【作者】 邢金燕；

【导师】 陈玉国；

【作者基本信息】 山东大学 ， 急诊医学， 2011， 博士

【摘要】 研究背景：脓毒症是是目前患病率和死亡率最高的疾病之一,欧美国家脓毒症的患病率在10%以上,我国缺乏相应资料,但根据抗生素使用情况判断,其患病率远在欧美国家之上,脓毒症成为严重威胁着人类健康的最主要疾病之一；为降低脓毒症的患病率和病死率,早在2002年就由多个国家学会共同提出了"surviving sepsis campaign",并根据大量循证医学证据和广泛论证,提出了"sepsis resuscitation bundles"和‘’Sepsis management bundle",从2008年起在世界各地进行推广；经过大量的努力,同时伴随诊疗技术的不断提高,2010年数据分析显示脓毒症的病死率仍在30.8%。如何降低其发病率和病死率是当今研究的热点和难点。研究证明,毛细血管内皮细胞功能异常及结构受损,导致内皮细胞脱落变形、凝血功能异常、毛细血管渗漏等在脓毒症的病情进展中其重要作用,成年内皮细胞的自我增殖和修复能力有限,内皮祖细胞是出生后血管内皮损伤修复的重要细胞,研究表明循环内皮祖细胞数量和功能直接影响到脓毒症患者的预后。细菌脂多糖是革兰氏阴性杆菌的主要致病原,实验中经常用LPS体内注射模拟机体感染过程；3-羟-3-甲基戊二酸辅酶A(3-hydroxy-3-methylglutaryl coenzyme A,HMG-CoA)还原酶抑制剂(他汀类药物)通过抑制HMG-CoA还原酶的活性,而抑制胆固醇的生成、降低血胆固醇的水平,最近研究表明,在有效地调节血脂水平外,还具有抑制平滑肌增生和血小板聚集以及抑制炎症反应等作用,有研究证明他汀类药物还能促进血管新生和内皮损伤后的修复过程,而且已经证实EPCs在该过程中扮演了重要角色,研究证实他汀类药物有动员内皮祖细胞、提高内皮功能等作用。自2000年Ando首先报道他汀类药物可以改善大鼠脓毒症的生存率后,多个大规模临床观察显示,他汀类药物能降低脓毒症的患病率和病死率,但对于LPS对EPCs数量及功能的影响及他汀类药物对LPS作用下的内皮祖细胞作用未见相关报道。本研究共分三部分进行：1、脐静脉血EPCs分离、培养、鉴定；2、LPS与EPC共孵育后加入辛伐他汀EPC迁移、黏附、增殖情况。3、EPC与辛伐他汀共孵育后加入LPS其迁移、黏附、增殖、凋亡情况。研究目的：1、从脐静脉血获得人EPCs,鉴定并进行体外传代培养。2、将脂多糖(LPS)与人脐血EPCs共孵育,在相应时间点添加不同剂量辛伐他汀,观察LPS对人脐血EPCs增殖、迁移、黏附、凋亡的影响及辛伐他汀干预作用；3、将不同浓度辛伐他汀与人脐血EPCs共孵育,相应时间点加入LPS,观察辛伐他汀对EPCs增殖、迁移、黏附功能的影响,以及预先应用辛伐他汀后LPS对人脐血EPCs增殖、迁移、黏附的影响；通过该研究明确在细胞水平单纯LPS及辛伐他汀对内皮细胞祖的影响,及两者的相互影响,为进一步进行作用机理探讨提供基础。方法：第一部分：采用密度梯度离心法从脐静脉血获取单个核细胞,将其接种在人纤维连接蛋白包被培养板上,诱导培养7天,收集贴壁细胞,通过细胞形态观察及DiI-acLDL、FITC-UEA-I染色激光共聚焦显微镜培养细胞。第二部分：将等量脐静脉血来源EPCs(UCBEPCs)接种到96孔培养板上,随机分为对照组：UCBEPCs与EGM-2MV标准液培养48小时；LPS组,UCBEPCs与GM-2MV标准液和LPS液(100ng/mL)共同培养48小时;LPS+ simv0.1组：UCBEPCs与GM-2MV标准液和LPS液(100ng/mL)共同培养24小时后,加入0.1μmol/L辛伐他汀培养至48小时；LPS+ simv1.0组：UCBEPCs与GM-2MV标准液和LPS液(100ng/mL)共同培养24小时后,加入1.0μmol/L辛伐他汀培养至48小时；LPS+ simv10.0组：UCBEPCs与GM-2MV标准液和LPS液(100ng/mL)共同培养24小时后,加入10.0μmol/L辛伐他汀培养至48小时。应用MTT比色法测定EPCs增殖功能和粘附功能,通过Transwell小室观察EPCs迁移功能。统计学处理：结果中数据以均数±标准差(x±s)表示,资料采用SPSS(16.0版)统计软件进行统计学分析,组间比较用随机取组设计的方差分析,以P<0.05为差异有显著性。第三部分：将等量EPCs接种到96孔培养板上,随机分为对照组：UCBEPCs与EGM-2MV标准液培养48小时；LPS组：UCBEPCs与GM-2MV标准液和LPS液(100ng/mL)共同培养48小时；Sim0.1+LPS组：UCBEPCs与GM-2MV标准液和0.1μmol/L辛伐他汀共同孵育24小时后,加入LPS液(100ng/mL)共同培养至48小时；Siml.O+LPS组：UCBEPCs与GM-2MV标准液和1.0μmol/L辛伐他汀共同孵育24小时后,加入LPS液(100ng/mL)共同培养至48小时；Sim10.0+LPS组：UCBEPCs与GM-2MV标准液和10.0μmol/L辛伐他汀共同孵育24小时后,加入LPS液(100ng/mL)共同培养至48小时；辛伐他汀组：UCBEPCs与GM-2MV标准液和1.0μmol/L辛伐他汀共同孵育48小时。应用MTT比色法测定EPCs增殖功能和粘附功能,通过Transwell小室观察EPCs迁移功能,应用流式细胞仪检测EPCs凋亡情况,细胞凋亡率(AP)计算公式：AP=凋亡细胞数/总检测细胞数。统计学处理：结果中数据以均数±标准差(x±s)表示,资料采用SPSS(16.0版)统计软件进行统计学分析,组间比较用随机取组设计的方差分析,以P<0.05为差异有显著性结果第一部分：经鉴定分离培养细胞为EPCs,经复苏传代见细胞生长良好。第二部分：脐静脉血来源EPCs与LPS共孵育后其增殖、迁移显著降低(P=0.009、P=0.026),黏附能力也下降,但差异无显著性(P=0.279)。脐静脉EPCs与LPS共孵育后加入不同浓度辛伐他汀,EPCs的增殖、迁移和黏附能力不同程度改善,随辛伐他汀浓度增加,该作用增强,辛伐他汀浓度为1.0μmol/L时EPCs增殖显著改善(P=0.017),浓度10.0μmol/L时EPCs增殖、黏附、迁移功能,较LPS组均显著改善(P=0.00,P=0.002,P=O.00),其中黏附和迁移功能较空白对照组显著改善(P=O.039,P=0.000)。第三部分：人EPCs与LPS共孵育后其凋亡率显著增加(12.632±0.5271 vs3.0266±0.5271, P<0.00)。辛伐他汀(1.0umol/L)与EPCs共孵育,EPCs各检测指标与对照组比较均无显著变化。UCBEPCs与不同浓度辛伐他汀孵育后再加入LPS,EPCs的增殖、迁移和黏附能力较LPS组不同程度改善,凋亡率降低,随辛伐他汀浓度增加,该作用增强,辛伐他汀浓度为0.1umol/L EPCs增殖、黏附、迁移功能较LPS组有改善,但无统计学差异(P=0.687、0.501、0.683),EPCs凋亡率显著降低(9.2750±0.5961 vs 12.632±0.5271,P=0.00),辛伐他汀浓度为10.0μmol/L时,EPCs增殖、黏附、迁移功能,较LPS组均显著改善(P=0.035,P=0.002,P=0.011),其中粘附功能较对照组也有显著改善(P=0.039),不同浓度辛伐他汀与EPCs作用24小时后再与LPS共孵育,其EPCs凋亡率较LPS组均显著改善,但仍高于对照组和辛伐他汀组。结论自脐静脉血可成功分离内皮祖细胞,并可在体外进行传代培养。LPS与UCBEPCs共孵育,可使UCBEPCs增殖和迁移功能显著降低,黏附能力下降,凋亡率显著增加。单纯应用辛伐他汀(1.0umol/L)与UCBEPCs共孵育,对UCBEPCs的增殖、粘附、迁移功能及细胞凋亡无显著影响。辛伐他汀可以改善LPS导致的EPCs增殖、黏附、迁移功能的降低,在研究范围内改善作用随浓度增加而增强。提前应用辛伐他汀与EPCs共孵育,可以显著降低LPS导致的EPCs增殖、黏附、迁移功能的降低,在研究范围内该作用随浓度增加而增强。更多还原

【Abstract】 Background:Sepsis continues to be a major cause of morbidity and mortality worldwide. Preclinical and clinical sepsis studies have shown that the acute systemic inflammatory and procoagulant response results in structural and functional alterations in the endothelium. The endothelial cells (ECs) play a pivotal role in the progression of sepsis; the ECs are not only the participant of inflammatory reaction, but also the first damaged target cells. The dysfunction of ECs is a critical event in the initiation of organ dysfunction caused by sepsis.Recent studies suggested that the injured ECs could be regenerated by circulating bone marrow-derived progenitor cells called endothelial progenitor cells (EPCs), which had capabilities to mobilize from bone marrow with homing to foci of injuries and to differentiate into mature ECs to ameliorate the dysfunction of ischemic organs caused by sepsis. The impairment of EPCs caused by severe inflammation may contribute to the progression of organ dysfunction. Circulating endothelial progenitor cells may be an important mechanism of vascular repair, and thus shows significant promise for prognostic and therapeutic strategies in critical illness, namely sepsis and sepsis-related organ dysfunction. Many studies have convincingly demonstrated that simvastatin benefit for sepsis,and could promote the function of EPCs.but there no evidence about the effects of simvastatin on the EPCs incubated with LPS.Objective:1、To separate EPCs from human umbilical cord blood (HUCB).2、To investigate the variation of the proliferation、adhention、migration and apoptosis of EPCs when incubated with LPS.3、The effects of simvastatin at different concentration on the functions of EPCs when added before or after the EPCs incubated with LPS in vitro.Methods:Total mononuclear cells(MNCs) were isolated from human umbilical cord blood by Ficoll density gradient centrifugation,and then the cells were plated on fibronectin-coated culture dishes. After 7 days culture, attached cells were isolated and assessed with a laser scanning confocal microscope. EPCs divided to different group:control group:EPCs were cultivated with standard EGM-2MW fluid for 48hr; LPS group:EPCs were cultivated with standard EGM-2MW fluid plus LPS(100 ng/Ml) for 48hr;simvastatin grup:EPCs were cultivated with standard EGM-2MW fluid add simvastatin 1.0μmol/L for 48hr;test group:first EPCs were incubated with standard EGM-2MW fluid and LPS(100 ng/ml)/simvastatin of different concentrations (simv0.1:add simvastatin 0.1μmol/L, simv1.0:add simvastatin 1.0μmol/L, simv10.0:add simvastatin 10.0μmol/L) for 24hrs then addde simvastatin of different concentrations (simv0.1:add simvastatin 0.1μmol/L, simv1.0:add simvastatin 1.0μmol/L, simv10.0:add simvastatin 10.0μmol/L)/LPS(100 ng/ml).The proliferation, adhesion and migration of EPCs were detected with MTT assay, Transwell chamber assay after 48hrs respectively.And the influences of LPS and simvastatin on EPCs’proliferation, adhesion、migration and apoptosis were evaluated.Results:Incubated with LPS the proliferation of EPCs were decline significantly (P=0.009),the adhesion and migration of EPCs were also descended,but there was no significance (P=0.279、P=0.056),the apoptosis rate of EPCs were increased significantly.When added simvastatin the proliferation, adhesion and migration of EPCs were all improved,there are some difference for difference concentration of simvastatin.There were hardly any improvment in the proliferation, adhesion and migration of EPCs when added simvastatin of 0.1μmol/L (P=0.687、P=0.501、P=0.683 respectively), EPCs’ proliferation was improve significantly when incubated with simvastatin of 1.0μmol/L compared with LPS group(P=0.017),while the adhesion and migration were also improved but there was no significance(P=0.064、P=0.069 respectively). When incubated with simvastatin at the concentration of 10.0μmol/L the the proliferation、adhesion and migration of EPCs were all improved significantly (P=0.00, P=0.002, P=0.00 respectively)compared with LPS group,and the adhesion and migration of EPCs were ameliorated significantly even compared with the EPCs cultivated with standard EGM-2MW fluid (P=0.039,P=0.000 respectivly).Incubated with simvastatin there were no significant changes on the EPCs function of proliferation, adhesion and migration. When EPCs were incubated with standard EGM-2MW fluid added simvastatin of different concentrations (simv0.1:add simvastatin 0.1μmol/L, simv1.0:add simvastatin 1.0μmol/L, simv10.0:add simvastatin 10.0μmol/L) for 24hrs then addde LPS(100 ng/ml).The proliferation, adhesion and migration of EPCs all improved compare with the LPS group,but lower than the control group;the apoptosis were all decreased compare with the LPS group,but higher than control group and simvastatin group still.Conclusion:LPS could cut down the proliferation of EPCs sharply while the adhesion and migration were decline in some degree when incubated with EPCs.Simvastatin could improve the declined proliferation, adhesion and migration of EPCs which incubated with LPS first.When incubated with simvastatin first there were some profect function on the effect of LPS on EPCs更多还原