。筛选出低、高2种浓度(3和9 g/L)的hUMSC-CM和低、中、高3种浓度(50、100和200µmol/L)的TMZ配伍,作用于胶质瘤细胞后检测细胞活力和病理形态学变化。TUNEL染色检测细胞凋亡;流式细胞术分析细胞周期变化;Western blot检测凋亡相关蛋白cleaved caspase-3、cleaved caspase-8和cleaved PARP1,以及自噬相关蛋白beclin-1和LC3的表达变化,探讨hUMSC-CM与TMZ体外联合给药协同增敏的作用机制。结果:3种胶质瘤细胞系对hUMSC-CM和TMZ的敏感度为RG-2>U251>LN-428。hUMSC-CM(3和9 g/L)与TMZ(50、100和200µmol/L)配伍给药对胶质瘤细胞生长的抑制作用比单独给药组显著增强(P<0.05),且随着配伍药物剂量的增加而增强。其中,9 g/L hUMSC-CM(C9)与50µmol/L TMZ(T50)配伍可有效抑制胶质瘤细胞生长。与C9或T50组相比,CCK-8实验显示C9+T50组细胞活力显著下降(P<0.05),HE染色和TUNEL检测结果显示C9+T50组细胞形态变化明显,出现典型凋亡形态学特征,流式细胞术结果显示C9+T50可诱导胶质瘤细胞周期发生阻滞,Western blot结果显示C9+T50组细胞中cleaved caspase-3、cleaved caspase-8、cleaved PARP1、beclin-1和LC3-II/LC3-I水平显著升高(P<0.01)。结论:(1)hUMSC-CM与TMZ配伍给药对胶质瘤细胞的抑制作用具有广谱性,且两者之间存在增敏作用,在不同细胞系中呈现不同AIM:To explore the synergistic sensitization effect of human umbilical cord mesenchymal stem cell culture supernatant(hUMSC-CM)combined with temozolomide(TMZ)on various glioma cell lines,and to elucidate the underlying mechanisms.METHODS:The hUMSC-CM was harvested using two different serum deprivation techniques at 24 and 48 h,and was converted into freeze-dried powder,which was then given to rat malignant glioma cell line RG-2,human astrocytoma cell line U251 and human glioblastoma cell line LN-428 at 5 concentrations(0,1,3,6 and 9 g/L).The effectiveness and sensitivity of hUMSC-CM for inhibiting growth of glioma cells at 24,48 and 72 h were assessed using CCK-8 assay.Hematoxylin-eosin(HE)staining combined with CCK-8 assay was employed to evaluate the chemotherapy sensitivity of glioma cells after 48 h of treatment with TMZ at 6 concentrations(0,25,50,100,200 and 400µmol/L).Two concentrations(3 and 9 g/L)of hUMSC-CM and 3 concentrations(50,100 and 200µmol/L)of TMZ were chosen for concurrent treatment of glioma cells to assess the proliferation and pathological alterations.TUNEL staining was utilized to detect apoptosis.Flow cytometry was utilized to analyze cell cycle modifications.The expression alterations of apoptosis-inducing proteins,cleaved caspase-3,cleaved caspase-8 and cleaved PARP1,as well as autophagy-inducing proteins beclin-1 and LC3,were examined using Western blot to investigate the synergistic sensitization mechanism of hUMSC-CM combined with TMZ in vitro.RESULTS:The susceptibility of glioma cell lines to hUMSC-CM and TMZ varied,with RG-2 showing the highest sensitivity,followed by U251,and then LN-428.The inhibitory effect of hUMSC-CM(3 and 9 g/L)and TMZ(50,100 and 200µmol/L)combined treatment on glioma cells was significantly greater than that that of single-agent treatments(P<0.05),demonstrating a dose-and concentration-dependent enhancement.Notably,the combination of 9 g/L hUMSC-CM(C9)with 50µmol/L TMZ(T50)effectively suppressed glioma cell growth.CCK-8 assay indicated a significant reduc

关 键 词：脐带间充质干细胞 替莫唑胺 胶质瘤 细胞凋亡 自噬 caspase-8/caspase-3/PARP1信号通路 

分 类 号：R739.4[医药卫生—肿瘤] R979.11[医药卫生—临床医学] R363.2