【作者】 赵丹；

【导师】 孟力；

【作者基本信息】 华中科技大学 ， 血液病学， 2011， 硕士

【摘要】 【目的】1.骨髓基质细胞(Bone Marrow Stromal cells,BMSCs)的分离及鉴定。2.检测骨髓基质细胞是否表达纤维连接蛋白(Fibronectin,Fn)。3.模拟体内肿瘤细胞与骨髓基质中纤维连接蛋白接触模型,验证纤维连接蛋白是否介导了血液肿瘤微小残留病灶的产生,即肿瘤耐药。【方法】1.收集血液科非恶性肿瘤患者骨髓液标本,梯度密度离心后,通过贴壁法培养骨髓基质细胞。直接在显微镜下观察分离培养的BMSCs细胞形态,应用流式细胞仪检测BMSCs的细胞标志。2.细胞免疫荧光法检测BMSCs表达Fn的表达情况。3.将包被有纤维连接蛋白的孔板与K562细胞共培养,MSCs细胞与K562细胞共培养,分别与伊马替尼、柔红霉素作用,然后通过流式细胞仪检测K562细胞周期阻滞和细胞凋亡情况。【结果】1.分离培养的BMSCs表面标志: CD44、CD105表达阳性(PE标记);CD19、CD34和CD45表达阴性(FITC标记)。2. MSCs细胞胞浆内表达Fn,且可以分泌Fn。3. Fn诱导K562细胞发生G1期阻滞,凋亡率降低。【结论】1.分离培养了BMSCs可以用于建立Fn介导的粘附耐药模型。分离培养的BMSCs分子标记与国际相关研究一致,可用于后期研究。2. BMSCs细胞胞浆内表达Fn,且可以分泌Fn。3.BMSCs细胞可能是通过分泌Fn蛋白诱导血液肿瘤细胞粘附耐药的。更多还原

【Abstract】 Objective:1. To Separate and identificate the bone marrow stromal cells.2. To detect whether fibronectin exist or is expressed in bone marrow stromal cells.3. To make the blood tumor cell contact with fibronectin simulating the bone marrow microenvironment in vivo, to validate if fibronectin has mediated the minimal residual disease of blood tumor cells, namely tumor resistance.Methods:1. To collect the bone marrow aspiration specimens of non-malignant tumor patients, by the method of density gradient centrifugalization to obtain bone marrow stromal cells and culture them which adhere on the cultural bottles’plastic surface . To observe the morphous of the BMSCs directly under the microscope and then to detect the symbols of the BMSCs by flow cytometric analysis.2. To detect the expression of fibronectin on or in BMSCs.3. To make the K562 cell-line co-cultured with the fibronectin wrapped plateand MSCs, respectively.Then to add imatinib or daunorubicin into those two co-cultural systems mentioned above.Finally to detect the cell cycle arrest and apoptosis. Results:1. We have separated and cultured MSCs , which expressed CD44,CD105 positive(PE marks), and CD19,CD45,CD34 negative(FITC marks).2. Fn has been detected in MSCs’cytoplasm,which can be secreted from them,either.3. Fn have induced K562 cell in G1 stage arrest and reduced apoptosis rate.Conclusions:1. We have separated and cultured MSCs ,which can be used to establish Fn-mediated adhesion resistance models. The molecular markers of these MSCs which can be used for further research are coincidence with international studies.2. Fn have been detected in MSCs’cytoplasm, and can be secreted from them either.3. MSCs might induce blood tumor chemotherapy adhesion resistance by secreting Fn.更多还原