骨髓基质细胞-白血病细胞共培养模型中主要微环境成分对化疗敏感性的影响

Effects of essential microenvironment components in the bone marrow stromal cell-leukemic cell co-culture model on chemotherapeutic sensitivity

背景：目前多数耐药机制研究均采用单细胞培养模型。目的：构建骨髓基质细胞．白血病共培养模型，探讨该模型中纤维连接蛋白、基质细胞膜蛋白及基质细胞因子等白血病微环境成分对白血病细胞化疗敏感性的影响。设计、时间及地点：开放性实验，于2006—03／10在解放军沈阳军区总医院中心实验室完成。材料：血常规、骨髓涂片细胞检查正常的骨髓标本11份，其中男6例，女5例，中位年龄34岁；经临床和血常规、骨髓涂片细胞检查确诊的未经治疗的急性淋巴细胞白血病患者骨髓标本13份，其中男7例，女6例，中位年龄28岁；每份骨髓1．0～2．0mL，50U／mL肝素抗凝。方法：①常规分离、培养骨髓基质细胞，Jurkat细胞与^60Co照射的基质细胞层黏附培养48h，收集上清，扫描电镜观。②2％戊二醛固定灭活基质细胞层，去除其细胞因子分泌功能，保留基质细胞膜蛋白。③通过MTT法测定柔红霉素的IC50、FITC-Annexin V／PI标记流式细胞仪定量细胞凋亡率。主要观察指标：采用倒置显微镜、扫描电镜及荧光显微镜对骨髓基质细胞、骨髓基质细胞-Jurkat细胞共培养模型及凋亡的Jurkat细胞进行形态学观察；通过流式细胞仪定量细胞凋亡率；MTT法测定IC50评价化疗药物敏感性。结果：①成功构建骨髓基质细胞-Jurkat共培养模型，扫描电镜发现Jurkat细胞通过伪足黏附于基质细胞层。②纤维连接蛋白黏附、灭活基质细胞黏附培养、活性基质细胞黏附及共培养上清均明显黏附抑制了柔红霉素对Jurkat细胞的细胞毒作用。③纤维连接蛋白黏附、灭活基质细胞黏附培养、活性基质细胞黏附及共培养上清均明显黏附抑制了柔红霉素对Jurkat的凋亡诱导效应。结论：骨髓基质细胞-Jurkat细胞共培养模型中微环境成分骨髓基质细胞膜蛋白、纤维连接蛋白和基质细胞因子均参与了Jurkat细胞耐药的形成。

BACKGROUD： Single-cell culture model is widely used in the studies of drug resistance mechanism currently. OBJECTIVE： To construct the bone marrow stromal cell （BMSCs）-leukemic cell co-culture model and investigate the influences of leukemia microenvironment components, such as fibronectin, BMSC membrane proteins and cytokines, in the co-culture system on chemotherapeutic sensitivity of leukemia cells. DESIGN, TIME AND SETTING： The open experiment was done at the Central Laboratory, General Hospital of Shenyang Military Area Command of Chinese PLA, between March and October 2006. MATERIALS： Eleven normal bone marrow samples were examined with blood routine tests and bone marrow smears, including 6 male and 5 female, the mean age of them was 34 years; thirteen bone marrow samples of patients who had definite acute lymphoblastic leukemia examined through clinical examination, blood routine tests and bone marrow smears and had not received treatement, including 7 male and 6 female, the mean age of them ＇was 28 years; each bone marrow sample was 1.0 -2.0 mL and was added 50 U/mL heparin for anticoagulation. METHODS： BMSCs were isolated and cultured routinely. Iurkat cells and ^60Co-irradiated BMSC layer were co-cultured for 48 hours, then supernatant was collected and cells observed under scanning electron microscope; BMSCs were inactivated by 2% glutaral to remove their function of cytokine secretion and remain membrane protein; dannomycin IC50 was detected through MTT and apoptosis rate was detected through FITC-Annexin V/PI-labeled flow cytometry, respectively. MAIN OUTCOME MEASURES： The cultured BMSCs, the co-cultured model of BMSCs-Jurkat cells and apoptosis Jurkat cells were observed with inverted microscope, scanning electron microscope and fluorescence microscope, respectively; apoptosis rate detected by flow cytometry; IC50 measured by MTT. RESULTS： BMSC-Jurkat co-culture model was constructed successfully, under scanning electron microscope we could see that Jurkat cells adhered to BMSC layer through pedes spurii. The fibronectin adhesion, adherent culture of inactivated BMSCs, adhesion of BMSCs and supernatant from the co-cultured system have a strong ability to inhibit daunomycin cytotoxic effects to Jurkat cells and daunomycin-induced apoptosis effect of Jurkat cells. CONCLUSION： The microenvironment components in the BMSC-Jurkat co-culture model, fibronectin, BMSC membrane proteins and cytokines, all participate in the formation of Jurkat cell drug resistance mechanism.