【作者】 陈文明；

【导师】 陈子兴；

【作者基本信息】 苏州大学 ， 内科学， 2007， 硕士

【摘要】 目的研究MDS来源的成骨细胞的生物学特性及其体外造血支持能力,探讨成骨细胞在MDS发病中的作用方法(1)第一部分采用酶联免疫夹心( ELISA)法检测12例MDS患者骨髓上清液中SDF-1α水平,探讨MDS骨髓微环境是否存在异常。(2)第二部分以人成骨细胞系hFOB1.19为对象,研究成骨细胞的生物学特性,初步探讨成骨细胞在造血调控中的作用。应用流式细胞术(Flow cytometry , FCM)分析人成骨细胞系hFOB1.19的免疫表型, RT-PCR检测生长因子的表达情况。(3)第三部分研究MDS来源的成骨细胞的生物学特性,探讨成骨细胞在MDS发病中的作用。采集MDS患者和正常供者骨髓标本,分离、培养间充质干细胞(Mesenchymal stem cell, MSC),进行细胞形态学观察、免疫表型及成纤维细胞集落(CFU-F)形成分析。取第3代MSC体外诱导为成骨细胞,丝裂霉素C处理后做为滋养层细胞。在无外源性细胞因子的条件下,将经Ficoll分离获取的正常供者来源的单个核细胞(MNC)接种到成骨细胞上共培养,研究成骨细胞体外支持造血祖细胞生存的作用;应用RT-PCR在mRNA水平上分析由骨髓间充质干细胞诱导分化的成骨细胞生长因子的表达情况。结果(1)第一部分MDS难治性贫血伴有原始细胞增多(RAEB)组患者骨髓上清液中SDF-1α水平明显低于难治性贫血(RA)+环形铁粒幼细胞性难治性贫血(RAS)组及对照组(均P<0.05)。MDS患者骨髓上清液中SDF-1α水平和MDS患者骨髓原始细胞比例成负相关(r = -0.585, P=0.046),和其国际预后评分系统(IPSS)积分成负相关(r=-0.657,P=0.02),但和外周血中的白细胞计数、血小板数量及血红蛋白浓度均无明显相关性(均P>0.05)。(2)第二部分FCM显示, hFOB1.19表达CD44、CD73(SH3)、CD105(SH2)及CD90 (Thy1),而不表达CD34、CD45、HLA-DR等造血细胞标记;RT-PCR显示hFOB1.19表达Oct-4、Rex-1等胚胎干细胞标志但不表达hTERT,并经证实具有多向分化的潜能,此外hFOB1.19还能表达SCF , IL-6,IL-11,SDF-1, GM-CSF和G-CSF等多种生长因子。(3)第三部分MDS患者MSC在体外呈典型的成纤维样细胞形态,细胞形态和生长模式与正常供者来源的MSCs无明显差异。MDS患者和正常供者骨髓细胞的成纤维集落形成能力(CFU-F)也没有差异(P>0.05)。和MNC共培养后,发现MDS患者来源的成骨细胞在无外源性细胞因子的情况下仍能够短期(3周)维持GM -CFC造血祖细胞的存活。MDS成骨细胞组培养上清中的细胞形成的CFU-GM集落数与对照组比较无明显差异(P > 0. 05)。RT-PCR发现,正常供者来源的MSC表达SCF,IL-6,IL-11,SDF-1等生长因子mRNA,当诱导分化为成骨细胞时开始表达G-CSF mRNA,但始终未见GM-CSF表达。MDS患者来源的MSCs也能表达上述生长因子的mRNA,当诱导分化为成骨细胞后开始表达G-CSF mRNA,同样未见GM-CSF表达。结论(1)MDS患者SDF-1/CXCR4系统存在异常,检测SDF-1α水平可能有助于MDS的诊断、判断其预后,并为治疗MDS提供新思路。(2)人成骨细胞系hFOB1.19可能是处于较早阶段的成骨祖细胞,表达多种造血相关的生长因子,成骨细胞可能对骨髓正常造血具有重要的调控作用。(3)MDS患者来源的成骨细胞表达多种造血相关的生长因子,并能维持GM-CFC造血祖细胞的存活,提示MDS的病变可能并不累及成骨细胞。更多还原

【Abstract】 Objective To study the biological characteristics of osteoblasts derived from patients with myelodysplastic syndrome (MDS) and their hematopoietic supportive function in vitro.Methods (1) PartⅠIn order to investigate the abnormal hematopoietic microenvironment in MDS, ELISA was used to detect levels of SDF-1αin bone marrow plasma from 12 MDS patients. (2) PartⅡUsing human fetal osteoblastic cell line 1.19 as a model to study the biological characteristics of osteoblasts, and their capacity in support of hematopoiesis. Flow cytometry was used to identify their immunophenotype,and RT-PCR was used to detect the expression of hematopoietic cytokines.(3) PartⅢTo study the biological characteristics of osteoblasts derived from patients with myelodysplastic syndrome (MDS) and their hematopoietic supportive function in vitro. MSCs isolated from bone marrow of MDS patients and normal donors were cultured. Morphology, immunophenotype and colony forming unit fibroblast (CFU-F) of MSC were measured and analyzed. The third generation of cultured MSCs were induced to osteoblasts. After treated with mitomycin C , they became a feeder layer. Ficoll-isolated bone marrow mononuclear cell from normal donors were then seeded on the feeder layer to culture in vitro without exogenous cytokines. RT-PCR was used to determine the expression of hematopoietic cytokines in osteoblasts induced from human marrow mesenchymal stem cells.Results (1) PartⅠThe level of SDF-1αin refractory anemia with excess blasts (RAEB) patients was significant lower than that in refractory anemia (RA) patients , refractory anemia with ring sideroblasts(RAS) patients and controls (P < 0.05). The SDF-1αlevel was negatively correlated with the percentage of BM myeloblasts(r = -0.585, P=0.046).The correlation of SDF-1αlevels with outcomes of International Prognostic Scoring System (IPSS) was also observed(r = -0.657, P=0.02). Whereas, the SDF-1αlevels of MDS patients had no correlation with peripheral white blood cells counts, platelet counts and hemoglobin level (P>0.05).(2) PartⅡFCM revealed that hFOB cells were positive for CD44, CD73(SH3), CD105(SH2) and CD90 (Thy1), but negative for CD34, CD45, HLA-DR. RT-PCR found that hFOB cells expressed the ESC pluripotency marker of Oct-4, Rex-1, except hTERT. Meanwhile, it has also been proved that hFOB cells’capacity of adipogenic differentiation. Moreover, hFOB cells expressed mRNA of SCF , IL-6 , IL-11, SDF-1, GM-CSF and G-CSF. (3) PartⅢMSCs obtained from MDS patients and normal donors were displaying fibroblastoid morphology. Their growth pattern, immunophenotype and colony forming unit fibroblast (CFU-F) number were similar (P >0.05). Without exogenous cytokines, the osteoblasts derived from MDS can sustain GM–CFC survival for at least 3 weeks. The CFU-GM yield in supernatants after co-culture was not different from those of control in hematopoiesis supportive experiments in vitro ( P>0.05). RT-PCR clearly showed that the cultured BM-MSC from normal donor expressed mRNA of SCF, SDF-1, IL-6, and IL-11. As the MSC differentiated toward osteoblasts , the expression of G-CSF could be detected,whereas GM-CSF remained undetectable. The same expression pattern of above cytokines were also seen from osteoblasts induced from BM-MSC of MDS patients.Conclusions (1) The SDF-1/CXCR4 system may play a potential role in the pathogenesis of MDS. Detection of SDF-1αmay be helpful for diagnosis and predicting its prognosis of MDS. In addition, it may provide new clues for the treatment of MDS. (2) Human fetal osteoblastic cell line 1.19 possess the capacity of multilinage differentiation and express many hematopoietic cytokines. In conclusion , osteoblasts may play a pivotal role in hematopoiesis.(3) The biological characteristics of osteoblasts from bone marrow of MDS patients were generally not different from those of osteoblasts in bone marrow of normal controls. Both of them could support GM -CFC hematopoietic progenitor cells survival in vitro, according to their expression of multiple cytokines. In conclusion, the osteoblasts derived from MDS patients may not be involved in the malignant process.更多还原