【作者】 邸春霞；

【导师】 郭文怡； 王海昌；

【作者基本信息】 第四军医大学 ， 内科学， 2008， 硕士

【摘要】 研究背景和目的近年来,包括内皮祖细胞(EPCs)在内的成体干细胞的研究为冠心病和糖尿病等心血管疾病的治疗开启了一个新的方向。EPCs来源于骨髓,表达AC133、CD34、VEGFR-2(flk-1)等表面标志,可以通过分泌生长因子或通过归巢到内皮损伤及缺血部位,直接分化、发育为内皮细胞并形成新生血管,因而在内皮损伤后修复及心肌梗死后血运重建中起着重要的作用[1-2]。研究表明,循环EPCs数量可作为预测血管功能和心血管疾病危险因素的指标,其数量减少提示血管内皮修复能力降低,心血管疾病发生率增高。冠心病和糖尿病(1型和2型)患者循环EPCs数量减少,其增殖、迁移、归巢和成血管能力降低[3-5]。故用药物增加EPCs数量并改善其功能,进而促进心血管疾病患者的血管内皮修复和血管新生,成为治疗心血管疾病的新策略。胰岛素可以激活内皮型一氧化氮合酶(eNOS),刺激一氧化氮(NO)产生,导致血管舒张。而eNOS对促进干细胞从骨髓动员是必不可少的因素,并且是调节干(祖)细胞活性和血管形成的重要因子[6]。吡格列酮是一种噻唑烷二酮类胰岛素增敏剂,用于治疗2型糖尿病有较好的降糖效果和耐受性。最新研究表明,吡格列酮可以促进EPCs介导的新生血管形成[7],但其具体作用机制尚不明确。本研究拟探讨体外培养条件下,体内不同浓度吡格列酮预处理、体外给予胰岛素对正常大鼠骨髓源性EPCs的数量、增殖、凋亡以及分泌NO功能的影响,以期从新的角度认识吡格列酮和胰岛素对心血管系统的保护作用。方法第一部分:EPCs的分离培养及表型鉴定:1.取大鼠四肢骨骨髓,以密度梯度离心法分离出单核细胞,于添加了VEGF和bFGF的M199培养液中培养并定期观察;2.DiI-ac-LDL和FITC-UEA-I免疫荧光双染法鉴定细胞。第二部分:不同浓度吡格列酮对大鼠骨髓内皮祖细胞增殖、凋亡及功能的影响:1.雄性SD大鼠随机分为四组:对照组和吡格列酮组(10、20、40 mg·kg-1·d-1)并给予相应灌胃处理10天;2.密度梯度离心法分离骨髓单核细胞并消化得EPCs;3.各组EPCs计数并继续培养;4. MTT法检测EPCs增殖能力;5.流式细胞双染法检测EPCs凋亡;6.硝酸还原酶法测NO分泌量。第三部分:吡格列酮和胰岛素对骨髓内皮祖细胞增殖、凋亡及NO分泌的影响:1.雄性SD大鼠随机分为吡格列酮组和非吡格列酮组并给予相应灌胃预处理10天;2.密度梯度离心法分离单核细胞并培养5天后消化得EPCs;3.将所得EPCs相应分为对照组、胰岛素组、吡格列酮组、吡格列酮+胰岛素(即联合用药组)并给予胰岛素干预;4. MTT法检测EPCs增殖能力;5.流式细胞双染法检测EPCs凋亡;6.硝酸还原酶法测NO分泌量。结果1.培养7天的细胞经DiI-ac-LDL和FITC-UEA-I双染后,激光共聚焦显微镜下示双染阳性的细胞为正在分化的内皮祖细胞。2.不同浓度吡格列酮均能提高EPCs数量和功能。3.中等浓度吡格列酮(20 mg·kg-1·d-1)处理组对EPCs的促增殖、促NO分泌作用较10,40 mg·kg-1·d-1组作用强,但其抑制EPCs凋亡作用与后二者差别无明显统计学意义。4.吡格列酮和胰岛素单独作用及联合作用均可促进EPCs增殖、抑制其凋亡,并促进其分泌NO。5.胰岛素促进EPCs增殖作用较吡格列酮强,但其抑制EPCs凋亡及促进EPCs分泌NO作用与吡格列酮无明显差别。6.吡格列酮和胰岛素二者联合作用促进EPCs增殖、抑制其凋亡,并促进其分泌NO的作用较两种药物单独作用强。结论1.不同浓度吡格列酮均能提高EPCs数量,促进EPCs增殖、抑制其凋亡,并促进其分泌NO。2.中等浓度吡格列酮(20 mg·kg-1·d-1)和胰岛素均可促进EPCs增殖、抑制其凋亡,并促进其分泌NO,且二者具有协同作用。更多还原

【Abstract】 Background and aimsRecently, more and more researches of stem cells including endothelial progenitor cells (EPCs) are turning to the treatment of coronary heart disease (CHD) and diabetes (DM). EPCs are bone marrow-derived progenitor cells that express surface markers such as AC133,CD34 and VEGFR-2(flk-1), and can differentiate into mature endothelial cells on the vascular wall by the way of secreting VEGF or homing to injured areas. So, EPCs play an important role in repair of endothelial injuries and revascularization of infarcted areas. Many researches indicate that the number of circulatory EPCs is the index of predicting cardiovascular functions and risk factor of cardiovascular diseases.Smaller number means lower capacity of repairing endothelia and higher morbidity of cardiovascular diseases. The number of EPCs in patients suffering from CHD and DM decreases, so does the capacity of proliferation, migration, homing and angiogenesis of EPCs. As a result, increasing the number and function of EPCs with drugs in order to promote the process of repairing endothelia and revascularization becomes a new method of treating cardiovascular diseases. One of these drugs mentioned above is insulin. Insulin can activate endothelial nitric oxide synthase (eNOS), which secretes nitric oxide (NO) and dilates blood vessels. Recent researches have showed that eNOS is an indispensable factor in mobilization and modulation of stem cells. Another drug is pioglitazone, which is a kind of thiazolidinediones (TZDs) and shows good effects on decreasing blood glucose and excellent tolerance. A recent study illustrates that pioglitazone could promote angiogenesis of EPCs, but the mechanism is still unknown.This study intends to explore the effects of pioglitazone given in vivo with different dosages and insulin given in vitro on the number, proliferation, apoptosis and secretion of NO of EPCs derived from bone marrow, and the results may help to understand the cardiovascular effects of pioglitazone and insulin from a new aspect.MethodsPartⅠ: Culture and identification of surface markers of EPCs. 1. Harvest bone marrow of rats, and collect mononuclear cells through density gradient centrifugation. Then culture them in Medium 199 supplemented with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). 2. Immunofluorescent co-stain with DiI-ac-LDL and FITC-UEA-I to identify EPCs.PartⅡ: Effects of pioglitazone with different dosages given in vivo on EPCs. 1. Sprague-Dawlay (SD) rats are divided into four groups randomly. The control group was given saline by intragastric administration, and the three other groups are given pioglitazone by 10, 20, 40 mg·kg-1·d-1 respectively for 10 days. 2. Mononuclear cells were collected from rats’bone marrow by density gradient centrifugation. 3. Once being harvested, EPCs of each group were calculated and cultured with Medium 199. 4. Test the proliferation ability of EPCs by MTT assay. 5. Detect the apoptosis level of EPCs by immunofluorescent co-staining. 6. Measure the secretion of NO by modified Griess reaction method after 24 h of incubation.PartⅢ: Effects of pioglitazone and insulin on EPCs. 1. SD rats are divided into two groups randomly. The non-pioglitazone group is given saline by intragastric administration, and the pioglitazone group is given pioglitazone (20 mg·kg-1·d-1 ) for 10 days. 2. Mononuclear cells were collected from rats’bone marrow by density gradient centrifugation. 3. Once being harvested, EPCs of each group were divided into two subgroups, with one subgroup being given solvent and the other insulin (1nmol/L). All EPCs of these four groups were calculated and cultured with Medium 199. 4. Test the proliferation ability of EPCs by MTT assay. 5. Detect the apoptosis level of EPCs by immunofluorescent co-staining. 6. Measure the secretion of NO by modified Griess reaction method after 24 h of incubation.Statistical analysis is performed with SPSS version 13.0. One-way analysis of variation and post hoc t (LSD-t) test are employed.Results1. After being cultured for 7 days, cells obtained showed double positive for DiI-ac-LDL and FITC-UEA-I, which indicated that they were EPCs.2. Pioglitazone of different dosages increased the number and function of EPCs.3. Pioglitazone of 20 mg·kg-1·d-1 showed better effects of promoting proliferation and secreting NO of EPCs than the groups of 10 mg·kg-1·d-1 and 40 mg·kg-1·d-1 but same effects of inhibiting apoptosis as these two groups.4. Both pioglitazone and insulin had effects of promoting proliferation and secreting NO and inhibiting apoptosis of EPCs. 5. Insulin showed better effect than pioglitazone in promoting proliferation but same effects of inhibiting apoptosis and promoting secretion of NO of EPCs as pioglitazone.6. Co-application of pioglitazone and insulin showed better effects of promoting proliferation and secreting NO and inhibiting apoptosis of EPCs than single use of each drug.Conclusion1. Pioglitazone of different dosages can increase the number, promote proliferation and secreting NO and inhibit apoptosis of EPCs.2. Both pioglitazone of 20 mg·kg-1·d-1 and insulin have effects of promoting proliferation and secretion of NO and inhibiting apoptosis of EPCs, and they have synergistic effects in these aspects.更多还原