【作者】 刘毅；

【导师】 李志超；

【作者基本信息】 第四军医大学 ， 病理生理学， 2010， 博士

【摘要】 研究目的:旨在证明骨髓来源的血管平滑肌祖细胞参与了低氧性肺血管重塑,这一过程受到SDF-1/CXCR4生物轴调控:低氧刺激肺血管内皮细胞产生SDF-1,与细胞表面的CXCR4结合介导了平滑肌祖细胞的向肺血管定向迁移。实验方法:从大鼠骨髓细胞中分离和培养平滑肌祖细胞(SPCs),以示踪剂标记后移植给SD大鼠,检测低氧性肺动脉高压时SPCs在肺血管壁中的分布,并在整体和细胞水平阻断SDF-1/CXCR4生物轴,观察SPCs分布和肺血管重塑的变化。1、骨髓来源SPCs的分离和培养通过构建以平滑肌细胞特异性启动的报告基因载体,转染培养的大鼠骨髓间质细胞,经过流式活细胞分选获得SPCs;体外扩大培养SPCs,通过流式细胞术和免疫荧光技术分析细胞是否表达前体细胞表面受体CXCR4和平滑肌细胞特异性蛋白α-SM-actin,并与内皮祖细胞(EPCs)和成熟的平滑肌细胞(SMCs)进行对比;以CXCR4和钙调蛋白(CaM)分别作为前体细胞和成熟细胞的标志,观察血小板衍生生长因子-BB(PDGF-BB)对体外培养的平滑肌祖细胞分化成熟的诱导作用。2、SPCs参与低氧性肺血管重塑形成的研究大鼠经尾静脉移植标记有CM-Dil荧光活细胞示踪剂的SPCs(1×106/只),复制低氧性肺动脉高压动物模型,观察SPCs在肺血管壁的分布;在细胞水平建立SPCs与肺血管内皮细胞(PVECs)粘附实验模型,以Trans-well装置为基础建立SPCs穿过内皮细胞层的迁移实验模型,观察低氧或氯化钴(CoCl2)对SPCs粘附和迁移能力的影响。3、SDF-1/CXCR4生物轴调控SPCs定向迁移的研究通过免疫组化分析和ELISA实验,分别在整体和细胞水平研究低氧刺激对肺血管内皮细胞表达SDF-1的影响;在SPCs粘附和迁移实验基础上,给予SDF-1中和抗体,或者以CXCR4中和抗体封闭配体结合位点,观察SPCs粘附和迁移能力的变化;在复制低氧性肺动脉高压动物模型的同时,给予大鼠注射SDF-1或者移植经CXCR4中和抗体处理的SPCs(5×107/只),观察对肺动脉压力、血管重塑指标、血管壁SPCs分布数量的影响。实验结果:1、体外培养的SPC单个细胞生长呈梭形,与SMC相似,融合成片后呈现出集落生长的前体细胞特征,连续传7代以内细胞生长形态无明显改变;2、SPCs表达前体细胞共有的表面受体CXCR4,同时表达平滑肌细胞特异性蛋白α-SM-actin,而不表达内皮细胞标志分子CD31和成熟平滑肌细胞标志CaM,借此可以鉴别SPC与EPC、SMC;3、PDGF-BB能诱导体外培养的SPCs的CXCR4蛋白含量逐渐减少,而表达CaM逐渐增多;4、大鼠移植标记红色荧光标记的SPCs,慢性低氧条件下动物肺血管荧光强度明显高于常氧组,且主要呈现在肺动脉中膜层;5、相对于常氧条件,低氧条件或有CoCl2存在时SPCs与PVEC的粘附率明显增高,SPCs穿过内皮细胞层的迁移数量也明显增多;6、低氧性肺动脉高压大鼠肺血管内皮层SDF-1的表达量较正常肺血管增多,低氧或CoCl2都能使体外培养的PVECs表达和分泌SDF-1增加;7、细胞模型上阻断SDF-1/CXCR4能降低SPCs与PVECs的粘附率,也能减少SPCs透过内皮细胞层的数量;8、与低氧组比较,阻断SDF-1/CXCR4生物轴的大鼠肺动脉压力明显减轻,肺血管壁相对厚度降低,到达肺血管壁的SPCs数量也显著减少。研究结论:低氧环境下肺循环氧分压降低,刺激肺血管内皮细胞产生SDF-1,趋化骨髓来源的血管平滑肌祖细胞经过血液循环向肺脏迁移,并通过与平滑肌祖细胞表面CXCR4受体结合,介导该细胞与肺血管内皮的粘附,最终穿过内皮细胞层进入肺动脉中膜,分化为成熟的平滑肌细胞,参与低氧性肺血管重塑的形成。此项研究揭示了低氧性肺血管重塑的新机制,为低氧性肺动脉高压的防治提供了新的药物作用靶点。更多还原

【Abstract】 AIM:To prove bone marrow-derived smooth muscle progenitor cells (SPCs) contribute to hypoxia-induced pulmonary vascular remodeling (PVR), which mediated by SDF-1/CXCR4 axis.METHODS:1. Isolation of bone marrow-derived SPCs.We constructed the smooth muscle specially promoted plasmid. Then transfered it to the bone marrow stromal cells and sorted the GFP positive ones (these were deemed to SPCs) by flow cytometry. SPCs were cultured 3 to 5 passages in vitro. Cell phenotype and growth characteristic were observed. Cell makers were identified by flow cytometry and immunofluorescence. SPCs were cultured with platelet derived growth factor-BB (PDGF-BB), and valuated whether those differentiate to smooth muscle-liked cells.2. SPCs involve in hypoxia-induced PVR.CM-Dil labeled SPCs (1×106 per rat) were injected into caudal vein of SD rats. Animals were placed in hypoxia cabin in 28 days, 8 hours per day, with setting barometric pressure to 380mmHg. Then SPCs in pulmonary arterial wall were measured. We established the models of SPCs adherent and penetrate to endothelial cells (ECs). Base on the models, the adherence and migration cof SPC were valued in the condition of hypoxia and presence of CoCl2.3. SDF-1/CXCR4 axis mediates SPCs directional migration.Through immunohistochemistry and ELISA assays, the SDF-1 expression was measured in pulmonary arterial wall or ECs under the condition of hypoxia. On cell models, the SPCs adherence and migration ability were valued with SDF-1 or CXCR4 neutral antibody. In vivo, SDF-1ab or SPCs (5×107 per rat) blocked CXCR4 binding site were injected to rats. Then the pulmonary artery pressure, indexes of vascular remodeling, and SPCs numbers in pulmonary arterial wall were measured.RESULT:1. In vitro, the cultured SPC was spindle-shape of single growth, and colony-liked in cells fusion.2. Both the precursor cell marker CXCR4 and SMC makerα-SM-actin were positive in SPC, but negative for the EC maker CD31 and the mature SMC maker CaM. 3. SPCs eliminated CXCR4, but expressed CaM gradually when cultured with PDGF-BB.4. Under the condition of hypoxia, more SPCs migrated into pulmonary arterial wall than normoxia, and most of those showed in media of the vascular.5. Contrast to normoxia, the adherence rate and migration quantity of SPCs were markedly increased under hypoxia or with CoCl2.6. SDF-1 expression rise in the pulmonary vascular endothelial and cultured PVECs supernatant the condition of hypoxia or cells cultured with CoCl2.7. On the cell models, inhibition of SDF-1 or blockade of CXCR4 could significantly decrease the adherence rate and migration quantity of SPCs.8. Blockade of SDF-1/CXCR4 axis could down regulate the pulmonary artery pressure, attenuate the PAs remodeling and reduce the number of SPCs in PAs wall.CONCLUSION:The PaO2 of pulmonary circulation is low under the hypoxic condition, which leads to the expression of SDF-1 in pulmonary endothelial, and homing the bone marrow–derived SPCs from blood. Once SDF-1 binding to CXCR4 on the SPC surface, cell adherents to pulmonary arterial ECs, migrate into vessel wall, and differentiate to matured SMCs, finally contribute to the vascular remodeling. These findings maybe proposal a new mechanism of hypoxia-induced PVR, and provide a new pharmacal target of HPAH.更多还原