自体内皮祖细胞移植对内毒素致兔急性肺损伤生物学作用的实验研究
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摘要
背景和目的：
急性肺损伤和急性呼吸窘迫综合症(ALI/ARDS)是各种致病因素导致的急性进行性呼吸衰竭,是临床常见急重症。其死亡率在20世纪70年代早期高达70%以上,尽管近年来对其认识及救治技术不断提高,但总体病死率仍然在40%以上。国内外学者对ARDS的治疗进行了大量研究并取得了一些进展,但相关治疗策略均只能在一定程度上缓解病情,并不能从根本上改善ARDS的不良预后。炎症反应介导的肺毛细血管内皮细胞与肺泡上皮细胞损伤是ARDS的特征性病理改变,因此对受损肺脏进行结构修复和功能重建才是降低ARDS死亡率的理想治疗方式。成体干细胞具有多向或单向分化潜能,取材方便,易于分离扩增,并且不涉及到医学伦理学的相关问题,因而在ALI/ARDS治疗中具有重要的应用价值。
内皮祖细胞(EPC)是血管内皮细胞的前体细胞,并且可参与出生后血管新生、再内皮化和内皮损伤后的修复。鉴于EPC作为内源性修复机制在维持内皮细胞层完整性方面发挥了重要的作用,并且内皮受损为ARDS早期的特征性病理改变之一,因此EPC移植有希望成为ALI/ARDS的有效治疗手段。本研究旨在了解ARDS动物模型不同发病阶段EPC的动态变化规律,观察EPC自体移植以及EPC培养液“无细胞移植”对ARDS的生物作用并探讨其可能的作用机制,为提出ALI/ARDS治疗新方法提供实验线索。
方法和结果：
1.兔外周循环EPC的分离培养和鉴定
从兔耳中央动脉抽取外周血(10mL/Kg),以密度梯度法分离出单个核细胞(PMC),并将其接种于培养皿内；使用添加了细胞因子和胎牛血清的内皮祖细胞专用培养液(EGM-2)进行诱导分化培养。首次换液时间为72小时,其后每48小时换液一次。结果显示,体外培养24小时后可见到单个核细胞粘附于培养皿底部。粘在第3天时,部分细胞呈现出纺锤形变化,并且细胞分裂增殖明显。此时更换培养液以除去未贴壁细胞和红细胞。从第5天起,一部分单核细胞转化为梭形的贴壁细胞,并且随着体外培养时间的延长,圆形细胞的数量逐渐减少,而梭形细胞的数量逐渐增多。在第7天左右可观察到成团细胞形成的细胞集落。在第14天左右,细胞形状变为长梭形,并且多角形细胞有融合趋势。吞噬功能和粘附功能实验显示,体外培养的EPC可同时摄取Dil标记的乙酰化低密度脂蛋白(Dil-acLDL)并粘附FITC标记的荆豆凝集素-1(FITC-UEA-1),荧光显微镜下双阳性细胞的比率接近100%。通过免疫荧光对表面标志物进行检测显示,95%以上的细胞同时表达血管内皮生长因子受体2(VEGFR2)和CD34。依据上述条件可认定通过密度梯度离心后体外培养扩增的细胞为早期EPC。
2.ALI动物模型建立
通过耳缘静脉将内毒素(ET055:B5; 500μg/Kg和700μg/Kg)注入新西兰大白兔体循环内,72小时后活杀各组动物并取肺组织进行大体病理观察。对两组动物的肺组织切片行HE染色显示,两组动物均表现为不同程度的肺组织充血、微血管内皮损伤、出血和以中性粒细胞浸润为主的大量白细胞渗出、聚集；同时两组动物还出现肺泡间隔增厚、破坏以及部分肺泡萎陷不张等病理改变。上述结果提示两组剂量内毒素均可成功建立ALI模型。与500μg/Kg剂量组相比,700μg/Kg剂量组动物的肺组织损伤程度相对较重,但早期死亡率过高,不利于进行后期分析。因此在后续实验中采用内毒素500μg/Kg建立ALI动物模型。
3.EPC在ALI早期的数量和功能变化
利用内毒素(500μg/Kg)建立兔ALI模型,同时以等量生理盐水注射设立对照组。分别在12小时、24小时、48小时、72小时和第5天抽取外周血。利用流式细胞仪测定外周血中VEGFR-2和CD34双阳性细胞(EPC)的比率。体外培养细胞后测定EPC的增殖、粘附和迁移功能。结果显示,对照组外周血EPC比率基本保持稳定；模型组外周血EPC比率在内毒素注射后即出现降低,24小时后降至最低点,其后有所上升,但第5天时仍低于对照组。EPC增值、粘附和迁移功能均在内毒素注射后出现降低,48小时后降至最低点,其后有所上升,第5天时仍低于对照组；模型组在各时间点与对照组相比均具有显著差异。
4.自体EPC移植对ALI治疗作用及机制探讨
将45只新西兰大白兔分为正常对照组(n=15)、肺损伤对照组(n=15)和EPC移植组(n=15)。模型建立前7天抽取外周血(10ml/Kg)进行EPC体外培养扩增,并于移植前利用CM-Dil标记EPC。内毒素耳缘静脉注入30分钟后,EPC移植组给予自体EPC(约105个)于耳缘静脉注入,肺损伤对照组给予等量生理盐水。正常对照组则均给予等量生理盐水耳缘静脉注入。分别于内毒素静脉注入后24小时、第3天和第5天处死动物,留取肺组织、支气管肺泡灌洗液(BALF)和血清进行相关检查。
结果显示,CM-Dil标记的EPC可定植于肺组织内。正常对照组各项指标在各时间点均无显著变化,并且与肺损伤对照组和EPC移植组相比均具有显著差异(P<0.01)。EPC移植组肺组织病理切片的病变程度轻于肺损伤对照组,但病理评分无统计学显著差异。与肺损伤对照组相比,EPC移植组的肺湿干比(W/D)和BALF蛋白含量降低,并且在3d和5d时具有显著差异(P<0.05)；BALF中性粒细胞计数降低,并且在24h和3d时具有显著差异(P<0.05)；肺组织髓过氧化物酶(MPO)活性降低,并且在3d时具有显著差异(P<0.05)；IL-1β水平降低,并且在3d和5d时具有显著差异(P<0.05)；肿瘤坏死因子α(TNF-a)水平降低,并且在24h和3d时具有显著差异(P<0.05)；IL-10水平升高,并且在3d时具有显著差异(P<0.05)；血清细胞间粘附分子1(ICAM-1)水平较低,并且在24h时具有显著差异(P<0.05)；P选择素mRNA水平降低,并且在3d时具有显著差异(P<0.05)。EPC移植组的肺组织细胞凋亡数量少于肺损伤对照组,并且在5d时最为明显。
5.自体EPC培养液对ALI的治疗作用
将15只新西兰大白兔分为正常对照组(n=5)、肺损伤对照组(n=5)和EPC培养液组(n=5)。模型建立前3天抽取外周血(10ml/Kg),行EPC体外培养获取培养液上清。内毒素耳缘静脉注入30分钟后,EPC移植组给予EPC培养液上清于耳缘静脉注入,肺损伤对照组给予等量生理盐水。正常对照组则均给予等量生理盐水耳缘静脉注入。48小时后处死动物,留取肺组织和BALF进行相关检查。结果显示,肺损伤对照组和EPC培养液组各项指标与正常对照组相比均具有显著差异(P<0.01)。EPC培养液组肺组织普通病理检查所示的病变程度稍轻于肺损伤对照组。与肺损伤对照组相比,EPC培养液组肺W/D、BALF总蛋白含量、BALF中性粒细胞数量和肺组织MPO活性均较低,但各指标均不具有统计学显著差异(P>0.05)。
结论：
1密度梯度离心联合贴壁选择法能够成功从外周血中分离中EPC,并可在体外进行培养扩增。
2在内毒素构建的兔ALI动物模型中,外周血EPC数量以及增殖、粘附和迁徙功能在ALI早期均出现显著降低；随着肺部炎症的消退,EPC数量和相关功能有所恢复。
3来源于外周血的自体EPC于移植后可归巢到受损肺脏,并且可减轻内毒素诱导的急性肺损伤病变严重程度。自体EPC移植可通过降低ICAM-1和P-选择素表达的方式减少ALI起始阶段的PMN肺内扣押,进而预防肺损伤。自体EPC移植可将内毒素诱导的促炎细胞因子反应部分转化为抗炎细胞因子反应,同时可还可通过减少肺组织细胞凋亡发挥治疗作用。EPC培养液移植可在一定程度上减轻内毒素诱导的肺损伤,具有用作辅助治疗的潜力,同时亦表明EPC旁分泌为EPC治疗ALI的作用机制之一。
Background and objectives
Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are progressive acute respiratory failure caused by various etiological factor, and they are common critical diseases in clinical practice. The mortality of ALI/ARDS was up to 70% in 1970's, and the current mortality is still about 40% despite the advances in supportive and pharmacologic treatment. Researchers have conducted a lot of studies and made some progress, but the treatment strategy can only reduce the injury and cannot totally change its poor outcomes. The injury of pulmonary capillary endothelial cells and alveolar epithelial cells mediated by inflammation is the characteristic pathological changes of ALI/ARDS, so the ideal treatment strategy of ALI/ARDS is the repairing of injuryed lungs and the reconstructing of function. Adult stem cells have important values in the treatment of ALI/ARDS.
Endothelial progenitor cells (EPCs) are immature cells capable of differentiating into mature endothelial cells. EPCs have been shown to display a higher proliferative potential and may migrate to regions of the circulatory system with injured endothelia. Since the EPCs have important role in maintaining the integrity of endothelial cells and the injury of endothelial cells is the characteristic lesion in early period of ALI/ARDS, EPCs could become a novel cell-based therapeutic strategy for prevention and treatment of ALI/ARDS. The aims of this study include observing the variation of peripheral blood endothelial progenitor cells in the early stage of ALI induced by lipopolysaccharide (LPS) in rabbit, exploring the protective effects of autotransplantation of circulating EPCs on lung injury and the underling mechanisms, and investigateing the therapeutic potential of EPC-derived conditioned medium (EPC-CM) on ALI.
Methods and results
1 Isolation, Culture and identification of EPC
Peripheral blood was obtained from New Zealand white rabbits via ear artery (10 ml/kg). Peripheral blood mononuclear cells were isolated by density gradient centrifugation with Ficoll-Paque Plus. Mononuclear cells were then washed and planted on culture dish coated with human plasma fibronectin and supplemented with endothelial growth medium 2 (EGM-2). Seven days after isolation, incorporation of acLDL and binding of isolectin were detected in early EPCs, and the percentage of double positive cell was nearly 100%. Expressions of VEGFR2 and CD34 of those cells were detected by immunofluorescent staining, and the percentage of double positive cell was over 95%. Formation of round or fusiform shape appearance of those cells was also observed 7 days after culturing in EGM-2.
2. Develpoment of animal model of ALI
Conscious rabbits were rapidly injected intravenously via one marginal ear vein with 500μg/Kg or 700μg/Kg body weight of purified LPS endotoxin (from Escherichia coli O55.B5), and the lung of two group rabbits were harvested for HE staining at 72h. In both groups of rabbits, HE staining showed typical ALI pathological traits, such as size decrease in the pulmonary alveolus cavity, thickening of alveolar wall, expanded mesenchyme with increased numbers of polymorphonuclear cells (PMNs), formation of hyaline membrane, and hemorrhage. The lung injury of 700μg/Kg group was more severe than 500μg/Kg group, but its mortality was too high and may produce the adverse effect to the subsequent analysis. We decided to use 500μg/Kg LPS to produce the rabbit model of ALI.
3. The changes of quantity and function of peripheral blood EPCs in early stage of ALI
Rabbit model of ALI was induced by LPS (500μg/Kg), and control group was injected with normal sodium (NS). Peripheral blood was drawn at 12h,24h, 48h,72h and 5d. The expression rate of VEGFR-2+/CD34+EPCs in rabbit peripheral blood was counted by flow cytometry. The proliferation, adherenee and metabasis function of EPCs were measured after in vitro culture. The rate of EPC in control group maintain stable at each time. The rate of EPC in ALI group immediately reduced after the injection of LPS. The rate reduced to the lowest point at 24h and gradually rised after that time, but it still lower than the rate in control group at 5d. The proliferation, adherenee and metabasis function of EPCs were impaired after the injection of LPS. The functional parameters reduced to the lowest point at 48h and gradually rised after that time, but it still lower than the functional parameters in control group at 5d. The differences between the control and ALI group were significant at each time point.
4. Protective effects of EPC autotransplantion on ALI and the possible mechanism
Conscious animals were rapidly injected intravenously via one marginal ear vein with 0.5 mg/kg body weight of LPS. Autologous early EPCs (105 cells in 200μL PBS; EPC group) or 200μL PBS alone (control group) were administered via another marginal ear vein of each rabbit 30 min after injection of LPS. To track the homing of EPCs in the pulmonary, a fluorescent cell tracker CM-DiI was used to label early EPCs immediately before transplantation. Sham-operated animals (sham group) received PBS without injection of LPS.1,3 and 5 days after EPCs transplantation, rabbits were killed and samples of lung tissue, serum and bronchoalveolar lavage fluid (BALF) were collected.
The CM-DiI labeled cells were observed in frozen lung sections of the rabbit treated with EPCs, although in less amount. HE staining of lung sections from sham group rabbit showed there was no obvious lesion in the lung tissues. HE staining of control group showed typical ALI pathological traits. At each time point, EPC treatment improved the lung injury compared with control. However, the lung injury of EPC group was still apparent compared with sham group at all time points. Autotransplantation with EPCs lowered the increased lung wet/dry ratio and BAL protein at all time point, and the differences were significant at 3d and 5d (P<0.05). In LPS-treated rabbit, a significant reduction in MPO activity was found following autotransplantation with EPCs compared with the group treated with LPS alone at 1 and 3d (P<0.05). Neutrophils in the BALF were reduced in LPS-treated rabbit that received autologous transplantation of EPCs compared with the group treated with LPS alone, and the differences were significant at 3d (P<0.05). Administration of EPCs abated the increase of IL-1βand TNF-a, and the differences were significant at 3d and 5d for IL-1βand at 24h and 3d for TNF-a (P<0.05). Administration of EPCs magnified the increasing amplitude of IL-10, and the difference beteween control group and EPC group was statistically significant by 3d (P<0.05). Rabbit given EPC showed a trend toward lower sICAM-1, and there was significant difference beteween control group and EPC group at 1d (P<0.05). The changes of P-selectin mRNA expression were similar with sICAM-1, and the differences were significant at 3d (P<0.05). Administration of EPCs decreased the number of TUNEL-positive cells and prevented LPS-mediated apoptosis.
5. Protective effects of EPC-derived conditioned medium autotransplantion on ALI
EPC-derived conditioned medium (EPC-CM) was obtained from culture expanded EPC for three days.15 rabbits were divided into three group:sham group (n=5), control group (n=5) and EPC-CM group (n=5). Conscious animals were rapidly injected intravenously via one marginal ear vein with 0.5 mg/kg body weight of LPS. EPC-CM or PBS with equal volume were administered via another marginal ear vein of each rabbit 30 min after injection of LPS. Rabbits were killed and samples of lung tissue and BALF were collected 48h after EPC-CM injection. HE staining of lung sections from sham group rabbit showed there was no obvious lesion in the lung tissues. HE staining of lung sections showed that EPC-CM treatment slightly improved the lung injury compared with control. Compared with the control group, lung wet/dry ratio, BAL protein, MPO activity and neutrophils in the BALF of the EPC-CM group were all improved, but the differences were not significant between control and EPC-CM group (P>0.05).
Conclusions
1. EPCs can be successfully isolated from peripheral blood by density gradient centrifugation and adherence selection, and can be cultured in vitro.
2. In rabbit model of ALI, the quantity and proliferation, adherenee and metabasis function of EPCs were significantly reduced in early stage of ALI. The quantity and fuctions of EPCs partly restored with the resolution of inflammation in lungs.
3. autotransplantation of EPCs from peripheral blood can home to injuryed lung and attenuate LPS-induced ALI in rabbit model. EPCs transplantation can reduce neutrophil recruitment by decreasing ICAM-1 and P-selectin in initial phase of ALI and subsequently prevent lung injury. Proinflammatory cytokine responses to LPS partly altering to anti-inflammatory response and decreased apoptosis of lung cells also make contribution to the therapeutic benefits of EPCs. Treatment with EPC-CM can slightly improve the lung injury induced by LPS, suggesting that paracine of EPCis one of the mechanisms.
急性肺损伤和急性呼吸窘迫综合症(ALI/ARDS)是各种致病因素导致的急性进行性呼吸衰竭,是临床常见急重症。其死亡率在20世纪70年代早期高达70%以上,尽管近年来对其认识及救治技术不断提高,但总体病死率仍然在40%以上。国内外学者对ARDS的治疗进行了大量研究并取得了一些进展,但相关治疗策略均只能在一定程度上缓解病情,并不能从根本上改善ARDS的不良预后。炎症反应介导的肺毛细血管内皮细胞与肺泡上皮细胞损伤是ARDS的特征性病理改变,因此对受损肺脏进行结构修复和功能重建才是降低ARDS死亡率的理想治疗方式。成体干细胞具有多向或单向分化潜能,取材方便,易于分离扩增,并且不涉及到医学伦理学的相关问题,因而在ALI/ARDS治疗中具有重要的应用价值。
内皮祖细胞(EPC)是血管内皮细胞的前体细胞,并且可参与出生后血管新生、再内皮化和内皮损伤后的修复。鉴于EPC作为内源性修复机制在维持内皮细胞层完整性方面发挥了重要的作用,并且内皮受损为ARDS早期的特征性病理改变之一,因此EPC移植有希望成为ALI/ARDS的有效治疗手段。本研究旨在了解ARDS动物模型不同发病阶段EPC的动态变化规律,观察EPC自体移植以及EPC培养液“无细胞移植”对ARDS的生物作用并探讨其可能的作用机制,为提出ALI/ARDS治疗新方法提供实验线索。
方法和结果：
1.兔外周循环EPC的分离培养和鉴定
从兔耳中央动脉抽取外周血(10mL/Kg),以密度梯度法分离出单个核细胞(PMC),并将其接种于培养皿内；使用添加了细胞因子和胎牛血清的内皮祖细胞专用培养液(EGM-2)进行诱导分化培养。首次换液时间为72小时,其后每48小时换液一次。结果显示,体外培养24小时后可见到单个核细胞粘附于培养皿底部。粘在第3天时,部分细胞呈现出纺锤形变化,并且细胞分裂增殖明显。此时更换培养液以除去未贴壁细胞和红细胞。从第5天起,一部分单核细胞转化为梭形的贴壁细胞,并且随着体外培养时间的延长,圆形细胞的数量逐渐减少,而梭形细胞的数量逐渐增多。在第7天左右可观察到成团细胞形成的细胞集落。在第14天左右,细胞形状变为长梭形,并且多角形细胞有融合趋势。吞噬功能和粘附功能实验显示,体外培养的EPC可同时摄取Dil标记的乙酰化低密度脂蛋白(Dil-acLDL)并粘附FITC标记的荆豆凝集素-1(FITC-UEA-1),荧光显微镜下双阳性细胞的比率接近100%。通过免疫荧光对表面标志物进行检测显示,95%以上的细胞同时表达血管内皮生长因子受体2(VEGFR2)和CD34。依据上述条件可认定通过密度梯度离心后体外培养扩增的细胞为早期EPC。
2.ALI动物模型建立
通过耳缘静脉将内毒素(ET055:B5; 500μg/Kg和700μg/Kg)注入新西兰大白兔体循环内,72小时后活杀各组动物并取肺组织进行大体病理观察。对两组动物的肺组织切片行HE染色显示,两组动物均表现为不同程度的肺组织充血、微血管内皮损伤、出血和以中性粒细胞浸润为主的大量白细胞渗出、聚集；同时两组动物还出现肺泡间隔增厚、破坏以及部分肺泡萎陷不张等病理改变。上述结果提示两组剂量内毒素均可成功建立ALI模型。与500μg/Kg剂量组相比,700μg/Kg剂量组动物的肺组织损伤程度相对较重,但早期死亡率过高,不利于进行后期分析。因此在后续实验中采用内毒素500μg/Kg建立ALI动物模型。
3.EPC在ALI早期的数量和功能变化
利用内毒素(500μg/Kg)建立兔ALI模型,同时以等量生理盐水注射设立对照组。分别在12小时、24小时、48小时、72小时和第5天抽取外周血。利用流式细胞仪测定外周血中VEGFR-2和CD34双阳性细胞(EPC)的比率。体外培养细胞后测定EPC的增殖、粘附和迁移功能。结果显示,对照组外周血EPC比率基本保持稳定；模型组外周血EPC比率在内毒素注射后即出现降低,24小时后降至最低点,其后有所上升,但第5天时仍低于对照组。EPC增值、粘附和迁移功能均在内毒素注射后出现降低,48小时后降至最低点,其后有所上升,第5天时仍低于对照组；模型组在各时间点与对照组相比均具有显著差异。
4.自体EPC移植对ALI治疗作用及机制探讨
将45只新西兰大白兔分为正常对照组(n=15)、肺损伤对照组(n=15)和EPC移植组(n=15)。模型建立前7天抽取外周血(10ml/Kg)进行EPC体外培养扩增,并于移植前利用CM-Dil标记EPC。内毒素耳缘静脉注入30分钟后,EPC移植组给予自体EPC(约105个)于耳缘静脉注入,肺损伤对照组给予等量生理盐水。正常对照组则均给予等量生理盐水耳缘静脉注入。分别于内毒素静脉注入后24小时、第3天和第5天处死动物,留取肺组织、支气管肺泡灌洗液(BALF)和血清进行相关检查。
结果显示,CM-Dil标记的EPC可定植于肺组织内。正常对照组各项指标在各时间点均无显著变化,并且与肺损伤对照组和EPC移植组相比均具有显著差异(P<0.01)。EPC移植组肺组织病理切片的病变程度轻于肺损伤对照组,但病理评分无统计学显著差异。与肺损伤对照组相比,EPC移植组的肺湿干比(W/D)和BALF蛋白含量降低,并且在3d和5d时具有显著差异(P<0.05)；BALF中性粒细胞计数降低,并且在24h和3d时具有显著差异(P<0.05)；肺组织髓过氧化物酶(MPO)活性降低,并且在3d时具有显著差异(P<0.05)；IL-1β水平降低,并且在3d和5d时具有显著差异(P<0.05)；肿瘤坏死因子α(TNF-a)水平降低,并且在24h和3d时具有显著差异(P<0.05)；IL-10水平升高,并且在3d时具有显著差异(P<0.05)；血清细胞间粘附分子1(ICAM-1)水平较低,并且在24h时具有显著差异(P<0.05)；P选择素mRNA水平降低,并且在3d时具有显著差异(P<0.05)。EPC移植组的肺组织细胞凋亡数量少于肺损伤对照组,并且在5d时最为明显。
5.自体EPC培养液对ALI的治疗作用
将15只新西兰大白兔分为正常对照组(n=5)、肺损伤对照组(n=5)和EPC培养液组(n=5)。模型建立前3天抽取外周血(10ml/Kg),行EPC体外培养获取培养液上清。内毒素耳缘静脉注入30分钟后,EPC移植组给予EPC培养液上清于耳缘静脉注入,肺损伤对照组给予等量生理盐水。正常对照组则均给予等量生理盐水耳缘静脉注入。48小时后处死动物,留取肺组织和BALF进行相关检查。结果显示,肺损伤对照组和EPC培养液组各项指标与正常对照组相比均具有显著差异(P<0.01)。EPC培养液组肺组织普通病理检查所示的病变程度稍轻于肺损伤对照组。与肺损伤对照组相比,EPC培养液组肺W/D、BALF总蛋白含量、BALF中性粒细胞数量和肺组织MPO活性均较低,但各指标均不具有统计学显著差异(P>0.05)。
结论：
1密度梯度离心联合贴壁选择法能够成功从外周血中分离中EPC,并可在体外进行培养扩增。
2在内毒素构建的兔ALI动物模型中,外周血EPC数量以及增殖、粘附和迁徙功能在ALI早期均出现显著降低；随着肺部炎症的消退,EPC数量和相关功能有所恢复。
3来源于外周血的自体EPC于移植后可归巢到受损肺脏,并且可减轻内毒素诱导的急性肺损伤病变严重程度。自体EPC移植可通过降低ICAM-1和P-选择素表达的方式减少ALI起始阶段的PMN肺内扣押,进而预防肺损伤。自体EPC移植可将内毒素诱导的促炎细胞因子反应部分转化为抗炎细胞因子反应,同时可还可通过减少肺组织细胞凋亡发挥治疗作用。EPC培养液移植可在一定程度上减轻内毒素诱导的肺损伤,具有用作辅助治疗的潜力,同时亦表明EPC旁分泌为EPC治疗ALI的作用机制之一。
Background and objectives
Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are progressive acute respiratory failure caused by various etiological factor, and they are common critical diseases in clinical practice. The mortality of ALI/ARDS was up to 70% in 1970's, and the current mortality is still about 40% despite the advances in supportive and pharmacologic treatment. Researchers have conducted a lot of studies and made some progress, but the treatment strategy can only reduce the injury and cannot totally change its poor outcomes. The injury of pulmonary capillary endothelial cells and alveolar epithelial cells mediated by inflammation is the characteristic pathological changes of ALI/ARDS, so the ideal treatment strategy of ALI/ARDS is the repairing of injuryed lungs and the reconstructing of function. Adult stem cells have important values in the treatment of ALI/ARDS.
Endothelial progenitor cells (EPCs) are immature cells capable of differentiating into mature endothelial cells. EPCs have been shown to display a higher proliferative potential and may migrate to regions of the circulatory system with injured endothelia. Since the EPCs have important role in maintaining the integrity of endothelial cells and the injury of endothelial cells is the characteristic lesion in early period of ALI/ARDS, EPCs could become a novel cell-based therapeutic strategy for prevention and treatment of ALI/ARDS. The aims of this study include observing the variation of peripheral blood endothelial progenitor cells in the early stage of ALI induced by lipopolysaccharide (LPS) in rabbit, exploring the protective effects of autotransplantation of circulating EPCs on lung injury and the underling mechanisms, and investigateing the therapeutic potential of EPC-derived conditioned medium (EPC-CM) on ALI.
Methods and results
1 Isolation, Culture and identification of EPC
Peripheral blood was obtained from New Zealand white rabbits via ear artery (10 ml/kg). Peripheral blood mononuclear cells were isolated by density gradient centrifugation with Ficoll-Paque Plus. Mononuclear cells were then washed and planted on culture dish coated with human plasma fibronectin and supplemented with endothelial growth medium 2 (EGM-2). Seven days after isolation, incorporation of acLDL and binding of isolectin were detected in early EPCs, and the percentage of double positive cell was nearly 100%. Expressions of VEGFR2 and CD34 of those cells were detected by immunofluorescent staining, and the percentage of double positive cell was over 95%. Formation of round or fusiform shape appearance of those cells was also observed 7 days after culturing in EGM-2.
2. Develpoment of animal model of ALI
Conscious rabbits were rapidly injected intravenously via one marginal ear vein with 500μg/Kg or 700μg/Kg body weight of purified LPS endotoxin (from Escherichia coli O55.B5), and the lung of two group rabbits were harvested for HE staining at 72h. In both groups of rabbits, HE staining showed typical ALI pathological traits, such as size decrease in the pulmonary alveolus cavity, thickening of alveolar wall, expanded mesenchyme with increased numbers of polymorphonuclear cells (PMNs), formation of hyaline membrane, and hemorrhage. The lung injury of 700μg/Kg group was more severe than 500μg/Kg group, but its mortality was too high and may produce the adverse effect to the subsequent analysis. We decided to use 500μg/Kg LPS to produce the rabbit model of ALI.
3. The changes of quantity and function of peripheral blood EPCs in early stage of ALI
Rabbit model of ALI was induced by LPS (500μg/Kg), and control group was injected with normal sodium (NS). Peripheral blood was drawn at 12h,24h, 48h,72h and 5d. The expression rate of VEGFR-2+/CD34+EPCs in rabbit peripheral blood was counted by flow cytometry. The proliferation, adherenee and metabasis function of EPCs were measured after in vitro culture. The rate of EPC in control group maintain stable at each time. The rate of EPC in ALI group immediately reduced after the injection of LPS. The rate reduced to the lowest point at 24h and gradually rised after that time, but it still lower than the rate in control group at 5d. The proliferation, adherenee and metabasis function of EPCs were impaired after the injection of LPS. The functional parameters reduced to the lowest point at 48h and gradually rised after that time, but it still lower than the functional parameters in control group at 5d. The differences between the control and ALI group were significant at each time point.
4. Protective effects of EPC autotransplantion on ALI and the possible mechanism
Conscious animals were rapidly injected intravenously via one marginal ear vein with 0.5 mg/kg body weight of LPS. Autologous early EPCs (105 cells in 200μL PBS; EPC group) or 200μL PBS alone (control group) were administered via another marginal ear vein of each rabbit 30 min after injection of LPS. To track the homing of EPCs in the pulmonary, a fluorescent cell tracker CM-DiI was used to label early EPCs immediately before transplantation. Sham-operated animals (sham group) received PBS without injection of LPS.1,3 and 5 days after EPCs transplantation, rabbits were killed and samples of lung tissue, serum and bronchoalveolar lavage fluid (BALF) were collected.
The CM-DiI labeled cells were observed in frozen lung sections of the rabbit treated with EPCs, although in less amount. HE staining of lung sections from sham group rabbit showed there was no obvious lesion in the lung tissues. HE staining of control group showed typical ALI pathological traits. At each time point, EPC treatment improved the lung injury compared with control. However, the lung injury of EPC group was still apparent compared with sham group at all time points. Autotransplantation with EPCs lowered the increased lung wet/dry ratio and BAL protein at all time point, and the differences were significant at 3d and 5d (P<0.05). In LPS-treated rabbit, a significant reduction in MPO activity was found following autotransplantation with EPCs compared with the group treated with LPS alone at 1 and 3d (P<0.05). Neutrophils in the BALF were reduced in LPS-treated rabbit that received autologous transplantation of EPCs compared with the group treated with LPS alone, and the differences were significant at 3d (P<0.05). Administration of EPCs abated the increase of IL-1βand TNF-a, and the differences were significant at 3d and 5d for IL-1βand at 24h and 3d for TNF-a (P<0.05). Administration of EPCs magnified the increasing amplitude of IL-10, and the difference beteween control group and EPC group was statistically significant by 3d (P<0.05). Rabbit given EPC showed a trend toward lower sICAM-1, and there was significant difference beteween control group and EPC group at 1d (P<0.05). The changes of P-selectin mRNA expression were similar with sICAM-1, and the differences were significant at 3d (P<0.05). Administration of EPCs decreased the number of TUNEL-positive cells and prevented LPS-mediated apoptosis.
5. Protective effects of EPC-derived conditioned medium autotransplantion on ALI
EPC-derived conditioned medium (EPC-CM) was obtained from culture expanded EPC for three days.15 rabbits were divided into three group:sham group (n=5), control group (n=5) and EPC-CM group (n=5). Conscious animals were rapidly injected intravenously via one marginal ear vein with 0.5 mg/kg body weight of LPS. EPC-CM or PBS with equal volume were administered via another marginal ear vein of each rabbit 30 min after injection of LPS. Rabbits were killed and samples of lung tissue and BALF were collected 48h after EPC-CM injection. HE staining of lung sections from sham group rabbit showed there was no obvious lesion in the lung tissues. HE staining of lung sections showed that EPC-CM treatment slightly improved the lung injury compared with control. Compared with the control group, lung wet/dry ratio, BAL protein, MPO activity and neutrophils in the BALF of the EPC-CM group were all improved, but the differences were not significant between control and EPC-CM group (P>0.05).
Conclusions
1. EPCs can be successfully isolated from peripheral blood by density gradient centrifugation and adherence selection, and can be cultured in vitro.
2. In rabbit model of ALI, the quantity and proliferation, adherenee and metabasis function of EPCs were significantly reduced in early stage of ALI. The quantity and fuctions of EPCs partly restored with the resolution of inflammation in lungs.
3. autotransplantation of EPCs from peripheral blood can home to injuryed lung and attenuate LPS-induced ALI in rabbit model. EPCs transplantation can reduce neutrophil recruitment by decreasing ICAM-1 and P-selectin in initial phase of ALI and subsequently prevent lung injury. Proinflammatory cytokine responses to LPS partly altering to anti-inflammatory response and decreased apoptosis of lung cells also make contribution to the therapeutic benefits of EPCs. Treatment with EPC-CM can slightly improve the lung injury induced by LPS, suggesting that paracine of EPCis one of the mechanisms.
引文
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