脐血干细胞向巨核细胞分化后信号转导基因的表达(英文)

Expression of signal transduction genes after differentiation of cord blood stem cells into megakaryocytes

背景:造血干细胞具有多向分化的潜能,在合适的造血生长因子和系特异性生长因子的诱导下,可分化成巨核细胞。在体外诱导CD34^+造血干细胞向巨核细胞分化可导致许多基因尤其是信号转导基因表达的变化。目的:观察脐血干细胞向巨核细胞分化后信号转导基因的表达,拟从基因水平验证其对巨核细胞分化发育的调控。设计:开放性实验。单位:福建省肿瘤医院肿瘤免疫学研究室。材料:无菌条件下应用血袋采集法收集足月正常分娩的胎儿脐带血60～145 mL,产妇及家属自愿捐献。VarioMACS免疫磁性吸附柱分离装置,CD34+Isolation Kit;SCGM无血清培养基;CD单抗;细胞因子。人类全基因组寡核苷酸微阵列芯片V2.0;LuxScan 10K/A双通道激光扫描仪。实验经医院伦理委员会批准。方法:实验于2005-07/2006-06在福建省肿瘤医院肿瘤免疫研究室及北京博奥生物芯片有限公司完成。分别收集同一份脐血分化培养前CD34^+细胞和培养后CD41^+细胞,提取总RNA。逆转录合成和纯化cDNA探针,以Cy3-dCTP标记培养前CD34^+细胞,以Cy5-dCTP标记培养后CD41^+细胞标记的DNA溶于30μL杂交液中,42℃过夜。主要观察指标:应用基因芯片技术比较造血干细胞与分化的巨核细胞信号转导相关的基因表达差异。根据基因芯片结果,选定17个表达差异基因用RT-PCR作进一步验证。结果:芯片分析结果显示,共筛选出3522个差异基因,其中上调基因1705个,下调基因1817个。3522个差异基因中,与细胞信号相关的基因有343个,与转录调节相关的有150个,与分化相关的有21个。其中,CD61基因的表达增加了369.83倍,CD41基因的表达增加27.38倍,PF4基因的表达增加24.06倍;促分裂原活化蛋白澈酶s、G蛋白偶联受体、RAS家族相关的基因多数表达上调;与STAT通路相关的基因中,SOCS1、JANUS激酶表达上调,STAT5A表达下调。对选定的17个表达差异基因用RT-PCR作进一步验证,以GAPDH为内对照,结果与芯片检测完全一致。结论:血小板生成素等造血生长因子可能主要通过G蛋白偶联受体-Ras-促分裂原活化蛋白激酶途径,促进脐血干细胞向巨核细胞系增殖、分化。

BACKGROUND： Hematopoietic stem cells have an ability of multi-directional differentiation, and can be differentiated into megakayocytes in the presence of suitable hematopoietic growth factors and lineage special growth factors. Inducing of CD34＋ stem cells to megakaryocytes in vitro involves many changes in gene expression, especially the signal transduction genes. OBJECTIVE： To study the expression of signal transduction genes after differentiation from stem cells into megakaryocytes. DESIGN ： Open experiment. SETTING： Department of Tumor Immunology, Fujian Provincial Tumor Hospita. MATERIALS： 60-145 mL of cord blood was collected from normal labor fetuses under sterile conditions by using the blood-bag collective method. Patients and their relatives provided the confirmed consent. Reagents and equipments were detailed as follows： VarioMACS segregation apparatus, CD34＋ Isolation Kit, SCGM serum-free medium, CD monoclonal antibodies, cytokines, human genome Oligo Set Version 2.1 from the CapitalBio Corporation, and LuxScan 10K/A bi-passage laser scanning apparatus. The researchers obtained consent from the local ethic committee. METHODS： This experiment was carded out in the Department of Tumor Immuonlogy, Fujian Provincial Tumor Hospital and the CaptialBio Corporation. from July 2005 to June 2006. RNA was extracted separately from CD34＋ cells purified from cord blood and CD41＋ cells induced from the same cord blood. The cDNA probes were synthesized by reverse transcription and purified. CD34＋ cells before culturing and CD41＋ cells after culturing were labeled with Cy3-dCTP and Cy5-dCTP respectively. Labeled DNA was dissolved in 30 μL hybridization fluid and hybndized at 42 ℃ over night. MAIN OUTCOME MEASURES; The differential gene expression between haematopoietic stem cells and megakaryocytes involving cell signal transduction were detected by using DNA microarray analysis. According to the gene chip results, 17 differentially expressed genes were selected for further analysis by RT-PCR. RESULTS ： In this experiment, 3 522 differentially expressed genes were screened, including 1705 up-regulated genes and 1 817 down-regulated genes. In the 3 522 differentially expressed genes, there were 343 genes involved in cell signaling, 150 genes involved in transcription regulation, 21 genes involved in cell differentiation. The expression of the CD61 gene increased 369.83 fold, the expression of the CD41 gene increased 27.38 fold, and the expression of PF4 gene increased 24.06 folds; The expression of mitogen-activated protein kinases （MAPKs）, G protein-coupled receptors （GPCRs） and members of RAS oncogene family increased mostly. The expression of the genes involved in STAT pathways, both SOCS1 and JAK2 were up-regulated, but STAT5A was down-regulated. As determined by the gene chip results, 17 differentially expressed genes were selected for further analysis by RT-PCR. GAPDH was used as the internal control, and RT-PCR results were in agreement and confirmed the finding from the gene chip expression results. CONCLUSION： Thrombopoeitin （TPO） and other hematopoietic growth factors may enhance the proliferation and differentiation of megakaryocytes derived from cord blood stem cells by GPCRs-Ras-MAPK pathway.