肿瘤相关成纤维细胞可介导急性髓系白血病细胞抵抗化疗

Carcinoma-associated fibroblasts mediates chemotherapy-resistance of acute myeloid leukemia cells

目的 :研究骨髓中肿瘤相关成纤维细胞(carcinoma-associated fi broblast,CAF)在急性髓系白血病(acute myeloid leukemia,AML)化疗抵抗中的作用,并初步探讨其作用机制。方法 :首先用重组人转化生长因子β1(recombinant human transforming growth factorβ1,rh TGFβ1)体外诱导AML患者的骨髓间充质干细胞(bone marrow-mesenchymal stem cells,BM-MSCs)形成CAF,蛋白质印迹法检测CAF中α-平滑肌肌动蛋白(alpha-smooth muscle actin,α-SMA)、成纤维细胞激活蛋白(f ibroblast activation protein,FAP)、Ⅰ型胶原蛋白(collagenⅠ)及Ⅲ型胶原蛋白(collagenⅢ)的分泌水平。然后构建CAF与AML细胞系THP-1或K562的共培养模型,锥虫蓝染色法检测经阿糖胞苷(cytosine arabinoside,Ara-C)化疗后白血病细胞的活性变化,FCM法分析白血病细胞的凋亡率和细胞周期分布情况。进一步应用TGFβ受体拮抗剂SB431542处理人白血病细胞系THP-1或K562,然后再与CAF共培养,采用锥虫蓝染色法检测CAF对白血病细胞抵抗Ara-C化疗的保护作用。结果 :TGFβ1可在体外诱导BM-MSCs分化形成CAF;与BM-MSCs相比,CAF高分泌α-SMA、FAP、collagenⅠ和collagenⅢ蛋白(P值均<0.01)。共培养实验结果表明,CAF细胞可明显减弱白血病THP-1和K562细胞对Ara-C的敏感性(P值均<0.01);此外,经Ara-C作用48 h后,CAF共培养组THP-1细胞的G0/G1期所占比例为(60.94±3.67)%,相比THP-1单独培养组的(37.20±3.91)%明显升高(P<0.05)。受体拮抗实验表明,SB431542处理能够显著减弱CAF对白血病细胞抵抗化疗的保护作用(P<0.001)。结论 :CAF可保护白血病细胞抵抗化疗,其机制可能与TGFβ通路密切相关,并通过维持白血病细胞处于静止状态而逃避化疗药物的杀伤作用。

Objective：To investigate the errect or carcinoma-associated fibroblasts（CAFs） on chemotherapy-resistance of acute myeloid leukemia（AML） cells,and to explore its mechanism.Methods：Recombinant human transforming growth factor beta 1（rhTGFβ1） was used in vitro to induce the bone marrow-mesenchymal stem cells（BM-MSCs） from AML patients into CAFs.The secretion levels of alpha-smooth muscle actin（a-SMA）,fibroblast activation protein（FAP）,collagen I and collagen Ⅲ in CAFs were detected by Western blotting.The co-cultured model was conducted by CAFs and AML cell line THP-1 or K562.Then the cell viability of the leukemia cells was measured by trypan blue staining after treatment with cytosine arabinoside（Ara-C）,and the apoptosis rate and cell cycle distribution of the leukemia cells were detected by flow cytometry.Furthermore,the leukemia THP-1 or K562 cells were treated by TGFpreceptor inhibitor SB431 542,then co-cultured with CAFs.The protective effect of CAFs on drug-resistance of the leukemia cells to Ara-C was measured by trypan blue staining.Results：BM-MSCs could be successfully induced into CAFs by rhTCFpi in vitro.Compared with the BM-MSCs,CAFs could secrete more a-SMA,FAP,collagen Ⅰ and collagen Ⅲ proteins（all P〈0.01）.The co-cultured experiments showed that the chemotherapy-sensitivity of the leukemic THP-1 or K562 cells was remarkably reduced after co-culture with CAFs（both P〈0.01）.In addition,under the treatment of Ara-C for 48 h,the percentage of THP-1cells in G0/G1 phase in CAFs co-culture group was significantly higher than that in THP-1cultured alone group[（60.94±3.67）%vs（37.20±3.91）%,P〈0.05].The TGFβ-receptor inhibitor experiment showed that SB431 542 could significantly decreased the chemotherapy protection effect of CAFs on leukemia cells（P〈0.001）.Conclusion：CAFs can protect leukemia cells against chemotherapy.Its mechanism may be closely related to TGFβ pathway,and maintaining the leukemia cells in a quiescent state to escape the killing effect of chemotherapeutic drugs.