骨髓间充质干细胞治疗呼吸机所致肺损伤的动物实验研究
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摘要
研究背景及目的
SARS、甲型H1N1流感病毒等引起的重症肺部感染可导致急性肺损伤(Acute lung injury, ALI)/急性呼吸窘迫综合征(Acute respiratory distress syndrome, ARDS)。由于ALI/ARDS病情危重、预后差,其救治已成为目前全世界的重点话题。目前对于ALI/ARDS在内的急、慢性呼吸衰竭患者的有效救治手段主要有机械通气、ECMO等。其中机械通气作为最主要的手段之一,其使用不当即可导致呼吸机所致的肺损伤(Ventilator-induced lung injury, VILI)。
虽然目前对于VILI的发生机制不完全清楚,但在VILI模型上可发现其病理上以弥漫肺泡损伤、肺泡—毛细血管渗透性增加并继发以中性粒细胞(Neutrophil, polymorphonuclear neutrophil, PMN)肺部浸润显著增加并伴随以BALF及全身循环中PMN升高为主的炎症反应等特征。既往研究显示PMN的过度激活可加重肺损伤,在VILI模型中PMN的过度激活与肺损伤程度密切相关,而针对PMN激活及其功能的各种干预手段[如中性粒细胞弹性蛋白酶(neutrophil elastase, NE)阻滞剂、PMN趋化因子MIP-2受体敲除等]可显著降低肺损伤的严重程度,故对PMN激活及其功能的干预有望成为防治VILI的有效手段。
骨髓间充质干细胞(Bone marrow-derived mesenchymal stem cells, MSCs)是一种多能分化的干细胞,其在特定环境下可诱导分化为多种细胞(如软骨细胞、肌肉细胞、内皮细胞、上皮细胞、神经细胞等)。在一些严重烧伤及损伤情况下使用MSCs时,MSCs可迅速趋向于损伤部位、获得一些损伤部位细胞的表型而发挥作用,且在肺损伤的研究中也显示其可获得肺部细胞表型、起到促进损伤修复等重要作用。此外,在脓毒症肺损伤的动物研究中显示MSCs使用可改善动物的预后,然而MSCs在VILI中使用是否也有类似作用目前仍缺乏相关报道。
MSCs具有抑制免疫活性的功能,这可能是其促进损伤动物组织损伤修复的一个重要原因。其中MSCs与循环中的PMN、巨噬细胞、淋巴细胞、DC细胞及自然杀细胞等的相互作用在MSCs发挥其损伤修复作用时具有重要意义。新近研究表面MSCs-PMN的体外细胞实验、动物实验结果均提示MSCs可抑制PMN的激活。然而,MSCs在VILI模型中是否也能抑制PMN的激活目前仍未知。
本研究假设MSC的输注可以通过调节PMN功能的激活在内的炎症反应和(或)通过MSC移植入肺并获得肺部细胞表型、重建损伤肺的结构及功能的途径进而减轻甚至预防大潮气量机械通气导致的肺损伤,对VILI模型起到保护作用。为此,本研究采用大潮气量机械通气来制备VILI模型,并在该模型中防治性使用MSCs以评价MSCs在VILI的作用。
第一章大鼠骨髓间充质干细胞的体外分离、培养及鉴定
目的体外分离、培养雄性SD大鼠骨髓间充质干细胞(MSCs),并研究其生物学特性、表型特征及分化能力。
方法在无菌条件下取出雄性SD大鼠股骨、胫骨,用冰L-DMEM培养基冲洗骨髓腔,离心取有核细胞,采用密度梯度离心法结合贴壁法分离、纯化大鼠MSCs,用含10%胎牛血清的L-DMEM培养基培养MSCs,去除非贴壁细胞。待细胞铺满瓶底面积约90%时,按1：2进行传代培养。用流式细胞仪检测细胞周期、凋亡率(Annexin-V/PI法),以明确体外培养目标细胞的生长特征；用MTT法通过描述细胞生长曲线以评价目标细胞的增殖活性；用流式细胞仪(FCM)检测培养的目标细胞表面CD分子CD34、CD45、CD29、CD44的表达,以鉴定目标细胞的纯度；用软骨细胞基础培养基加转化生长因子—β诱导目标细胞分化为软骨细胞,用免疫细胞化学法观察诱导的细胞表达Ⅱ型胶原的情况,以明确目标细胞的定向分化能力。
结果从骨髓分离到的目标有核细胞大部分呈圆形,接种于培养基后72 h大部分细胞贴培养瓶壁、呈集落性生长,细胞形状变为梭形。培养10d-15d后,集落增多大部分融合成片并达到传代标准。细胞传代之后,细胞生长速度增快,约1周即可进行再次传代。在细胞传3代后,细胞形态仍为梭形、且形态较为均一。P3细胞中大部分细胞处于合成前期(G1期),其比例>80.0%；其次为处于DNA复制期(S期,约10.0%-12.0%)及DNA合成后期(G2期,约占6.0%-7.5%)的细胞,而静止期(G0期)及有丝分裂期(M期)的细胞占比例极少(G0+M期,约占0.0%-0.4%)；Annexin V/PI法检测目标细胞凋亡率均<5.0%。细胞在传代后第3d进入指数生长期,到第5d达到高峰；之后进入生长平台期。培养P3细胞表面CD29阳性表达率均大于99.50%、CD44阳性表达率均大于99.90%、CD34阳性表达率小于2.50%、CD45阳性表达率小于2.00%。在软骨细胞基质培养基内培养14d后,细胞外形变为多角形,胞浆中出现Ⅱ型胶原表达的占50%。
结论本研究采用密度梯度离心法结合贴壁法可在体外分离、培养出SD大鼠来源的MSCs。用该方法培养出的MSC大部分处于增殖期,具有增殖活跃、存活率高、纯度高的特点,且其具有较高的分化能力。由于其生物学性状、表型稳定、分化能力较好,可用于进一步的动物实验研究。
第二章大潮气量机械通气所致肺损伤大鼠模型的制备
目的用大潮气量机械通气制备呼吸机所致肺损伤的SD大鼠模型。
方法将250～260g体重的成年雌性SD大鼠腹腔内注射苯巴比妥钠麻醉后行气管切开插入气管导管,右颈内动脉留置头皮针。插管完毕后将动物分为三组：大潮气量机械通气组(VT20组,VT=20 ml/kg体重)、小潮气量机械通气组(VT8组,VT=8 ml/kg体重)、非机械通气对照组(不行机械通气,在插气管导管后大鼠自主呼吸)。各组n=12,以上机械通气组机械通气设置为：PEEP=2 cm H2O、RR=40次/分,吸呼比(I：E)均为1：2,吸入的气体均为空气,即吸入氧浓度(FiO2)均为21%。机械通气时间均为2h。机械通气组在导管置入完毕时(T0)、机械通气结束时(T2)、机械通气结束后2h时(T4)、机械通气结束后4h时(T6)等4个时间点分批取血标本后放血处死动物,而非机械通气对照组则在插管完毕时(T0)、插管完毕后2h(T2)、4h(T4)、6h(T6)分批取血标本后放血处死动物。各组各时点均取3只动物。观察并比较各组各时点动脉血气、肺干湿比、肺泡灌洗液内PMN数量、肺组织病理。
结果1)各组大鼠在整个实验过程中pH值进行性下降,各时间点间差异有统计学意义(F=9.029,P=0.001),但组间差异无统计学差异(F=1.347,P=0.329)。VT20组在T2时动脉血PaO2/氧合指数(OI)较TO时显著下降(均P=0.016),但机械通气后组内各时间点前后动脉血PaO2/OI差异均无统计学意义(T2比T4,P=0.775;T4比T6,P=0.338)；其它各组组内PaO2/OI差异也均无统计学意义(对照组F=0.603,P=0.531；VT8组F=2.901,P=0.206)。动脉血PaCO2的变化上,分组、时间因素均无显著影响(时间因素：F=3.503,P=0.082；分组因素：F=0.874,P=0.464)。VT20组在T4时动脉血[HCO3-]浓度较T0、T2均下降,差异有统计学意义(均P=0.022)；之后维持在T4水平(T6比T4,P=0.497)；而对照组及VT8组在实验过程中[HCO3-]浓度组内差异均无统计学意义(对照组F=4.200,P=0.064；VT8组F=4.659,P=0.149)。2)对照组及VT8组动物肺湿干比组内各时点间差异无统计学意义(对照组F=0.336,P=0.658；VT8组F=2.017,P=0.272)；而VT20组动物肺湿干比在机械通气后随时间增加而增加,T6时与T0时比较差异有统计学意义(P=0.019)。3)对照组及VT8组动物肺组织病理随时间变化不大,但VT20组出现双上肺明显肿胀、包膜下血性渗出,弹性较前明显降低,肺间质水肿、PMN浸润,肺泡腔内出现大量血性液体渗出、且肺泡腔内出现以PMN为主的细胞渗出,部分肺组织实变及肺泡出血等损伤改变。且VT20组动物肺泡灌洗液内PMN数量也随着时间推移而增加,组内各时点差异有统计学意义(F=93.782,P=0.000)。
结论用20ml/kg体重的潮气量对SD大鼠进行为期2h的机械通气可导致大鼠氧合障碍、肺部出现间质水肿、肺泡渗出及PMN浸润为主等急性肺损伤的病理改变,可见本研究采用20ml/kg潮气量对大鼠行机械通气2h可成功制备VILI大鼠模型。该研究可为下一步研究提供VILI动物模型。
第三章MSC给予对于VILI模型肺损伤及炎症反应的影响
目的明确MSC对VILI模型肺组织损伤及局部、全身炎症反应的影响。
方法将30只体重为250～260g的成年雌性SD大鼠麻醉后气管切开插管,右颈内动脉留置头皮针。插管完毕后将动物分为5组：正常对照组(control组)、MSC给予组(MSC组)、大潮气量机械通气组(VT20组)、MSCs预给予+大潮气量机械通气组(MSC+VT20组,在大潮气量机械通气前给予MSCs)、大潮气量机械通气+MSCs后给予组(VT20+MSC组,在大潮气量机械通气结束后立即给予MSCs)。各组n=6,机械通气组机械通气参数为：VT=20 ml/kg体重,PEEP=2 cm H2O, RR=40次/分,吸呼比(I：E)均为1：2,FiO2=21%。机械通气时间均为2h,机械通气结束后给予去除机械通气自主呼吸4h,之后处死动物。非机械通气组在插管完毕后6h处死动物。MSCs给予组均静脉给予每只大鼠数量为3×106/L的MSCs,而未给予MSC组在插管2h后给予静脉注射等体积生理盐水(0.5ml)。动态观察各组大鼠血气变化、肺组织病理、对肺组织损伤进行评分(Smith评分)、肺组织内PMN浸润数量、BALF中PMN数量、BALF及循环中促炎因子(TNF-α、IL-6、MIP-2)及抗炎因子(IL-10)的变化情况,并将以上指标进行组间比较。
结果1)机械通气各组(包括VT20组、MSC+VT20组及VT20+MSC组)在机械通气后动脉血PaO2较T0时下降,组内差异只有VT20组有统计学意义(P分别为0.001、0.359、0.147)；机械通气后MSC+VT20组各时点Pa02均较VT20组增高,但只有T2时差异才有统计学意义(T2、T4及T6,P分别为0.035、0.050、0.053)。2)给予MSC对正常大鼠肺部病理组织无显著影响,而VT20可导致大鼠肺部出现肺间质水肿、PMN浸润,肺泡腔内出现大量的血性液体渗出、且肺泡腔内出现以PMN为主的细胞渗出,部分肺组织实变及肺泡出血等损伤改变。而MSC+VT20组肺组织病理仅见部分肺间质稍增厚及较少量PMN浸润,而VT20+MSC组部分肺间质进一步增厚、PMN浸润明显减少等改变。肺损伤评分组间差异有统计学意义(F=68.131,P=0.000)。而肺组织病理高倍视野下PMN数量、BALF中PMN计数量组间差异同样也有统计学意义(肺组织：F=43.039,P=0.000：BALF:F=134.171,P=0.000)。3)各组动物BALF中TNF-α、IL-6、MIP-2及IL-10的浓度组间差异有统计学意义(TNF-α:F=69.706, P=0.000; IL-6:F=155.816,P=0.000；MIP-2:F=120.529,P=0.000；IL-10：F=42.154,P=0.000),其中VT20组BALF中TNF-α、IL-6、MIP-2及IL-10的浓度均较对照组高,且组间差异有统计学意义(均P=0.000);MSC+VT20组BALF中TNF-α、H-6、MIP-2浓度较VT20组低,组间差异有统计学意义(P分别为0.000、0.025、0.009);而VT20+MSC组与VT20组比较时,BALF的TNF-α、MIP-2水平组间差异有统计学意义(P分别为0.031、0.012),但组间BALF的IL-6差异无统计学意义(P=0.995)；此外,MSC+VT20组、VT20+MSC组的IL-10水平分别与VT20组IL-10比较时,组间差异也无统计学意义(P分别为0.117、0.556)。4)机械通气损伤后(包括T2、T4、T6)大鼠血浆TNF-α、IL-6、MIP-2及IL-10的浓度均较T0时升高,且各指标组内差异均有统计学意义(均P=0.000),而MSC+VT20组机械通气后(T4、T6)血浆TNF-α、IL-6、MIP-2的浓度较VT20组的低(TNF-α：P分别为0.000、0.003；IL-6：P分别为0.003、0.011；MIP-2：P分别为0.997、0.002)；VT20+MSC组机械通气后与VT20组比较时,T6时组间血浆TNF-α、MIP-2的浓度差异有统计学意义(P分别为0.039、0.033),而IL-6浓度组间差异无统计学意义(P分别为1.000、0.469、0.978)。而MSC+VT20组、VT20+MSC组在机械通气后血浆IL-10水平与同一时点的VT20组血浆工L-10水平比较,组间差异均无统计学意义(P分别为0.804、0.995、1.000,0.557、1.000、0.966)。
结论MSCs给予可在不同程度上减轻大潮气量机械通气所致的肺部组织损伤、氧合恶化、肺部PMN浸润、局部及全身与PMN有关的炎症介质的释放量,且MSCs给予时间越早,这些作用就越明显；但MSCs给予但对VILI模型抗炎介质IL-10的释放量无显著影响。MSCs使用在降低肺损伤程度的同时也伴随着肺部浸润PMN数量的降低。这提示MSCs输注可为VILI模型提供一个相对稳定的内环境,这有助于提高动物对不当机械通气的耐受程度及防治VILI后继的以PMN为主的炎症及其所致的肺部进一步损伤。
第四章MSC处理对VILI大鼠PMN功能的影响
目的明确MSC对VILI模型SD大鼠PMN功能的影响。
方法将对照组(control组)、MSC给予组(MSC组)、大潮气量机械通气组(VT20组)、MSCs预给予+大潮气量机械通气组(MSC+VT20组,在大潮气量机械通气前给予MSCs)、大潮气量机械通气+MSCs后给予组(VT20+MSC组,在大潮气量机械通气结束后立即给予MSCs)大鼠在机械通气后4h后取的血标本及处死后取BALF的标本,用流式细胞仪检测其中PMN的细胞内ROS含量及表面分子CDllb的表达；分离血标本、BALF标本中的PMN,用流式细胞术进行凋亡率检测；利用化学发光法进行血浆、BALF中NE活性定量检测；用Fenton反应检测血浆及BALF中的ROS (·OH含量),以确定细胞外ROS含量；并将NE活性、细胞表面CD11b表大量、细胞内ROS含量、细胞外ROS含量、PMN细胞凋亡率资料进行组间比较。
结果1)血及BALF中PMN细胞表面CDllb表达量组间差异均无统计学意义(血：F=1.008,P=0.442;BALF:F=0.328,P=0.856)。2)血及BALF中NE活性各组间差异有统计学意义(F分别为39.813、31.061,均P=0.000)。其中VT20组血及BALF中NE活性均较对照组高,差异有统计学意义(均P=0.000)；MSC+VT20组血及BALF中NE活性均较VT20组降低,差异有统计学意义(均P=0.000)；且VT20+MSC组与VT20组组间血及BALF中的NE活性差异均有统计学意义(血：P=0.035,BALF:P=0.040)；但MSC+VT20组与VT20+MSC组组血浆及BALF的NE活性间差异无统计学意义(血：P=0.927,BALF:P=1.000)。3)血及BALF中PMN内ROS含量各组间差异均有统计学意义(F分别为986.470、118.612,均P=0.000)。VT20组血及BALF中PMN内ROS含量较对照组增高,且差异有统计学意义(均P=0.000);MSCs使用组(包括MSC+VT20组、VT20+MSC组)细胞内ROS产量均较VT20组低,与VT20组比较差异均有统计学意义(除BALF中MSC+VT20组与VT20组比较P=0.001外,其它均P=0.000)；且MSC+VT20组、VT20+MSC组比较时,组间BALF来源的PMN内ROS含量的差异也有统计学意义(P=0.000)。4)血浆及BALF中ROS含量各组间差异有统计学意义(F分别为19.597、198.991,均P=0.000)。其中VT20组血浆及BALF中ROS含量均较对照组低,组间差异均有统计学意义(均P=0.000)；MSC+VT20组血浆及BALF中的ROS含量较VT20组降低,组间差异均有统计学意义(均P=0.000)；而VT20+MSC组只有BALF中ROS含量较VT20组低,组间差异有统计学意义(P=0.001);且MSC+VT20组、VT20+MSC组比较时,血浆、BALF中ROS含量组间差异均有统计学意义(P分别为0.001、0.021)。5)血及BALF来源的PMN细胞凋亡率各组间差异有统计学意义(F分别为41.869、89.661,均P=0.000)。其中VT20组血及BALF来源的PMN凋亡率均较对照组降低,且组间差异有统计学意义(均P=0.000)；而MSC给予组中只有MSC+VT20组血中PMN的凋亡率较VT20组高,差异有统计学意义(P=0.001)。
结论大潮气量机械通气所致的肺损伤大鼠模型中存在PMN的过度激活,其主要表现在NE活性增高、细胞内外ROS产量增加、细胞凋亡减少及延迟等。而MSCs的给予在不同程度上降低NE活性及ROS的产生量,尤其是损伤前给予MSCs时PMN功能激活相关指标降低量更为明显。这表面MSCs有调节PMN功能的能力,且MSC给予时间越早则对PMN功能调节作用越明显。
第五章MSC静脉输注对于VILI模型预后的影响及MSCs输注后在肺部转化情况观察
目的明确MSCs静脉内注射能否改善VILI模型的预后与及其是否能迁移到VILI大鼠的肺部、替代损伤组织细胞、甚至获得肺部损伤细胞的表型及发挥相应功能。
方法取45只体重为250～260g的成年雌性SD大鼠麻醉、气管切开插管后随机分为大潮气量机械通气组b(VT20组b)、MSCs预给予+大潮气量机械通气组(MSC+VT20组b,在大潮气量机械通气前给予MSCs)、大潮气量机械通气+MSCs后给予组(VT20+MSC组b,在大潮气量机械通气结束后立即给予MSCs),各组n=15,机械通气组机械通气参数为：VT=20ml/kg体重,PEEP= 2 cm H20,RR=40次/分,吸呼比(I：E)为1：2,吸入气氧浓度(FiO2)为21%,机械通气时间为2h。机械通气结束后拔除呼吸机接头、缝合颈部切口后放置回实验笼中,让动物自主呼吸空气。在随后的14d内每24h观察并记录一次动物的存亡情况。取6只体重相近的成年雌性SD大鼠作MSC长期观察组(MSC组b)经尾静脉注射MSCs,之后放回养殖笼中继续养殖14d。MSCs均为雄性大鼠来源的,数量均为3×106/L每只大鼠。取MSC组、MSC+VT20组、VT20+MSC组、MSC组b、MSC+VT20组b及VT20+MSC组b动物各6只处死后取肺行免疫组织化学检测抗SRY抗体阳性的细胞、PCR检测肺组织中大鼠Y染色体上的Sry基因片段以明确静脉注射雄性大鼠来源的MSCs后是否能滞留在肺部、较长时期后肺部是否有雄性大鼠来源的细胞存在及其存在的部位。
结果MSC+VT20组b、VT20+MSC组b及VT20组b动物致伤后48h内生存率分别为73.33%、73.33%、66.67%,三组组间差异无统计学意义(χ2=0.216,P=0.897)；14d内生存率分别为53.33.00%、40.00%、40.00%,各组动物14d内死亡率差异也无统计学意义(χ2=0.720,P=0.698)。免疫组化、PCR结果提示MSCs输注后短期实验结束时各组动物均有MSCs滞留在肺部,其中在MSC组MSCs较均匀分布在肺毛细血管床内；而MSC+VT20组MSCs分布较MSC组较为局限,量较MSC组少,且差异有统计学意义(P=0.000)；而VT20+MSC组MSCs则分布在肺间质明显增厚的毛细血管床内,分布成片带状,量较MSC+VT20组要少,差异有统计学意义(P=0.000)。而在MSC输注后14d后MSC组b、MSC+VT20组b及VT20+MSC组b三组动物的肺组织中均未检出抗Y染色体特异性结合蛋白抗体阳性细胞及Y染色体Sry片段。
结论MSCs静脉内输注短期内可出现在VILI动物肺部,而组织损伤可减少MSCs在肺部的滞留数量,而MSCs并未移植到肺部、也未分化为肺部细胞并取代损伤细胞的功能。MSC细胞静脉内输注虽可将VILI大鼠14d生存率从40%提高到53.33%,但由于例数较少,仍需进一步扩大样本方可确认MSCs干预能否改善VILI模型14d内生存率。
全文总结
利用密度梯度离心法联合离心法可分离、体外培养出增殖能力强、纯度高、分化能力强、性状稳定的MSCs.而大潮气量短期机械通气可制备VILI模型,该模型病理上以PMN肺部浸润增加为主要特征,同时伴随与PMN相关的局部及全身炎症反应。在VILI大鼠模型中进行静脉内输注MSCs可减轻VILI大鼠继发的以PMN肺部浸润及PMN功能激活在内的炎症反应及肺损伤,而使用时机越早其保护作用越明显。而MSC在VILI动物模型中的保护作用并非通过MSCs移植入肺、获得肺部细胞表型、重建损伤肺细胞的结构和功能,而是通过调节PMN的功能激活在内的炎症反应。这提示临床有望使用MSCs来调节PMN功能激活为主要特征的炎症反应模式为有发生VILI风险的机械通气者提供稳定的内环境及提高其对不当机械通气的耐受能力,以达到防治VILI的目的。
Background
The acute lung injury (ALI) / acute respiratory distress syndrome (ARDS) induced by severe respiratory infection (such as SARS, severe H1N1 flu infection) is known to the whole world as its severity and poor outcome. Until recently, mechanical ventilation and ECMO and etc. were used as effective salvage method in the patients with acute or chronic respiratory failure (ALI/ARDS). Mechanical ventilation, a crucial supported and therapeutic technique for surviving patients with respiratory failure (include ALI/ARDS) could induced lung injury, which was known as ventilator-induced lung injury (VILI).
Subsequent neutrophil (PMN)-predominant inflammatory response as well as diffuse alveolar damage and pulmonary and systemic vascular leak was the key features of VILI, which had been observed in different animal models of VILI, although the mechanisms of VILI with different impropriate ventilation parameters are not fully understood. The excess activation of PMNs aggravates the severity of injury. Therapeutic methods referred to modulate the activation of PMNs, such as specific inhibitor of neutrophil elastase (NE) (Sivelestat) administration, MIP-2 (one of neutrophil chemotactic factors) receptor knockout were seemed to attenuate the severity of VILI. Hence, the management of PMN activation could be an important therapeutic approach to VILI.
Bone marrow-derived mesenchymal stem cells (MSC), a type of stem cells, could differentiate into several cell lineages, such as chondrocytes, myoblasts, endothelial cells, epithelial cells, and neurocytes, under certain circumstances. It had been used to heal severe wounded or damage tissue as it preferentially localize to the injured sites as well as adopting the phenotype of some kinds of cells in the local tissue after administration. Several studies showed that MSCs can immigrate into the lung and adopt the phenotype of lung cells and play positive roles in lung injury. Also it was reported that intrapulmonary delivery of MSC could improves mice survival model of endotoxin-induced acute lung injury. However, whether the MSC could immigrate into the injured lung and adopted the phenotype of lung cells, and improve the outcome of animals in VILI model is still unknown.
The active immunosuppressive ability of MSC, which partially attributed to the repair function of MSC in injured models, has been well documented. The interaction of MSCs with PMNs, besides macrophages, lymphocytes, dendritic cells and natural killer cells, was especially important in certain types of injury. Recently MSCs-PMNs coculture tests and animal trials showed that MSC could suppress the PMNs activation. However, whether MSC infusion could suppress the excess activation of PMN and subsequent inflammation induced by high tidal volume mechanical ventilation is still unknown.
The aims of this study were to test the hypothesis that infusion of MSC may be beneficial for VILI which is linked these effects to the regulation of inflammatory response which includes PMNs'activation, or linked these effects to the reconstruction of structure and function by MSCs transplantation; we used the in vivo high tidal volume ventilation induced lung injury model to evaluate the effect of infusive MSC.
Chapter 1 Isolation, expansion and identification of rat MSCs
Objective To separate and culture the rat bone marrow derived mesenchymal stem cells in vitro, and to explore the biological characteristics, phenotype and differentiation of the cultured cells.
Methods The femurs and tibia were removed from specific pathogen-free (SPF) male Sprague-Dawley after decapitated sacrifice. The whole marrow was flushed from the tibia and femur of rats with ice-cold L-Dulbecco's Modified Eagle Medium (L-DMEM), and the nucleated cells were adopted for isolation. And the isolation and purification of rat MSCs were finished with Percoll density gradient centrifugation combined with adherent method. The isolated MSCs were cultured with L-DMEM cultured media containing 10% fetal bovine serum and the non-adhesive cells were removed. At confluence, the cells were harvested for passage at the ratio of 1:2. In order to determine the target-cellular growth characteristics, the flow cytometer was used to determine cellular cell cycle and apoptotic ratio. And in order to determine the cellular proliferating ability of the target-cell, the cell growth curved was conducted using MTT kids. In order to determine the purification of the target cells, the surface markers of CD34, CD45, CD29 and CD44 were evaluated by flow cytometer. In order to determine the differentiation ability of cultured cells, the cultured MSCs were cultured in basic chondrocyte media with transformed growth factor-beta, and the collagen type II expression were determined by immunocytochemistry.
Results The primary isolated nucleated cells were mostly round and it converted to adherent, colony growth and fusiform-shaped 72 hours after innoculation. The cells were passaged after 10～15 days while mostly colonies melted into one. The cell growth faster after passaged and they could be repassaged about 1 w after inoculation. After the third passage, morphology of mostly cells was uniform and remains the fusiform-shaped. And more than 80% cells were at the G1 stage of the third passage of cultured MSCs, about 6.0% to 7.5% were at G2 stage and about 0 to 0.4% were at stage of GO and M. The apoptotic ratio of target cells were less than 5.0% which measured used Annexin-V measurement. The cells went into logarithmic growth phage from third days after passaged, it reached peak growth phage at the fifth days and later went into late stationary phage. The MSCs we cultured contained a small number of cells expressing CD45 (< 2.0%) and CD34 (<2.5%), and mostly cells expressing CD29 (>99.5%) and CD44 (>99.9%). And the basic chondrocyte media induced the transformation of mesenchymal cells into chondrocytes as the type II collagen could be detected in 50% of the transformed cells after 14 days of culture.
Conclusions SD rats-derived MSCs can be obtained by Percoll' density gradient centrifugation combined with adherent method. The mostly MSCs we cultured were at proliferating phage. And the MSCs we cultured with a characteristics of active proliferation, mostly survival and high purify, and active property of differentiation. As the MSCs we cultured with stable characteristics of biology, phenotypes, it can be used in the further animal research.
Chapter 2 Establishment of ventilator-induced lung injury model with high tidal volume ventilation in SD rats
Objective To establish ventilator-induced lung injury model induced by high tidal volume ventilation in SD rats.
Methods The rats weighting 250g to 260g were anesthetized with pentobarbital sodium (40mg/kg body weight) intra-peritoneal. A tracheotomy was performed, and a canula was inserted into the trachea. The animals were subgroup into 3 groups after canula insertion:high tidal volume mechanical ventilation group (VT20 group, VT= 20 ml/kg, PEEP=2 cmH2O), low tidal volume mechanical ventilation group (VT8 group, VT=8ml/kg, PEEP=2cmH2O) and non mechanical ventilation group (control group, breath spontaneously after tracheal canula insertion). The ventilator parameter was RR= 40 beats per minute, I:E=1:2, FiO2=0.21 and the ventilation lasted for 2 hours. The animals in ventilation groups were sacrificed after canula insertion(TO), at the end of ventilation(T2),2h and 4h after mechanical ventilation (T4 and T6 respectively). And the animals in control group were sacrificed after canula insertion (T0),2h,4h and 6h after insertion (T2, T4 and T6 respectively). Three animals were sacrificed in one time point in each group. The gas exchange, wet to dry lung ratio, PMN counts in BALF and lung pathologic change in different group were observed and compared.
Results 1) The pH of animals in different groups were decreased in the whole trial. A significantly statistical difference was found among different time points (F=9.029, P=0.001), but no significantly statistical difference were found within groups (F=1.347, P=0.329). The PaO2 and oxygenation index (01) of arterial blood were significantly decreased at the T2 as compared with those at TO (both P=0.016), after that the PaO2 and 01 keep in the similar level with those in T2 in VT20 group (T2 vs. T4, P=0.775; T4 vs. T6, P=0.338), while the PaO2/OI changed within group with no significantly difference either in control and VT8 group (control group:F= 0.603, P=0.531; VT8:group, F=2.901, P=0.206).No significantly difference was found in PaCO2 either within group or among groups during the trial (different time point among groups:F=3.503,P= 0.082; within group:F=0.874, P=0.464). The concentration of HCO3 was significantly decreased at T4 as compared to that at TO or at T2 (both P=0.022), and after that it remains in the same level at T4 (T6 vs.T4, P=0.497); however, the concentration of HCO3 remains in the same level during the whole trial in control group and VT8 group (Control group:F=4.200, P=0.064; VT8 group:F=4.659, P=0.149).2) The wet to dry lung weight ratios of animals in control group and VT8 group change with no significantly difference within group during the trial (control group:F=0.336,P=0.658; VT8 group:F=2.017, P=0.272) while it increased regularly after mechanical ventilation, but there was a significance difference until T6 as compared with TO within group. (P=0.019).3) No detectable pathological alteration was observed in control group and VT8 group. Lungs from VT20 group showed swelling, bloody under pleural, decreasing elasticity in the upper lobe. And edema and PMNs infiltrated in interstitial, bloody edema and PMNs appeared in the alveoli, alveolar consolidation and blooding occurred in the lung. The PMNs in the BALF significantly increased after ventilation in animals from VT20 group (F=93.782, P=0.000).
Conclusions The gas oxygenation deterioration, pathologic change of typical lung injury (included swelled interstitial, alveolar bloody effusion and neutrophils infiltration) occurred in animals underwent 2h' mechanical ventilation with a tidal volume equals to 20 ml/kg. It indicates that VILI rat model could be established with high tidal volume mechanical ventilation.
Chapter 3 Effects of MSC administration on lung injury and inflammatory response in VILI model
Objective To determine the effects of MSCs systemic administration on lung injury, local and systemic inflammatory response in an in vivo VILI model.
Methods 30 SD rats weighting 250g to 260g were anesthetized with pentobarbital sodium (40mg/kg body weight) intra-peritoneal. A tracheotomy was performed, and a canula was inserted into the trachea and a catheter was inserted into the right carotid artery. The animals were subgroup into 5 groups after canula insertion:normal control group (control group), MSC administration group (MSC group), high tidal volume mechanical ventilation group (VT20 group), high tidal volume mechanical ventilation plus MSCs pretreated group (MSC+VT20 group, MSCs were administrated just before mechanical ventilation) and high tidal volume mechanical ventilation plus MSCs post-treated group (VT20+MSC group, MSCs were administrated just after mechanical ventilation). N equals to 6 in each group. The parameters of mechanical ventilation were:VT=20 ml/kg body weight, PEEP=2 cmH2O, RR=40 bmp, I:E=1:2, FiO2=0.21. And the mechanical ventilation lasted for 2 hours and then the animals were breathed spontaneously for 4 hours. After that the animals were sacrificed. The animals in non mechanical ventilation groups were sacrificed 6h after catheter insertion. The numbers of MSC infused was 3×106 per animals with 0.5 ml normal saline, and the same volume of normal saline was infused into animals 2h after catheter insertion in groups without MSCs infusion. The blood gas, pathologic change of lung tissue and its injury score (scored with smith criteria), PMN lung infiltrated, PMN counts in the BALF and pro-inflammatory mediators (include TNF-a, IL-6 and MIP-2) and anti-inflammatory mediator (IL-10) in BALF and circulation in different groups were observed and compared.
Results 1)The PaO2 were decreased after mechanical ventilation as compared with that at TO in groups with mechanical ventilation (included VT20 group, MSC+VT20 group and VT20+MSC group, P equals to 0.001,0.359 and 0.147 respectively). The PaO2 were significantly higher in MSC+VT20 group as compared to VT20 group after mechanical ventilation at the same time point, especially at the timepoint T2 (P equals to 0.035,0.050 and 0.053 respectively at T0, T2 and T4).2) No detectable pathological alteration was observed as MSC administrated to normal animals in MSC group. Lungs from VT20 group showed swelling, edema and neutrophils infiltrated in interstitial, bloody edema and neutrophils appeared in the alveoli, alveolar consolidation and blooding occurred in part of the lung. Lightly interstitial thicken and small amount of PMN lung infiltrate were found in lung tissue form animals from MSC+VT20 group. However, partially interstitial was thickend in VT20+MSC group companying with decreasing PMN lung infiltration as compared to VT20 group. There was a significantly difference in injury score among groups (F =68.131, P=0.000). And there was significantly difference either the PMN amounts lung infiltrated or the PMN amounts in BALF among groups (PMN amounts lung infiltrated:F= 43.039, P=0.000;PMN amounts in BALF:F=134.171, P=0.000). 3) There was a significantly difference in the level of TNF-a, or IL-6, or MIP-2, or IL-10 in BALF among groups(TNF-a:F=69.706, P=0.000; IL-6:F=155.816, P=0.000; MIP-2:F=120.529, P=0.000; IL-10:F=42.154,P=0.000). Mechanical ventilation caused increased in the level of TNF-a,IL-6,MIP-2 and IL-10 in BALF significantly (VT20 group vs. control group, all P=0.000).The levels of TNF-a,IL-6 and MIP-2 in MSC+VT20 group were significantly lower than those in VT20 group (P equaled to 0.000,0.025 and 0.009 respectively) while the levels of TNF-αand MIP-2 in VT20+MSC group were significantly lower than those in VT20 group (P equaled to 0.031 and 0.012 respectively)as the level of IL-6 in VT20+MSC group was similar to VT20 group as no significantly difference were found between the two groups (P equaled to 0.995). However, no significantly difference was found either between MSC+VT20 group and VT20 group or between VT20+MSC group in the level of IL-10 (P equaled to 0.117 and 0.556 respectively).4) The levels of serum TNF-alpha, IL-6, MIP-2 and IL-10 were significantly increased after mechanical ventilation (included T2、T4、T6)as compared to that at TO (all P=0.000). The levels of serum TNF-a, IL-6 and MIP-2 in MSC+VT20 group at T4 and at T6 were lower than those in VT20 group at the same time point (TNF-a, P equaled to 0.000 and 0.003 respectively; IL-6, P equaled to 0.003 and 0.011 respectively; MIP-2, P equaled to 0.997 and 0.000 respectively). The level of serum TNF-a and MIP-2 in VT20+MSC group at T6 was significantly lower than those in VT20 group (P equaled to 0.039 and 0.033 respectively) while the level of serum IL-6 in VT20+MSC group was similar to that in the VT20 group as no significantly difference were found between VT20+MSC group and VT20 group (P equaled to 1.000,0.469 and 0.978 respectively). However, there was no significantly difference in the serum IL-10 level at the same time point either between MSC+VT20 group and VT20 group, or between VT20+MSC group and VT20 group after mechanical ventilation intervention (MSC+VT20 group vs. VT20 group, P equaled to 0.804,0.995 and 1.000 respectively, VT20+MSC group vs. VT20 group, P equaled to 0.557,1.000 and 0.966 respectively).
Conclusions MSCs infusion could attenuate the degree of lung tissue injury, oxygenic deterioration, PMN lung infiltration, and production of local and systemic pro-inflammatory mediators while it had no significant effect on the production of local and systemic anti-inflammatory mediator-IL-10. And the protective effect favors given earlier. Among them, the attenuation of lung injury was associated with decreasing PMN lung infiltrated. That indicates that MSC infusion could provide a homeostatic environment for those patients undergoing impropriated mechanical ventilation and it could in some extent prevent and lighten the subsequent PMN predominant inflammatory injury in VILI model.
Chapter 4 Effects of MSCs administration on PMN function in VILI rat model.
Objective To determine the effects of MSCs administration on PMN functions in the VILI SD rat model.
Methods The blood and BALF samples 4h after mechanical ventilation were collected from animals in control group, MSC group, mechanical ventilation with large tidal volume group (VT20 group), MSCs pretreatment + mechanical ventilation with large tidal volume group (MSC+VT20 group, the MSCs were systemic given just before ventilation), MSCs post-treatment + mechanical ventilation with large tidal volume group (VT20+MSC group, the MSCs were systemic given immediately after ventilation). The intercellular ROS production and surface CDllb expression were analyzed with flow cytometer. The neutrophil elastase (NE) activity of serum and BALF was measured. The PMNs were isolated from blood and BALF samples, and the apoptosis of PMNs was analyzed with flow cytometer. The fenton reaction was adopted to determined the extracellular ROS production of PMN. The data of CD11b expression, NE activity, intracellular and extracellular ROS production, and apoptotic rate of PMN in different groups were compared.
Results 1) There was no significantly difference among different groups about the surface molecular CD11b expression of PMN either in blood or in BALF (Blood:F =1.008, P= 0.442; BALF:F=0.328, P= 0.856).2) There was a significantly difference among different groups about the intracellular NE activity of PMN either in blood or in BALF (F=39.813 and 31.061 respectively, both P=0.000). The NE activity of PMN in VT20 group was significantly higher than that in control group either in blood or in BALF (all P=0.000). The NE activity of PMN in MSC+VT20 group was significantly lower than that in VT20 group in either blood or BALF (all P=0.000) while significantly difference was found between VT20 + MSC group and VT20 group either in blood or in BALF (blood, P=0.035; BALF, P=0.040). However, no significantly difference was found between VT20 + MSC group and MSC+VT20 group either in blood or in BALF (blood, P=0.927; BALF, P=1.000).3) There was a significantly difference among different groups about the intracellular ROS production of PMN either in blood or in BALF (F=986.470 and 118.612 respectively, both P=0.000). Mechanical ventilation with a large tidal volume increased the intracellular ROS production of PMN either in blood or in BALF (vs. control group, all P=0.000). The intracellular ROS production in groups with MSCs administration were significantly decreased as compared to that in VT20 group (all P=0.000, except P=0.001 as MSC+VT20 group vs. VT20 group in BALF). And there was significantly difference between MSC+VT20 group and VT20+MSC group in the intracellular ROS production of PMN from BALF (P=0.000).4) There was a significantly difference among different groups about the extracellular ROS production of PMN either in serum or in BALF (F=19.597 and 198.991 respectively,both P=0.000). The serum and BALF ROS production of PMN in VT20 group were significantly higher than that in control group either in blood or in BALF (all P=0.000). MSCs pretreatment (MSC+VT20 group) significantly decreased the ROS production either in serum or in BALF as compared to VT20 group (both P=0.000) while MSCs posttreatment decreased the ROS production in BALF as compared to VT20 group (P=0.000). And there was a significantly difference between MSC + VT20 group and VT20+MSC group in the serum ROS production or in the ROS production in BALF (P=0.001 and 0.021 respectively).5) There was a significantly difference among different groups about the apoptotic index of PMN either form blood or from BALF (F=41.869 and 89.661 respectively, both P=0.000). And MSC pretreatment significantly increased the apoptotic index of PMN from blood (P=0.001) while the ventilation mechanical ventilation significantly decreased the apoptotic index of PMN either blood-derived or BALF-derived (both P=0.000).
Conclusions The excessive activated PMN function existed in VILI rat model which caused by mechanical ventilation with a large tidal volume, which was illustrated with NE activity increasing, ROS production increasing and apoptosis decreased. MSCs administration decreased NE activity and ROS production in a certain extant, especially when MSCs was given before injured. Which indicates earlier MSCs administration are hope to limit the injury degree of ALI while the body in the risk of VILI via regulation the function of PMN, as MSCs posses the regulative ability of PMN function and that favors earlier given.
Chapter 5 Effects of systemic MSCs infusion on the prognosis and the fate observation of MSCs in the lung after infusion in VILI SD rats
Objective To determine whether MSCs systemic infusion could improve the outcome of VILI animal and identify whether the MSCs could immigrate into the injured lung, adopt the phenotype of lung cells, and replace the function of injured cells.
Methods 45 female rats weighting 250g to 260g were anesthetized and a canula was inserted into the trachea via tracheotomy. And then the animals were subgroup into 3 groups:high tidal volume mechanical ventilation group (VT20 group b), MSCs pretreated + high tidal volume mechanical ventilation group (MSC+VT20 group b, MSCs were treated before mechanical ventilation) and high tidal volume mechanical ventilation + MSCs post-treatment group (VT20+MSC group b, MSCs were given immediately after mechanical ventilation). N equaled to 15 each group. The ventilator parameters were:VT=20ml/kg body weight, PEEP=2cmH2O, RR=40 bmp, I:E= 1:2 and FiO2=0.21, and the ventilation lasted for 2 hours. The tracheotomy was closed and sutured after ventilation, and were sent back to their cages breathing room air. Long-term survival was observed every 24h within 14d after ventilation in these rats. The MSCs were male-SD rat-derived and its number given to the animal was 3x106/L per animal. The lungs were adopted from 6 animals from each group of MSC group, MSC+VT20 group, VT20+MSC group, MSC group b, MSC+VT20 group b and VT20+MSC group b after animals were scarified.The immunohistochemistry and PCR technique were used for fate observation of MSCs in the lung.
Results The survival rate of animals 48h after mechanical ventilation in MSC group b, MSC+VT20 group b and VT20+MSC group b was 73.33%,73.33% and 66.67% respectively, and there was no significantly difference among three groups (χ2=0.216, P=0.897). The survival rate of animals within 14d after mechanical ventilation in MSC group b, MSC+VT20 group b and VT20+MSC group b was 60.00%,46.67% and 46.67% respectively, and there was no significantly difference among three groups (χ2=0.720,P=0.698).The immonohistochemistry and PCR results showed that MSCs emerged in the lung shortly after infusion. MSCs distributed in pulmonary capillaries uniformly in MSC group, and it distributed locally in pulmonary capillaries and less in MSC+VT20 group as compared to MSC group(P =0.000). The MSCs distributed in the thicken pulmonary interstitial and even less in the VT20+MSC group as compared with MSC+VT20 group (P=0.000).However, there was no SRY-positive cells and no Sry sequence of Y chromosome was detected in the lung from animals from either MSC group b, or MSC+VT20 group b or VT20+MSC group b.
Conclusions MSCs emerged in the lung shortly after infusion in the VILI animal model, and the numbers of MSCs located in the lung was decreased as the lung injured. However, the MSCs did not transplant into the lung, adopt the phenotype of lung cells and replace the function of the injured lung cells. Although MSC pretreatment improved the 2w survival rate from 40% (6/15) to 53.33% (8/15) in VILI model, the difference did not reach statistically significance as the sample we observed was relatively small (just 15 each group) in the current study. Hence, a trial with large sample was needed to ascertain the benefit effect on the 2w survival rate of MSC treatment in the current VILI model.
Summary
The MSCs from SD rats could be cultured with combining Percoll density gradient centrifugation with adherent method. And the MSCs we cultured with characteristics of active proliferation, mostly survival and high purify active property of differentiation and stable morphology. And the VILI model could be established with mechanical ventilation with a large tidal volume (i.e.20 ml/kg body weight) lasted for a short time (2h) in SD rats. The model was with a main characteristics of PMN lung infiltrated predominant, accompany with local and systemic inflammatory response related to PMN activation. MSCs systemic infusion could attenuate the subsequent PMN predominant infiltration, subsequent acute inflammatory response includes PMN activation and lung injury, the protective effects favors given earlier, especially preventive given. Although the preventive effects of PMN infusion could not be resulted from transplanting into the lung, adopting the phenotype of lung cells and replacing the function of injured lung cells, which may be explained by modulating the inflammatory response (includes the PMN activity). The current study indicates that MSCs are hoped to prevent and cure the VILI by modulating the activated PMN-predominant inflammatory response, hence providing a related homeostasis environment for patients at risk of VILI and enhancing the patients' resistance to impropriate mechanical ventilation.
SARS、甲型H1N1流感病毒等引起的重症肺部感染可导致急性肺损伤(Acute lung injury, ALI)/急性呼吸窘迫综合征(Acute respiratory distress syndrome, ARDS)。由于ALI/ARDS病情危重、预后差,其救治已成为目前全世界的重点话题。目前对于ALI/ARDS在内的急、慢性呼吸衰竭患者的有效救治手段主要有机械通气、ECMO等。其中机械通气作为最主要的手段之一,其使用不当即可导致呼吸机所致的肺损伤(Ventilator-induced lung injury, VILI)。
虽然目前对于VILI的发生机制不完全清楚,但在VILI模型上可发现其病理上以弥漫肺泡损伤、肺泡—毛细血管渗透性增加并继发以中性粒细胞(Neutrophil, polymorphonuclear neutrophil, PMN)肺部浸润显著增加并伴随以BALF及全身循环中PMN升高为主的炎症反应等特征。既往研究显示PMN的过度激活可加重肺损伤,在VILI模型中PMN的过度激活与肺损伤程度密切相关,而针对PMN激活及其功能的各种干预手段[如中性粒细胞弹性蛋白酶(neutrophil elastase, NE)阻滞剂、PMN趋化因子MIP-2受体敲除等]可显著降低肺损伤的严重程度,故对PMN激活及其功能的干预有望成为防治VILI的有效手段。
骨髓间充质干细胞(Bone marrow-derived mesenchymal stem cells, MSCs)是一种多能分化的干细胞,其在特定环境下可诱导分化为多种细胞(如软骨细胞、肌肉细胞、内皮细胞、上皮细胞、神经细胞等)。在一些严重烧伤及损伤情况下使用MSCs时,MSCs可迅速趋向于损伤部位、获得一些损伤部位细胞的表型而发挥作用,且在肺损伤的研究中也显示其可获得肺部细胞表型、起到促进损伤修复等重要作用。此外,在脓毒症肺损伤的动物研究中显示MSCs使用可改善动物的预后,然而MSCs在VILI中使用是否也有类似作用目前仍缺乏相关报道。
MSCs具有抑制免疫活性的功能,这可能是其促进损伤动物组织损伤修复的一个重要原因。其中MSCs与循环中的PMN、巨噬细胞、淋巴细胞、DC细胞及自然杀细胞等的相互作用在MSCs发挥其损伤修复作用时具有重要意义。新近研究表面MSCs-PMN的体外细胞实验、动物实验结果均提示MSCs可抑制PMN的激活。然而,MSCs在VILI模型中是否也能抑制PMN的激活目前仍未知。
本研究假设MSC的输注可以通过调节PMN功能的激活在内的炎症反应和(或)通过MSC移植入肺并获得肺部细胞表型、重建损伤肺的结构及功能的途径进而减轻甚至预防大潮气量机械通气导致的肺损伤,对VILI模型起到保护作用。为此,本研究采用大潮气量机械通气来制备VILI模型,并在该模型中防治性使用MSCs以评价MSCs在VILI的作用。
第一章大鼠骨髓间充质干细胞的体外分离、培养及鉴定
目的体外分离、培养雄性SD大鼠骨髓间充质干细胞(MSCs),并研究其生物学特性、表型特征及分化能力。
方法在无菌条件下取出雄性SD大鼠股骨、胫骨,用冰L-DMEM培养基冲洗骨髓腔,离心取有核细胞,采用密度梯度离心法结合贴壁法分离、纯化大鼠MSCs,用含10%胎牛血清的L-DMEM培养基培养MSCs,去除非贴壁细胞。待细胞铺满瓶底面积约90%时,按1：2进行传代培养。用流式细胞仪检测细胞周期、凋亡率(Annexin-V/PI法),以明确体外培养目标细胞的生长特征；用MTT法通过描述细胞生长曲线以评价目标细胞的增殖活性；用流式细胞仪(FCM)检测培养的目标细胞表面CD分子CD34、CD45、CD29、CD44的表达,以鉴定目标细胞的纯度；用软骨细胞基础培养基加转化生长因子—β诱导目标细胞分化为软骨细胞,用免疫细胞化学法观察诱导的细胞表达Ⅱ型胶原的情况,以明确目标细胞的定向分化能力。
结果从骨髓分离到的目标有核细胞大部分呈圆形,接种于培养基后72 h大部分细胞贴培养瓶壁、呈集落性生长,细胞形状变为梭形。培养10d-15d后,集落增多大部分融合成片并达到传代标准。细胞传代之后,细胞生长速度增快,约1周即可进行再次传代。在细胞传3代后,细胞形态仍为梭形、且形态较为均一。P3细胞中大部分细胞处于合成前期(G1期),其比例>80.0%；其次为处于DNA复制期(S期,约10.0%-12.0%)及DNA合成后期(G2期,约占6.0%-7.5%)的细胞,而静止期(G0期)及有丝分裂期(M期)的细胞占比例极少(G0+M期,约占0.0%-0.4%)；Annexin V/PI法检测目标细胞凋亡率均<5.0%。细胞在传代后第3d进入指数生长期,到第5d达到高峰；之后进入生长平台期。培养P3细胞表面CD29阳性表达率均大于99.50%、CD44阳性表达率均大于99.90%、CD34阳性表达率小于2.50%、CD45阳性表达率小于2.00%。在软骨细胞基质培养基内培养14d后,细胞外形变为多角形,胞浆中出现Ⅱ型胶原表达的占50%。
结论本研究采用密度梯度离心法结合贴壁法可在体外分离、培养出SD大鼠来源的MSCs。用该方法培养出的MSC大部分处于增殖期,具有增殖活跃、存活率高、纯度高的特点,且其具有较高的分化能力。由于其生物学性状、表型稳定、分化能力较好,可用于进一步的动物实验研究。
第二章大潮气量机械通气所致肺损伤大鼠模型的制备
目的用大潮气量机械通气制备呼吸机所致肺损伤的SD大鼠模型。
方法将250～260g体重的成年雌性SD大鼠腹腔内注射苯巴比妥钠麻醉后行气管切开插入气管导管,右颈内动脉留置头皮针。插管完毕后将动物分为三组：大潮气量机械通气组(VT20组,VT=20 ml/kg体重)、小潮气量机械通气组(VT8组,VT=8 ml/kg体重)、非机械通气对照组(不行机械通气,在插气管导管后大鼠自主呼吸)。各组n=12,以上机械通气组机械通气设置为：PEEP=2 cm H2O、RR=40次/分,吸呼比(I：E)均为1：2,吸入的气体均为空气,即吸入氧浓度(FiO2)均为21%。机械通气时间均为2h。机械通气组在导管置入完毕时(T0)、机械通气结束时(T2)、机械通气结束后2h时(T4)、机械通气结束后4h时(T6)等4个时间点分批取血标本后放血处死动物,而非机械通气对照组则在插管完毕时(T0)、插管完毕后2h(T2)、4h(T4)、6h(T6)分批取血标本后放血处死动物。各组各时点均取3只动物。观察并比较各组各时点动脉血气、肺干湿比、肺泡灌洗液内PMN数量、肺组织病理。
结果1)各组大鼠在整个实验过程中pH值进行性下降,各时间点间差异有统计学意义(F=9.029,P=0.001),但组间差异无统计学差异(F=1.347,P=0.329)。VT20组在T2时动脉血PaO2/氧合指数(OI)较TO时显著下降(均P=0.016),但机械通气后组内各时间点前后动脉血PaO2/OI差异均无统计学意义(T2比T4,P=0.775;T4比T6,P=0.338)；其它各组组内PaO2/OI差异也均无统计学意义(对照组F=0.603,P=0.531；VT8组F=2.901,P=0.206)。动脉血PaCO2的变化上,分组、时间因素均无显著影响(时间因素：F=3.503,P=0.082；分组因素：F=0.874,P=0.464)。VT20组在T4时动脉血[HCO3-]浓度较T0、T2均下降,差异有统计学意义(均P=0.022)；之后维持在T4水平(T6比T4,P=0.497)；而对照组及VT8组在实验过程中[HCO3-]浓度组内差异均无统计学意义(对照组F=4.200,P=0.064；VT8组F=4.659,P=0.149)。2)对照组及VT8组动物肺湿干比组内各时点间差异无统计学意义(对照组F=0.336,P=0.658；VT8组F=2.017,P=0.272)；而VT20组动物肺湿干比在机械通气后随时间增加而增加,T6时与T0时比较差异有统计学意义(P=0.019)。3)对照组及VT8组动物肺组织病理随时间变化不大,但VT20组出现双上肺明显肿胀、包膜下血性渗出,弹性较前明显降低,肺间质水肿、PMN浸润,肺泡腔内出现大量血性液体渗出、且肺泡腔内出现以PMN为主的细胞渗出,部分肺组织实变及肺泡出血等损伤改变。且VT20组动物肺泡灌洗液内PMN数量也随着时间推移而增加,组内各时点差异有统计学意义(F=93.782,P=0.000)。
结论用20ml/kg体重的潮气量对SD大鼠进行为期2h的机械通气可导致大鼠氧合障碍、肺部出现间质水肿、肺泡渗出及PMN浸润为主等急性肺损伤的病理改变,可见本研究采用20ml/kg潮气量对大鼠行机械通气2h可成功制备VILI大鼠模型。该研究可为下一步研究提供VILI动物模型。
第三章MSC给予对于VILI模型肺损伤及炎症反应的影响
目的明确MSC对VILI模型肺组织损伤及局部、全身炎症反应的影响。
方法将30只体重为250～260g的成年雌性SD大鼠麻醉后气管切开插管,右颈内动脉留置头皮针。插管完毕后将动物分为5组：正常对照组(control组)、MSC给予组(MSC组)、大潮气量机械通气组(VT20组)、MSCs预给予+大潮气量机械通气组(MSC+VT20组,在大潮气量机械通气前给予MSCs)、大潮气量机械通气+MSCs后给予组(VT20+MSC组,在大潮气量机械通气结束后立即给予MSCs)。各组n=6,机械通气组机械通气参数为：VT=20 ml/kg体重,PEEP=2 cm H2O, RR=40次/分,吸呼比(I：E)均为1：2,FiO2=21%。机械通气时间均为2h,机械通气结束后给予去除机械通气自主呼吸4h,之后处死动物。非机械通气组在插管完毕后6h处死动物。MSCs给予组均静脉给予每只大鼠数量为3×106/L的MSCs,而未给予MSC组在插管2h后给予静脉注射等体积生理盐水(0.5ml)。动态观察各组大鼠血气变化、肺组织病理、对肺组织损伤进行评分(Smith评分)、肺组织内PMN浸润数量、BALF中PMN数量、BALF及循环中促炎因子(TNF-α、IL-6、MIP-2)及抗炎因子(IL-10)的变化情况,并将以上指标进行组间比较。
结果1)机械通气各组(包括VT20组、MSC+VT20组及VT20+MSC组)在机械通气后动脉血PaO2较T0时下降,组内差异只有VT20组有统计学意义(P分别为0.001、0.359、0.147)；机械通气后MSC+VT20组各时点Pa02均较VT20组增高,但只有T2时差异才有统计学意义(T2、T4及T6,P分别为0.035、0.050、0.053)。2)给予MSC对正常大鼠肺部病理组织无显著影响,而VT20可导致大鼠肺部出现肺间质水肿、PMN浸润,肺泡腔内出现大量的血性液体渗出、且肺泡腔内出现以PMN为主的细胞渗出,部分肺组织实变及肺泡出血等损伤改变。而MSC+VT20组肺组织病理仅见部分肺间质稍增厚及较少量PMN浸润,而VT20+MSC组部分肺间质进一步增厚、PMN浸润明显减少等改变。肺损伤评分组间差异有统计学意义(F=68.131,P=0.000)。而肺组织病理高倍视野下PMN数量、BALF中PMN计数量组间差异同样也有统计学意义(肺组织：F=43.039,P=0.000：BALF:F=134.171,P=0.000)。3)各组动物BALF中TNF-α、IL-6、MIP-2及IL-10的浓度组间差异有统计学意义(TNF-α:F=69.706, P=0.000; IL-6:F=155.816,P=0.000；MIP-2:F=120.529,P=0.000；IL-10：F=42.154,P=0.000),其中VT20组BALF中TNF-α、IL-6、MIP-2及IL-10的浓度均较对照组高,且组间差异有统计学意义(均P=0.000);MSC+VT20组BALF中TNF-α、H-6、MIP-2浓度较VT20组低,组间差异有统计学意义(P分别为0.000、0.025、0.009);而VT20+MSC组与VT20组比较时,BALF的TNF-α、MIP-2水平组间差异有统计学意义(P分别为0.031、0.012),但组间BALF的IL-6差异无统计学意义(P=0.995)；此外,MSC+VT20组、VT20+MSC组的IL-10水平分别与VT20组IL-10比较时,组间差异也无统计学意义(P分别为0.117、0.556)。4)机械通气损伤后(包括T2、T4、T6)大鼠血浆TNF-α、IL-6、MIP-2及IL-10的浓度均较T0时升高,且各指标组内差异均有统计学意义(均P=0.000),而MSC+VT20组机械通气后(T4、T6)血浆TNF-α、IL-6、MIP-2的浓度较VT20组的低(TNF-α：P分别为0.000、0.003；IL-6：P分别为0.003、0.011；MIP-2：P分别为0.997、0.002)；VT20+MSC组机械通气后与VT20组比较时,T6时组间血浆TNF-α、MIP-2的浓度差异有统计学意义(P分别为0.039、0.033),而IL-6浓度组间差异无统计学意义(P分别为1.000、0.469、0.978)。而MSC+VT20组、VT20+MSC组在机械通气后血浆IL-10水平与同一时点的VT20组血浆工L-10水平比较,组间差异均无统计学意义(P分别为0.804、0.995、1.000,0.557、1.000、0.966)。
结论MSCs给予可在不同程度上减轻大潮气量机械通气所致的肺部组织损伤、氧合恶化、肺部PMN浸润、局部及全身与PMN有关的炎症介质的释放量,且MSCs给予时间越早,这些作用就越明显；但MSCs给予但对VILI模型抗炎介质IL-10的释放量无显著影响。MSCs使用在降低肺损伤程度的同时也伴随着肺部浸润PMN数量的降低。这提示MSCs输注可为VILI模型提供一个相对稳定的内环境,这有助于提高动物对不当机械通气的耐受程度及防治VILI后继的以PMN为主的炎症及其所致的肺部进一步损伤。
第四章MSC处理对VILI大鼠PMN功能的影响
目的明确MSC对VILI模型SD大鼠PMN功能的影响。
方法将对照组(control组)、MSC给予组(MSC组)、大潮气量机械通气组(VT20组)、MSCs预给予+大潮气量机械通气组(MSC+VT20组,在大潮气量机械通气前给予MSCs)、大潮气量机械通气+MSCs后给予组(VT20+MSC组,在大潮气量机械通气结束后立即给予MSCs)大鼠在机械通气后4h后取的血标本及处死后取BALF的标本,用流式细胞仪检测其中PMN的细胞内ROS含量及表面分子CDllb的表达；分离血标本、BALF标本中的PMN,用流式细胞术进行凋亡率检测；利用化学发光法进行血浆、BALF中NE活性定量检测；用Fenton反应检测血浆及BALF中的ROS (·OH含量),以确定细胞外ROS含量；并将NE活性、细胞表面CD11b表大量、细胞内ROS含量、细胞外ROS含量、PMN细胞凋亡率资料进行组间比较。
结果1)血及BALF中PMN细胞表面CDllb表达量组间差异均无统计学意义(血：F=1.008,P=0.442;BALF:F=0.328,P=0.856)。2)血及BALF中NE活性各组间差异有统计学意义(F分别为39.813、31.061,均P=0.000)。其中VT20组血及BALF中NE活性均较对照组高,差异有统计学意义(均P=0.000)；MSC+VT20组血及BALF中NE活性均较VT20组降低,差异有统计学意义(均P=0.000)；且VT20+MSC组与VT20组组间血及BALF中的NE活性差异均有统计学意义(血：P=0.035,BALF:P=0.040)；但MSC+VT20组与VT20+MSC组组血浆及BALF的NE活性间差异无统计学意义(血：P=0.927,BALF:P=1.000)。3)血及BALF中PMN内ROS含量各组间差异均有统计学意义(F分别为986.470、118.612,均P=0.000)。VT20组血及BALF中PMN内ROS含量较对照组增高,且差异有统计学意义(均P=0.000);MSCs使用组(包括MSC+VT20组、VT20+MSC组)细胞内ROS产量均较VT20组低,与VT20组比较差异均有统计学意义(除BALF中MSC+VT20组与VT20组比较P=0.001外,其它均P=0.000)；且MSC+VT20组、VT20+MSC组比较时,组间BALF来源的PMN内ROS含量的差异也有统计学意义(P=0.000)。4)血浆及BALF中ROS含量各组间差异有统计学意义(F分别为19.597、198.991,均P=0.000)。其中VT20组血浆及BALF中ROS含量均较对照组低,组间差异均有统计学意义(均P=0.000)；MSC+VT20组血浆及BALF中的ROS含量较VT20组降低,组间差异均有统计学意义(均P=0.000)；而VT20+MSC组只有BALF中ROS含量较VT20组低,组间差异有统计学意义(P=0.001);且MSC+VT20组、VT20+MSC组比较时,血浆、BALF中ROS含量组间差异均有统计学意义(P分别为0.001、0.021)。5)血及BALF来源的PMN细胞凋亡率各组间差异有统计学意义(F分别为41.869、89.661,均P=0.000)。其中VT20组血及BALF来源的PMN凋亡率均较对照组降低,且组间差异有统计学意义(均P=0.000)；而MSC给予组中只有MSC+VT20组血中PMN的凋亡率较VT20组高,差异有统计学意义(P=0.001)。
结论大潮气量机械通气所致的肺损伤大鼠模型中存在PMN的过度激活,其主要表现在NE活性增高、细胞内外ROS产量增加、细胞凋亡减少及延迟等。而MSCs的给予在不同程度上降低NE活性及ROS的产生量,尤其是损伤前给予MSCs时PMN功能激活相关指标降低量更为明显。这表面MSCs有调节PMN功能的能力,且MSC给予时间越早则对PMN功能调节作用越明显。
第五章MSC静脉输注对于VILI模型预后的影响及MSCs输注后在肺部转化情况观察
目的明确MSCs静脉内注射能否改善VILI模型的预后与及其是否能迁移到VILI大鼠的肺部、替代损伤组织细胞、甚至获得肺部损伤细胞的表型及发挥相应功能。
方法取45只体重为250～260g的成年雌性SD大鼠麻醉、气管切开插管后随机分为大潮气量机械通气组b(VT20组b)、MSCs预给予+大潮气量机械通气组(MSC+VT20组b,在大潮气量机械通气前给予MSCs)、大潮气量机械通气+MSCs后给予组(VT20+MSC组b,在大潮气量机械通气结束后立即给予MSCs),各组n=15,机械通气组机械通气参数为：VT=20ml/kg体重,PEEP= 2 cm H20,RR=40次/分,吸呼比(I：E)为1：2,吸入气氧浓度(FiO2)为21%,机械通气时间为2h。机械通气结束后拔除呼吸机接头、缝合颈部切口后放置回实验笼中,让动物自主呼吸空气。在随后的14d内每24h观察并记录一次动物的存亡情况。取6只体重相近的成年雌性SD大鼠作MSC长期观察组(MSC组b)经尾静脉注射MSCs,之后放回养殖笼中继续养殖14d。MSCs均为雄性大鼠来源的,数量均为3×106/L每只大鼠。取MSC组、MSC+VT20组、VT20+MSC组、MSC组b、MSC+VT20组b及VT20+MSC组b动物各6只处死后取肺行免疫组织化学检测抗SRY抗体阳性的细胞、PCR检测肺组织中大鼠Y染色体上的Sry基因片段以明确静脉注射雄性大鼠来源的MSCs后是否能滞留在肺部、较长时期后肺部是否有雄性大鼠来源的细胞存在及其存在的部位。
结果MSC+VT20组b、VT20+MSC组b及VT20组b动物致伤后48h内生存率分别为73.33%、73.33%、66.67%,三组组间差异无统计学意义(χ2=0.216,P=0.897)；14d内生存率分别为53.33.00%、40.00%、40.00%,各组动物14d内死亡率差异也无统计学意义(χ2=0.720,P=0.698)。免疫组化、PCR结果提示MSCs输注后短期实验结束时各组动物均有MSCs滞留在肺部,其中在MSC组MSCs较均匀分布在肺毛细血管床内；而MSC+VT20组MSCs分布较MSC组较为局限,量较MSC组少,且差异有统计学意义(P=0.000)；而VT20+MSC组MSCs则分布在肺间质明显增厚的毛细血管床内,分布成片带状,量较MSC+VT20组要少,差异有统计学意义(P=0.000)。而在MSC输注后14d后MSC组b、MSC+VT20组b及VT20+MSC组b三组动物的肺组织中均未检出抗Y染色体特异性结合蛋白抗体阳性细胞及Y染色体Sry片段。
结论MSCs静脉内输注短期内可出现在VILI动物肺部,而组织损伤可减少MSCs在肺部的滞留数量,而MSCs并未移植到肺部、也未分化为肺部细胞并取代损伤细胞的功能。MSC细胞静脉内输注虽可将VILI大鼠14d生存率从40%提高到53.33%,但由于例数较少,仍需进一步扩大样本方可确认MSCs干预能否改善VILI模型14d内生存率。
全文总结
利用密度梯度离心法联合离心法可分离、体外培养出增殖能力强、纯度高、分化能力强、性状稳定的MSCs.而大潮气量短期机械通气可制备VILI模型,该模型病理上以PMN肺部浸润增加为主要特征,同时伴随与PMN相关的局部及全身炎症反应。在VILI大鼠模型中进行静脉内输注MSCs可减轻VILI大鼠继发的以PMN肺部浸润及PMN功能激活在内的炎症反应及肺损伤,而使用时机越早其保护作用越明显。而MSC在VILI动物模型中的保护作用并非通过MSCs移植入肺、获得肺部细胞表型、重建损伤肺细胞的结构和功能,而是通过调节PMN的功能激活在内的炎症反应。这提示临床有望使用MSCs来调节PMN功能激活为主要特征的炎症反应模式为有发生VILI风险的机械通气者提供稳定的内环境及提高其对不当机械通气的耐受能力,以达到防治VILI的目的。
Background
The acute lung injury (ALI) / acute respiratory distress syndrome (ARDS) induced by severe respiratory infection (such as SARS, severe H1N1 flu infection) is known to the whole world as its severity and poor outcome. Until recently, mechanical ventilation and ECMO and etc. were used as effective salvage method in the patients with acute or chronic respiratory failure (ALI/ARDS). Mechanical ventilation, a crucial supported and therapeutic technique for surviving patients with respiratory failure (include ALI/ARDS) could induced lung injury, which was known as ventilator-induced lung injury (VILI).
Subsequent neutrophil (PMN)-predominant inflammatory response as well as diffuse alveolar damage and pulmonary and systemic vascular leak was the key features of VILI, which had been observed in different animal models of VILI, although the mechanisms of VILI with different impropriate ventilation parameters are not fully understood. The excess activation of PMNs aggravates the severity of injury. Therapeutic methods referred to modulate the activation of PMNs, such as specific inhibitor of neutrophil elastase (NE) (Sivelestat) administration, MIP-2 (one of neutrophil chemotactic factors) receptor knockout were seemed to attenuate the severity of VILI. Hence, the management of PMN activation could be an important therapeutic approach to VILI.
Bone marrow-derived mesenchymal stem cells (MSC), a type of stem cells, could differentiate into several cell lineages, such as chondrocytes, myoblasts, endothelial cells, epithelial cells, and neurocytes, under certain circumstances. It had been used to heal severe wounded or damage tissue as it preferentially localize to the injured sites as well as adopting the phenotype of some kinds of cells in the local tissue after administration. Several studies showed that MSCs can immigrate into the lung and adopt the phenotype of lung cells and play positive roles in lung injury. Also it was reported that intrapulmonary delivery of MSC could improves mice survival model of endotoxin-induced acute lung injury. However, whether the MSC could immigrate into the injured lung and adopted the phenotype of lung cells, and improve the outcome of animals in VILI model is still unknown.
The active immunosuppressive ability of MSC, which partially attributed to the repair function of MSC in injured models, has been well documented. The interaction of MSCs with PMNs, besides macrophages, lymphocytes, dendritic cells and natural killer cells, was especially important in certain types of injury. Recently MSCs-PMNs coculture tests and animal trials showed that MSC could suppress the PMNs activation. However, whether MSC infusion could suppress the excess activation of PMN and subsequent inflammation induced by high tidal volume mechanical ventilation is still unknown.
The aims of this study were to test the hypothesis that infusion of MSC may be beneficial for VILI which is linked these effects to the regulation of inflammatory response which includes PMNs'activation, or linked these effects to the reconstruction of structure and function by MSCs transplantation; we used the in vivo high tidal volume ventilation induced lung injury model to evaluate the effect of infusive MSC.
Chapter 1 Isolation, expansion and identification of rat MSCs
Objective To separate and culture the rat bone marrow derived mesenchymal stem cells in vitro, and to explore the biological characteristics, phenotype and differentiation of the cultured cells.
Methods The femurs and tibia were removed from specific pathogen-free (SPF) male Sprague-Dawley after decapitated sacrifice. The whole marrow was flushed from the tibia and femur of rats with ice-cold L-Dulbecco's Modified Eagle Medium (L-DMEM), and the nucleated cells were adopted for isolation. And the isolation and purification of rat MSCs were finished with Percoll density gradient centrifugation combined with adherent method. The isolated MSCs were cultured with L-DMEM cultured media containing 10% fetal bovine serum and the non-adhesive cells were removed. At confluence, the cells were harvested for passage at the ratio of 1:2. In order to determine the target-cellular growth characteristics, the flow cytometer was used to determine cellular cell cycle and apoptotic ratio. And in order to determine the cellular proliferating ability of the target-cell, the cell growth curved was conducted using MTT kids. In order to determine the purification of the target cells, the surface markers of CD34, CD45, CD29 and CD44 were evaluated by flow cytometer. In order to determine the differentiation ability of cultured cells, the cultured MSCs were cultured in basic chondrocyte media with transformed growth factor-beta, and the collagen type II expression were determined by immunocytochemistry.
Results The primary isolated nucleated cells were mostly round and it converted to adherent, colony growth and fusiform-shaped 72 hours after innoculation. The cells were passaged after 10～15 days while mostly colonies melted into one. The cell growth faster after passaged and they could be repassaged about 1 w after inoculation. After the third passage, morphology of mostly cells was uniform and remains the fusiform-shaped. And more than 80% cells were at the G1 stage of the third passage of cultured MSCs, about 6.0% to 7.5% were at G2 stage and about 0 to 0.4% were at stage of GO and M. The apoptotic ratio of target cells were less than 5.0% which measured used Annexin-V measurement. The cells went into logarithmic growth phage from third days after passaged, it reached peak growth phage at the fifth days and later went into late stationary phage. The MSCs we cultured contained a small number of cells expressing CD45 (< 2.0%) and CD34 (<2.5%), and mostly cells expressing CD29 (>99.5%) and CD44 (>99.9%). And the basic chondrocyte media induced the transformation of mesenchymal cells into chondrocytes as the type II collagen could be detected in 50% of the transformed cells after 14 days of culture.
Conclusions SD rats-derived MSCs can be obtained by Percoll' density gradient centrifugation combined with adherent method. The mostly MSCs we cultured were at proliferating phage. And the MSCs we cultured with a characteristics of active proliferation, mostly survival and high purify, and active property of differentiation. As the MSCs we cultured with stable characteristics of biology, phenotypes, it can be used in the further animal research.
Chapter 2 Establishment of ventilator-induced lung injury model with high tidal volume ventilation in SD rats
Objective To establish ventilator-induced lung injury model induced by high tidal volume ventilation in SD rats.
Methods The rats weighting 250g to 260g were anesthetized with pentobarbital sodium (40mg/kg body weight) intra-peritoneal. A tracheotomy was performed, and a canula was inserted into the trachea. The animals were subgroup into 3 groups after canula insertion:high tidal volume mechanical ventilation group (VT20 group, VT= 20 ml/kg, PEEP=2 cmH2O), low tidal volume mechanical ventilation group (VT8 group, VT=8ml/kg, PEEP=2cmH2O) and non mechanical ventilation group (control group, breath spontaneously after tracheal canula insertion). The ventilator parameter was RR= 40 beats per minute, I:E=1:2, FiO2=0.21 and the ventilation lasted for 2 hours. The animals in ventilation groups were sacrificed after canula insertion(TO), at the end of ventilation(T2),2h and 4h after mechanical ventilation (T4 and T6 respectively). And the animals in control group were sacrificed after canula insertion (T0),2h,4h and 6h after insertion (T2, T4 and T6 respectively). Three animals were sacrificed in one time point in each group. The gas exchange, wet to dry lung ratio, PMN counts in BALF and lung pathologic change in different group were observed and compared.
Results 1) The pH of animals in different groups were decreased in the whole trial. A significantly statistical difference was found among different time points (F=9.029, P=0.001), but no significantly statistical difference were found within groups (F=1.347, P=0.329). The PaO2 and oxygenation index (01) of arterial blood were significantly decreased at the T2 as compared with those at TO (both P=0.016), after that the PaO2 and 01 keep in the similar level with those in T2 in VT20 group (T2 vs. T4, P=0.775; T4 vs. T6, P=0.338), while the PaO2/OI changed within group with no significantly difference either in control and VT8 group (control group:F= 0.603, P=0.531; VT8:group, F=2.901, P=0.206).No significantly difference was found in PaCO2 either within group or among groups during the trial (different time point among groups:F=3.503,P= 0.082; within group:F=0.874, P=0.464). The concentration of HCO3 was significantly decreased at T4 as compared to that at TO or at T2 (both P=0.022), and after that it remains in the same level at T4 (T6 vs.T4, P=0.497); however, the concentration of HCO3 remains in the same level during the whole trial in control group and VT8 group (Control group:F=4.200, P=0.064; VT8 group:F=4.659, P=0.149).2) The wet to dry lung weight ratios of animals in control group and VT8 group change with no significantly difference within group during the trial (control group:F=0.336,P=0.658; VT8 group:F=2.017, P=0.272) while it increased regularly after mechanical ventilation, but there was a significance difference until T6 as compared with TO within group. (P=0.019).3) No detectable pathological alteration was observed in control group and VT8 group. Lungs from VT20 group showed swelling, bloody under pleural, decreasing elasticity in the upper lobe. And edema and PMNs infiltrated in interstitial, bloody edema and PMNs appeared in the alveoli, alveolar consolidation and blooding occurred in the lung. The PMNs in the BALF significantly increased after ventilation in animals from VT20 group (F=93.782, P=0.000).
Conclusions The gas oxygenation deterioration, pathologic change of typical lung injury (included swelled interstitial, alveolar bloody effusion and neutrophils infiltration) occurred in animals underwent 2h' mechanical ventilation with a tidal volume equals to 20 ml/kg. It indicates that VILI rat model could be established with high tidal volume mechanical ventilation.
Chapter 3 Effects of MSC administration on lung injury and inflammatory response in VILI model
Objective To determine the effects of MSCs systemic administration on lung injury, local and systemic inflammatory response in an in vivo VILI model.
Methods 30 SD rats weighting 250g to 260g were anesthetized with pentobarbital sodium (40mg/kg body weight) intra-peritoneal. A tracheotomy was performed, and a canula was inserted into the trachea and a catheter was inserted into the right carotid artery. The animals were subgroup into 5 groups after canula insertion:normal control group (control group), MSC administration group (MSC group), high tidal volume mechanical ventilation group (VT20 group), high tidal volume mechanical ventilation plus MSCs pretreated group (MSC+VT20 group, MSCs were administrated just before mechanical ventilation) and high tidal volume mechanical ventilation plus MSCs post-treated group (VT20+MSC group, MSCs were administrated just after mechanical ventilation). N equals to 6 in each group. The parameters of mechanical ventilation were:VT=20 ml/kg body weight, PEEP=2 cmH2O, RR=40 bmp, I:E=1:2, FiO2=0.21. And the mechanical ventilation lasted for 2 hours and then the animals were breathed spontaneously for 4 hours. After that the animals were sacrificed. The animals in non mechanical ventilation groups were sacrificed 6h after catheter insertion. The numbers of MSC infused was 3×106 per animals with 0.5 ml normal saline, and the same volume of normal saline was infused into animals 2h after catheter insertion in groups without MSCs infusion. The blood gas, pathologic change of lung tissue and its injury score (scored with smith criteria), PMN lung infiltrated, PMN counts in the BALF and pro-inflammatory mediators (include TNF-a, IL-6 and MIP-2) and anti-inflammatory mediator (IL-10) in BALF and circulation in different groups were observed and compared.
Results 1)The PaO2 were decreased after mechanical ventilation as compared with that at TO in groups with mechanical ventilation (included VT20 group, MSC+VT20 group and VT20+MSC group, P equals to 0.001,0.359 and 0.147 respectively). The PaO2 were significantly higher in MSC+VT20 group as compared to VT20 group after mechanical ventilation at the same time point, especially at the timepoint T2 (P equals to 0.035,0.050 and 0.053 respectively at T0, T2 and T4).2) No detectable pathological alteration was observed as MSC administrated to normal animals in MSC group. Lungs from VT20 group showed swelling, edema and neutrophils infiltrated in interstitial, bloody edema and neutrophils appeared in the alveoli, alveolar consolidation and blooding occurred in part of the lung. Lightly interstitial thicken and small amount of PMN lung infiltrate were found in lung tissue form animals from MSC+VT20 group. However, partially interstitial was thickend in VT20+MSC group companying with decreasing PMN lung infiltration as compared to VT20 group. There was a significantly difference in injury score among groups (F =68.131, P=0.000). And there was significantly difference either the PMN amounts lung infiltrated or the PMN amounts in BALF among groups (PMN amounts lung infiltrated:F= 43.039, P=0.000;PMN amounts in BALF:F=134.171, P=0.000). 3) There was a significantly difference in the level of TNF-a, or IL-6, or MIP-2, or IL-10 in BALF among groups(TNF-a:F=69.706, P=0.000; IL-6:F=155.816, P=0.000; MIP-2:F=120.529, P=0.000; IL-10:F=42.154,P=0.000). Mechanical ventilation caused increased in the level of TNF-a,IL-6,MIP-2 and IL-10 in BALF significantly (VT20 group vs. control group, all P=0.000).The levels of TNF-a,IL-6 and MIP-2 in MSC+VT20 group were significantly lower than those in VT20 group (P equaled to 0.000,0.025 and 0.009 respectively) while the levels of TNF-αand MIP-2 in VT20+MSC group were significantly lower than those in VT20 group (P equaled to 0.031 and 0.012 respectively)as the level of IL-6 in VT20+MSC group was similar to VT20 group as no significantly difference were found between the two groups (P equaled to 0.995). However, no significantly difference was found either between MSC+VT20 group and VT20 group or between VT20+MSC group in the level of IL-10 (P equaled to 0.117 and 0.556 respectively).4) The levels of serum TNF-alpha, IL-6, MIP-2 and IL-10 were significantly increased after mechanical ventilation (included T2、T4、T6)as compared to that at TO (all P=0.000). The levels of serum TNF-a, IL-6 and MIP-2 in MSC+VT20 group at T4 and at T6 were lower than those in VT20 group at the same time point (TNF-a, P equaled to 0.000 and 0.003 respectively; IL-6, P equaled to 0.003 and 0.011 respectively; MIP-2, P equaled to 0.997 and 0.000 respectively). The level of serum TNF-a and MIP-2 in VT20+MSC group at T6 was significantly lower than those in VT20 group (P equaled to 0.039 and 0.033 respectively) while the level of serum IL-6 in VT20+MSC group was similar to that in the VT20 group as no significantly difference were found between VT20+MSC group and VT20 group (P equaled to 1.000,0.469 and 0.978 respectively). However, there was no significantly difference in the serum IL-10 level at the same time point either between MSC+VT20 group and VT20 group, or between VT20+MSC group and VT20 group after mechanical ventilation intervention (MSC+VT20 group vs. VT20 group, P equaled to 0.804,0.995 and 1.000 respectively, VT20+MSC group vs. VT20 group, P equaled to 0.557,1.000 and 0.966 respectively).
Conclusions MSCs infusion could attenuate the degree of lung tissue injury, oxygenic deterioration, PMN lung infiltration, and production of local and systemic pro-inflammatory mediators while it had no significant effect on the production of local and systemic anti-inflammatory mediator-IL-10. And the protective effect favors given earlier. Among them, the attenuation of lung injury was associated with decreasing PMN lung infiltrated. That indicates that MSC infusion could provide a homeostatic environment for those patients undergoing impropriated mechanical ventilation and it could in some extent prevent and lighten the subsequent PMN predominant inflammatory injury in VILI model.
Chapter 4 Effects of MSCs administration on PMN function in VILI rat model.
Objective To determine the effects of MSCs administration on PMN functions in the VILI SD rat model.
Methods The blood and BALF samples 4h after mechanical ventilation were collected from animals in control group, MSC group, mechanical ventilation with large tidal volume group (VT20 group), MSCs pretreatment + mechanical ventilation with large tidal volume group (MSC+VT20 group, the MSCs were systemic given just before ventilation), MSCs post-treatment + mechanical ventilation with large tidal volume group (VT20+MSC group, the MSCs were systemic given immediately after ventilation). The intercellular ROS production and surface CDllb expression were analyzed with flow cytometer. The neutrophil elastase (NE) activity of serum and BALF was measured. The PMNs were isolated from blood and BALF samples, and the apoptosis of PMNs was analyzed with flow cytometer. The fenton reaction was adopted to determined the extracellular ROS production of PMN. The data of CD11b expression, NE activity, intracellular and extracellular ROS production, and apoptotic rate of PMN in different groups were compared.
Results 1) There was no significantly difference among different groups about the surface molecular CD11b expression of PMN either in blood or in BALF (Blood:F =1.008, P= 0.442; BALF:F=0.328, P= 0.856).2) There was a significantly difference among different groups about the intracellular NE activity of PMN either in blood or in BALF (F=39.813 and 31.061 respectively, both P=0.000). The NE activity of PMN in VT20 group was significantly higher than that in control group either in blood or in BALF (all P=0.000). The NE activity of PMN in MSC+VT20 group was significantly lower than that in VT20 group in either blood or BALF (all P=0.000) while significantly difference was found between VT20 + MSC group and VT20 group either in blood or in BALF (blood, P=0.035; BALF, P=0.040). However, no significantly difference was found between VT20 + MSC group and MSC+VT20 group either in blood or in BALF (blood, P=0.927; BALF, P=1.000).3) There was a significantly difference among different groups about the intracellular ROS production of PMN either in blood or in BALF (F=986.470 and 118.612 respectively, both P=0.000). Mechanical ventilation with a large tidal volume increased the intracellular ROS production of PMN either in blood or in BALF (vs. control group, all P=0.000). The intracellular ROS production in groups with MSCs administration were significantly decreased as compared to that in VT20 group (all P=0.000, except P=0.001 as MSC+VT20 group vs. VT20 group in BALF). And there was significantly difference between MSC+VT20 group and VT20+MSC group in the intracellular ROS production of PMN from BALF (P=0.000).4) There was a significantly difference among different groups about the extracellular ROS production of PMN either in serum or in BALF (F=19.597 and 198.991 respectively,both P=0.000). The serum and BALF ROS production of PMN in VT20 group were significantly higher than that in control group either in blood or in BALF (all P=0.000). MSCs pretreatment (MSC+VT20 group) significantly decreased the ROS production either in serum or in BALF as compared to VT20 group (both P=0.000) while MSCs posttreatment decreased the ROS production in BALF as compared to VT20 group (P=0.000). And there was a significantly difference between MSC + VT20 group and VT20+MSC group in the serum ROS production or in the ROS production in BALF (P=0.001 and 0.021 respectively).5) There was a significantly difference among different groups about the apoptotic index of PMN either form blood or from BALF (F=41.869 and 89.661 respectively, both P=0.000). And MSC pretreatment significantly increased the apoptotic index of PMN from blood (P=0.001) while the ventilation mechanical ventilation significantly decreased the apoptotic index of PMN either blood-derived or BALF-derived (both P=0.000).
Conclusions The excessive activated PMN function existed in VILI rat model which caused by mechanical ventilation with a large tidal volume, which was illustrated with NE activity increasing, ROS production increasing and apoptosis decreased. MSCs administration decreased NE activity and ROS production in a certain extant, especially when MSCs was given before injured. Which indicates earlier MSCs administration are hope to limit the injury degree of ALI while the body in the risk of VILI via regulation the function of PMN, as MSCs posses the regulative ability of PMN function and that favors earlier given.
Chapter 5 Effects of systemic MSCs infusion on the prognosis and the fate observation of MSCs in the lung after infusion in VILI SD rats
Objective To determine whether MSCs systemic infusion could improve the outcome of VILI animal and identify whether the MSCs could immigrate into the injured lung, adopt the phenotype of lung cells, and replace the function of injured cells.
Methods 45 female rats weighting 250g to 260g were anesthetized and a canula was inserted into the trachea via tracheotomy. And then the animals were subgroup into 3 groups:high tidal volume mechanical ventilation group (VT20 group b), MSCs pretreated + high tidal volume mechanical ventilation group (MSC+VT20 group b, MSCs were treated before mechanical ventilation) and high tidal volume mechanical ventilation + MSCs post-treatment group (VT20+MSC group b, MSCs were given immediately after mechanical ventilation). N equaled to 15 each group. The ventilator parameters were:VT=20ml/kg body weight, PEEP=2cmH2O, RR=40 bmp, I:E= 1:2 and FiO2=0.21, and the ventilation lasted for 2 hours. The tracheotomy was closed and sutured after ventilation, and were sent back to their cages breathing room air. Long-term survival was observed every 24h within 14d after ventilation in these rats. The MSCs were male-SD rat-derived and its number given to the animal was 3x106/L per animal. The lungs were adopted from 6 animals from each group of MSC group, MSC+VT20 group, VT20+MSC group, MSC group b, MSC+VT20 group b and VT20+MSC group b after animals were scarified.The immunohistochemistry and PCR technique were used for fate observation of MSCs in the lung.
Results The survival rate of animals 48h after mechanical ventilation in MSC group b, MSC+VT20 group b and VT20+MSC group b was 73.33%,73.33% and 66.67% respectively, and there was no significantly difference among three groups (χ2=0.216, P=0.897). The survival rate of animals within 14d after mechanical ventilation in MSC group b, MSC+VT20 group b and VT20+MSC group b was 60.00%,46.67% and 46.67% respectively, and there was no significantly difference among three groups (χ2=0.720,P=0.698).The immonohistochemistry and PCR results showed that MSCs emerged in the lung shortly after infusion. MSCs distributed in pulmonary capillaries uniformly in MSC group, and it distributed locally in pulmonary capillaries and less in MSC+VT20 group as compared to MSC group(P =0.000). The MSCs distributed in the thicken pulmonary interstitial and even less in the VT20+MSC group as compared with MSC+VT20 group (P=0.000).However, there was no SRY-positive cells and no Sry sequence of Y chromosome was detected in the lung from animals from either MSC group b, or MSC+VT20 group b or VT20+MSC group b.
Conclusions MSCs emerged in the lung shortly after infusion in the VILI animal model, and the numbers of MSCs located in the lung was decreased as the lung injured. However, the MSCs did not transplant into the lung, adopt the phenotype of lung cells and replace the function of the injured lung cells. Although MSC pretreatment improved the 2w survival rate from 40% (6/15) to 53.33% (8/15) in VILI model, the difference did not reach statistically significance as the sample we observed was relatively small (just 15 each group) in the current study. Hence, a trial with large sample was needed to ascertain the benefit effect on the 2w survival rate of MSC treatment in the current VILI model.
Summary
The MSCs from SD rats could be cultured with combining Percoll density gradient centrifugation with adherent method. And the MSCs we cultured with characteristics of active proliferation, mostly survival and high purify active property of differentiation and stable morphology. And the VILI model could be established with mechanical ventilation with a large tidal volume (i.e.20 ml/kg body weight) lasted for a short time (2h) in SD rats. The model was with a main characteristics of PMN lung infiltrated predominant, accompany with local and systemic inflammatory response related to PMN activation. MSCs systemic infusion could attenuate the subsequent PMN predominant infiltration, subsequent acute inflammatory response includes PMN activation and lung injury, the protective effects favors given earlier, especially preventive given. Although the preventive effects of PMN infusion could not be resulted from transplanting into the lung, adopting the phenotype of lung cells and replacing the function of injured lung cells, which may be explained by modulating the inflammatory response (includes the PMN activity). The current study indicates that MSCs are hoped to prevent and cure the VILI by modulating the activated PMN-predominant inflammatory response, hence providing a related homeostasis environment for patients at risk of VILI and enhancing the patients' resistance to impropriate mechanical ventilation.
引文
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