PDGF-BB在巨核系造血及调控骨髓间充质细胞TPO生成的研究

发布时间：2018-04-08 11:35

本文选题：血小板衍生因子　切入点：巨核细胞　出处：《南方医科大学》2017年硕士论文

【摘要】：研究背景与研究目的血小板衍生生长因子(PDGF)是一种多肽生长因子,其储存于人血小板α颗粒中。PDGF家族有5个不同的二聚体,PDGF-AA,AB,BB,CC和DD,其通过结合PDGFα受体和β受体发挥其生物学功能。PDGF受体(PDGFR)是一种跨膜糖蛋白,由α和β两种酪氨酸蛋白激酶亚基构成,具有酪氨酸蛋白激酶活性。研究发现,PDGF-BB与两种受体高亲和力结合后,促增殖作用比其他亚型更强。PDGF-BB可以促进多种结缔组织细胞如成纤维细胞,内皮细胞和平滑肌细胞等的生长和分化。除此以外,PDGF-BB对造血也具有重要的作用,如促进红系爆式集落形成单位(BFU-E)和粒系-红系-单核-巨核细胞集落形成单位(CFU-GEMM)的形成。然而,PDGF-BB在造血中的具体机制尚不明确,可能与骨髓微环境中的骨髓间充质细胞TPO的生成有关。因此,本研究旨在探索PDGF-BB对CFU-MK形成的作用,并将其与IL-3、IL-6、TPO和GM-CSF的作用进行对比,以及加入抗IL-3、IL-6或TPO单克隆抗体后对CFU-MK生成的影响。进一步探讨PDGF-BB促进巨核系造血的作用机制:PDGF作用于骨髓间充质细胞表面的PDGF受体,激活下游相关的转录因子调控TPO产生,从而间接调节骨髓巨核系造血。研究内容与研究方法第一部分PDGF-BB促进巨核细胞生成一、PDGF-BB对小鼠CFU-MK集落形成的影响1.检测不同浓度的 PDGF-BB(0ng/ml、5ng/ml、10ng/ml、20ng/ml、50ng/ml、100ng/ml)对小鼠CFU-MK形成的影响;2.检测不同细胞因子(PDGF-BB、IL-3、IL-6、GM-CSF及TPO)及其不同组合对小鼠CFU-MK的作用;3.加入抗IL-3、IL-6、或TPO抗体后研究PDGF-BB对小鼠CFU-MK形成的影响。二、PDGF-BB对人CFU-MK集落形成的作用检测不同浓度 PDGF-BB(0 ng/ml、5 ng/ml、20 ng/ml、50 ng/ml、100 ng/ml)对人类 CFU-MK的作用。第二部分PDGF作用于骨髓间充质细胞调控TPO产生1.CCK-8法检测PDGF对人MSCs的增殖作用;2.Annexin-V/PI染色法检测PDGF对人MSCs凋亡的影响;3.流式细胞术检测人MSCs细胞表面PDGFR-β的表达;4.Western blot检测PDGF-BB对骨髓间充质细胞MAPK/ERK、STAT信号通路的影响;5.Q-PCR 检测 PDGF-BB 对人 MSCs TPO mRNA 表达水平。研究结果第一部分PDGF-BB促进巨核细胞生长一、PDGF-BB对小鼠CFU-MK集落形成的影响1.PDGF-BB以剂量依赖的方式促进小鼠CFU-MK的形成,其中50 ng/ml作用最为明显(n=5,P0.001);2.PDGF-BB 对 CFU-MK 的促增殖作用比 GM-CSF 强(n=5,P =0.0002),但低于 IL-3、IL-6 和 TPO(n=5,P0.05);3.PDGF-BB+IL-3 或 PDGF-BB+IL-6 与单独使用 IL-3 或IL-6 无明显差异(n=5,P0.05),但是IL-3+IL-6对CFU-MK的增殖具有明显的协同作用,是单独作用的2倍(n=5,P0.00001);4.PDGF-BB+IL-3 MoAb组与单用PDGF-BB组相比,CFU-MK计数无显著差异(n=4,P=0.127);而 PDGF-BB+IL-6MoAb 组的 CFU-MK 数量减少了53.8%(n=4,P=0.003);PDGF-BB+TPOMoAb 组减少了 51.2%(n=4,P=0.002)。二、PDGF-BB对人CFU-MK形成的作用PDGF-BB以剂量依赖的方式促进人CFU-MK的增殖,其中50 ng/ml作用最为明显(n=4,P0.001)。第二部分PDGF作用于骨毮间充质细胞调控TPO产生间接促进骨髓造血生成1.PDGF-BB促进人MSCs的增殖,最适浓度为50 ng/ml(n=8,P=0.0027);2.PDGF-BB能够显著减少MSCs的凋亡(n=5,P=0.00004);3.Q-PCR结果显示,MSCs高表达PDGFR-β mRNA;流式结果显示,MSCs表达PDGFR-β;4.Western blot结果显示,PDGF-BB处理组的骨髓间充质细胞P-ERK、P-STAT3蛋白表达增加;5.PDGF-BB 处理 MSCs 后,TPO mRNA 表达水平显著增高(n=7,P=0.021)。研究结论1.PDGF-BB可以显著促进小鼠和人CFU-MK的形成;2.PDGF-BB对MSCs有促增殖和抗凋亡作用,MSCs高表达PDGFR-β,其与PDGF-BB结合后激活下游MAPK/ERK、STAT信号通路;3.PDGF-BB作用于骨髓微环境中的骨髓间充质细胞,在转录水平促进TPO产生,间接促进巨核系造血。
[Abstract]:The research background and research purpose of platelet derived growth factor (PDGF) is a polypeptide growth factor, which is stored in the.PDGF granule family has 5 different two mer, PDGF-AA, AB, BB, CC and DD, the PDGF by binding receptor alpha and beta receptor (.PDGF receptor exerts its biological functions PDGFR) is a transmembrane glycoprotein composed of alpha and beta two subunit protein tyrosine kinase, a protein tyrosine kinase activity. The study found that high affinity binding of PDGF-BB and two kinds of receptors, proliferation is stronger than the other subtypes of.PDGF-BB can promote a variety of connective tissue cells such as fibroblasts, endothelial cells and smooth muscle cell growth and differentiation. In addition, PDGF-BB also plays an important role in hematopoiesis, such as promoting the erythroid burst forming unit (BFU-E) and myeloid erythroid - monocyte megakaryocyte colony forming unit (CFU-GEMM) of the form . however, the specific mechanism of PDGF-BB in hematopoiesis is not clear, and may be generated in the microenvironment of bone marrow mesenchymal stem cells TPO. Therefore, this study aims to explore the role of PDGF-BB on the formation of CFU-MK, and IL-3, IL-6, TPO and GM-CSF were compared and the effect, adding anti IL-3, CFU-MK, IL-6 or TPO effects on the formation of monoclonal antibody. After further explore the mechanism of PDGF-BB in promoting megakaryocytopoiesis: the effect of PDGF on bone marrow mesenchymal cell surface PDGF receptor, activation of downstream related transcription factor TPO, thus indirectly regulate bone marrow megakaryocytopoiesis. The research contents and methods the first part of PDGF-BB promoting megakaryocytopoiesis, PDGF-BB on mouse CFU-MK colony forming PDGF-BB 1. detection of different concentrations (0ng/ml, 5ng/ml, 10ng/ml, 20ng/ml, 50ng/ml, 100ng/ml) influence on the formation of mouse CFU-MK; 2. Detection of different cytokines (PDGF-BB, IL-3, IL-6, GM-CSF and TPO) on mice of different combinations of CFU-MK and IL-6; 3. joined the anti IL-3 TPO antibody, or after PDGF-BB on mice CFU-MK formation. The effects of two PDGF-BB on CFU-MK colony formation test of different concentrations of PDGF-BB (0 ng/ml, 5 ng/ml, 20 ng/ml, 50 ng/ml, 100 ng/ml) of human CFU-MK. The second part of the PDGF effect on bone marrow mesenchymal cells to produce 1.CCK-8 TPO control method was used to detect PDGF on MSCs proliferation; effect of detection of PDGF 2.Annexin-V/PI staining on MSCs apoptosis; detect the expression of human MSCs cell surface PDGFR- beta 3. flow cytometry FCM; 4.Western blot detection of PDGF-BB of bone marrow mesenchymal stem cells MAPK/ERK, STAT signaling pathway; the level of 5.Q-PCR to detect the expression of PDGF-BB on MSCs TPO mRNA. The research results of the first part of PDGF-BB to promote megakaryocyte growth, P DGF-BB set 1.PDGF-BB in a dose dependent manner of mice CFU-MK promote the formation of CFU-MK of mice, of which 50 ng/ml the most obvious function (n=5, P0.001); the proliferation effect of 2.PDGF-BB on CFU-MK GM-CSF (n=5, P, =0.0002) but lower than IL-3, IL-6 and TPO (n=5, P0.05); 3.PDGF-BB+IL-3 or PDGF-BB+IL-6 and IL-3 alone or IL-6 showed no significant difference (n=5, P0.05), but IL-3+IL-6 on the proliferation of CFU-MK have obvious synergistic effect, is 2 times the individual effect (n=5, P0.00001); 4.PDGF-BB+IL-3 MoAb group compared with PDGF-BB group, there was no significant difference between CFU-MK count (n=4, P=0.127); and the number of CFU-MK in PDGF-BB+IL-6MoAb group was reduced by 53.8% (n=4, P=0.003); group PDGF-BB+TPOMoAb decreased 51.2% (n=4, P=0.002). Two effects of PDGF-BB PDGF-BB on CFU-MK formation in a dose-dependent manner promote the proliferation of CFU-MK, of which 5 0 ng/ml the most obvious function (n=4, P0.001). In the second part, the effect of PDGF on the regulation of mesenchymal cells produce TPO bone between Sha indirectly promote bone marrow hematopoietic 1.PDGF-BB promote the proliferation of MSCs, the optimal concentration is 50 ng/ml (n=8, P=0.0027); 2.PDGF-BB can significantly reduce the apoptosis of MSCs (n=5, P=0.00004); 3.Q-PCR the results showed that MSCs high expression of PDGFR- beta mRNA; flow cytometry showed that MSCs expression of PDGFR- beta 4.Western; blot results showed that PDGF-BB treatment group and bone marrow mesenchymal stem cells P-ERK, P-STAT3 protein expression was increased; 5.PDGF-BB after MSCs treatment, the expression level of TPO mRNA increased significantly (n=7, P=0.021). Conclusion 1.PDGF-BB can significantly promote the the formation of mouse and human CFU-MK; 2.PDGF-BB proliferation and anti apoptosis effect of MSCs, MSCs high expression of PDGFR- beta and its combination with PDGF-BB after activation of downstream MAPK/ERK, STAT signal pathway; the effect of 3.PDGF-BB on bone marrow microenvironment in Bone marrow mesenchymal cells, at the transcriptional level, promote TPO production and indirectly promote megakaryocyte hematopoiesis.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R331

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