【作者】 许靖；

【导师】 封志纯；

【作者基本信息】 南方医科大学 ， 儿科学， 2013， 博士

【摘要】 支气管肺发育不良(Bronchopulmonary dysplasia, BPD)又叫慢性肺疾病(Chronic lung disease, CLD),是以慢性呼吸窘迫及肺功能不良为主要特征的疾病,随着超早产儿(extremly preterm,胎龄24周至27+6周)及极早产儿(very preterm,胎龄28周至32周)救治成功率及新生儿存活率的提高,BPD的诊断率及发生率也有逐渐增加的趋势,尤其在极低出生体重儿(VLBW)人群中的发生率高达20～40%。BPD发生于新生儿时期,病程长,对大部分患者来说,可伴随终身,严重影响生命安全及生活质量。临床上常见于新生儿,尤其早产儿发生呼吸窘迫综合征(respiratory distress syndrome, RDS)、吸入综合症、新生儿肺炎等疾病后,表现出呼吸增快、低氧血症、酸中毒、三凹征、发绀等呼吸衰竭症状,给予呼吸支持治疗之后,较长时期不能脱氧甚至依赖呼吸机。患者于新生儿时期即出现生长缓慢或停滞,病程短者数周内死亡,即使存活,迁延至数月至数年,对呼吸系统的损害也将伴随终身,再次住院率极高,多合并肺功能障碍,死因常为肺部感染、心肺功能不全等。典型临床病理表现为：早期出现肺泡上皮细胞和毛细血管内皮细胞破坏,间质及血管周围水肿,细小支气管损伤坏死,鳞状上皮细胞化生,纤毛上皮细胞凋亡坏死,平滑肌增生肥厚；病程进展很快,随后即出现炎症细胞侵润,巨噬细胞、浆细胞、纤维母细胞增加,纤维组织增生,胶原沉积,间质增厚,支气管细小支气管受损阻塞,肺泡数目减少,弹力纤维变性,肺部血管内膜增厚,各级支气管内壁增厚,腺体增生,发展为局部肺不张及肺气肿。BPD是一个多因素致病的综合征,基本可以概括为：在肺组织发育不成熟的基础上,氧中毒、气压伤、容量伤以及炎症因素引起的肺泡化进程阻滞,导致肺失去正常结构及正常的呼吸功能。呼吸系统的发生发育分为胚胎期和出生后两个时期。胎龄24周肺的发育进入小管期,开始形成终末囊泡(肺泡前体)、细支气管、毛细血管前体、粘液腺、支气管等主要支架性结构。胚胎28周以后,肺进入囊期,形成肺泡,并开始分泌肺表面活性物质,具有呼吸功能的肺开始形成。但肺泡完全的发育与功能的成熟,要到8岁左右才能完成。因此,任何能够阻滞这个过程的因素,均能导致对肺结构的破坏及功能的严重影响,尤其在新生儿阶段,所有能造成肺损伤、阻碍肺发育正常过程的因素,均能成为BPD的病因。小鼠肺组织的发展具有类似的时序性：假腺体时期肺基本支架,即各级主支气管形成；小管期上述结构继续发展,细支气管、毛细支气管开始发育,并伴随附属微血管形成；囊泡期为肺泡前体细胞形成时期,即肺泡化时期；最后是21日龄以后的肺泡成熟期。不同的是,人类在胚胎时期已完成肺的大体结构和基本功能结构的发育,如肺泡在小管期已经开始少量发育,胎龄28周以后肺泡正式发育形成,而新生小鼠的肺组织仅完成了其大体形态的建立,肺泡化的完成主要发生于出生后直到生后21天。因此小鼠可较好地成为BPD的研究工具。目前在研究肺损伤的动物实验中,主要有以下几种造模手段：博来霉素诱导、脂多糖诱导、石棉诱导、辐射诱导、高氧暴露诱导等,其中高氧暴露是BPD起决定性作用的危险因素,因此我们采取高氧暴露的方法制作小鼠BPD模型。骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells, BMSC)是来源于骨髓基质的干细胞,属于非造血干细胞,对后者具有机械支持的作用,并通过分泌多种生长因子支持其造血功能。最重要的是,BMSC具有多向分化潜能,包括骨细胞、软骨细胞及脂肪细胞；在一定的诱导条件下,还可以分化为神经细胞、肺泡上皮细胞、心肌细胞、肝脏细胞等内脏细胞。同时BMSC具有MHCl+、MHC2-、CD40-、CD80-等免疫表型,但均不具有免疫原性；通过激活诱导外周T细胞,发挥免疫调节作用。综上所述,BMSC具有多向分化潜能以及免疫调节作用,且不具免疫原性,同种异体移植不会产生免疫抑制,因此有理由认为,BMSC对组织损伤、免疫缺陷性疾病可以发挥治疗作用。BMSC获取较简单,无论是动物实验,还是临床自体取材,BMSC体外培养传代后的细胞同质性可高达95%以上,能保证细胞特性的稳定遗传,经过特定的诱导刺激,仍具有多向分化的功能,培养及纯化技术易于操作,成本低廉,因此BMSC具有广阔的应有前景。文献报道BMSC在器官再生修复中,能通过诱导分化达到良好的效果。在一些急性肺损伤动物模型的治疗研究中,证实外源性BMSC能减轻肺部炎症反应,改善急性炎症引起的胶原蛋白的沉积；在各种慢性肺损伤、肺纤维化的动物模型的治疗实验中,也发现了外源性BMSC的移植,能使受体肺部结构得到修复,能促进Ⅱ型肺泡上皮细胞的增生,改善肺纤维化的程度。通过不同的给药途径的研究,有些研究者认为BMSC通过迁移、分化潜能达到对损伤肺的靶向修复,还有研究者认为BMSC通过旁分泌的方式达到对损伤肺的修复。基于上述发现,我们将新生小鼠暴露于60%氧,对照组小鼠暴露于21%氧(空气),对一般情况、体重等进行观察与记录,至21日龄时取小鼠肺组织检测。在饲养小鼠制作BPD模型的过程中,我们发现,包括健康小鼠(即空气对照组小鼠)在内,小鼠在生后前3天内体格生长较为缓慢,在整个21天喂养周期内,高氧暴露组与空气对照组的小鼠都表现出了体重增长：高氧组2日龄、7日龄、21日龄体重分别为(5.75±0.13)g、(8.14±0.18)g、(21.02±0.22)g,差异有统计学意义(F=3168,P=0.000),空气组2日龄、7日龄、21日龄体重分别为(6.09±0.15)g、(8.92±0.16)g、(27.74±1.27)g,差异有统计学意义(F=3045,P=0.000)；但高氧暴露组小鼠体重总体上低于正常空气对照组：2日龄,高氧组：(5.75±0.13)g,空气组：(6.09±0.15)g,7日龄,高氧组：(8.14±0.18)g,空气组：(8.92±0.16)g；t分别为6.698,12.497,20.156,P均为0.000,差异均有统计学意义；随着日龄增加,逐渐差异增大,21日龄2组小鼠体重,高氧组：(21.02±0.22)g,空气组：(27.74±1.27)g,差异有统计学意义(t=20.156,P=0.000)；同时高氧暴露组小鼠缺乏活力,一般情况较差,7日龄高氧暴露组与空气对照组已出现较为明显的差异,高氧组小鼠皮下脂肪不如对照组丰满,反应一般,离氧不耐受,皮毛较粗糙,而对照组正常生长,较活泼；21日龄高氧组体格较小,吃奶差,皮毛粗糙,拱背,歪头,呼吸时可闻及鼻音,离氧或低梯度氧疗下明显不耐受,出现呼吸急促、皮肤发绀、苍白,甚至痉挛,而对照组正常生长,活泼好动。这和人类BPD婴儿营养不良较为符合,随着肺泡化的阻滞,气体交换不能满足机体对氧的需求,不能完成正常的新陈代谢,同时活性氧的细胞毒性也对细胞代谢造成损害,导致营养不良与生长受限。高氧组肺组织P2、P7、P21均出现结构性改变。2日龄的高氧组与空气组相比,形态上未见明显的量化改变,仅见远端过度充气的肺泡或者间质组织较肥厚。而7日龄高氧组小鼠的增大的肺泡增多,肺实质出现局灶性增厚。随着病情的进展,至21日龄,即P21,高氧暴露组小鼠已经表现出类似人类新生儿BPD的组织病理特征。进行RAC的量化评估发现,2日龄两组RAC计数：空气组3.77±0.12、高氧组3.61±0.12,差异有统计学意义(t=3.729,P=0.001)；7日龄两组RAC计数：空气组6.75±0.14、高氧组4.58±0.17,差异有统计学意义(t=37.492,P=0.000)；21日龄两组RAC计数：空气组12.82±0.11、高氧组8.67±0.11,差异有统计学意义(分别为t=100.686,P=0.000)。因此肺泡增大,肺表面积减少导致氧交换及动脉氧合减少,导致BPD肺功能障碍的发生。我们从雄性小鼠四肢骨髓腔分离出细胞,通过贴壁培养、纯化,得到BMSC。从小鼠股骨骨髓采集的原代BMSC尚未纯化,细胞核难以辨认,各系细胞混杂,BMSC透亮圆形,大小不等；培养24小时后光学显微镜下观察见少量圆形或不规则细胞贴壁,随后48～72h贴壁细胞增多,小集落形成,细胞生长较缓慢,形态不均一、散落分布,可见球形、椭圆形、梭形细胞,其中拉长梭形细胞逐渐占优势；培养接近1周时间,细胞生长加快,约1周后,经过换液弃悬浮细胞,纯化过程基本完成,贴壁的细胞形态均一,胞体增大呈纺锥形、梭形；换代培养的细胞生长迅速,形态进一步拉长呈长梭形,融合度高,紧密贴壁,呈旋涡状沿胞体长轴排列；消化后呈小而饱满的圆形。流式细胞检测显示分离培养的BMSC均一性良好。通过对细胞表面免疫表型的分析,CD105、CD106显示阴性对照与实验组呈双峰波形,CD34、CD45两封叠加重合,表明BMSC细胞群对CD105、CD106呈阳性反应,对CD34、CD45呈阴性反应,提示BMSC分离培养成功。BMSC经成骨诱导培养后,1周后逐渐形成铺路石状细胞形态,并逐渐形成局部重叠,形成多个结节的矿盐沉积,并随培养时间的延长,逐渐融合；2周后形成棕褐色结节矿化中心,随着时间延长,可融合成片。BMSC经成脂肪诱导培养约1周后,细胞呈无序排列,胞浆内可见细小脂滴,随着时间推移,脂滴增大,可推挤细胞核,折光度高,甚至肉眼可见呈白色的脂肪细胞集落。说明在一定的诱导条件下,BMSC具有向脂肪细胞、成骨细胞定向分化的能力。将BMSC与高氧损伤肺组织共培养,Real time qPCR检测可见SP-C表达,荧光显微镜下可见Ⅱ型肺泡上皮细胞特异性蛋白质,而单独培养、与正常对照组小鼠肺组织共培养则未见上述表达。BMSC与高氧损伤肺组织共培养后,激光共聚焦显微镜下可见出现微细丝的再生修复,表明损伤肺可促进BMSC的体外迁移,而单独培养、与正常对照组小鼠肺组织共培养则未见上述表达。BPD模型小鼠肺组织的原位杂交实验结果显示,在肺泡部位,以及植入BMSC的呼吸道的周围Y染色质染色的存在,而PBS对照组则无类似发现。这些结果表面,腹膜内外源性BMSC具有向损伤肺转移的能力。免疫荧光染色结果证实,与阴性对照组相比,使用BMSC干预治疗的BPD小鼠肺中SP-C与BMSC都有表达,表明外源性BMSC能提高AEC2在损伤肺中的含量。空气对照组(阴性对照)、BMSC干预治疗的高氧暴露组、PBS干预的高氧暴露组(阴性对照)小鼠肺组织SP-C通过real time qPCR检测的表达量也显示：空气对照组(阴性对照)与BMSC干预治疗的高氧暴露组的SP-C表达量较高,分别为4.72±1.15、4.45±1.18,PBS干预的高氧暴露组(阴性对照)表达量低,为1.77±1.54,总体比较,差异有统计学意义(F=23.407,P=0.000)；空气对照组与BMSC治疗组的SP-C表达量较高(P=0.581),两组差异无统计学意义；BMSC治疗组的SP-C表达量比PBS阴性对照组高(P=0.000),差异有统计学意义。肺组织病理表现为：空气对照组小鼠肺组织结构规整,肺泡数量较多,大小均匀,肺间隔较薄；高氧暴露组注射PBS阴性对照组小鼠肺组织,表现为正常肺组织结构消失,肺泡腔隙增大,数量少,肺泡融合,肺间隔较厚,高氧暴露组注射BMSC的小鼠肺肺部结构接近空气对照组。RAC结果显示,与PBS阴性对照组相比,高氧暴露组小鼠腹膜内注射BMSC的BPD小鼠肺泡化增加,差异有统计学意义(F=47.257,P=0.000)；组间两两比较显示,BMSC治疗组与空气组接近(P=0.404),差异无统计学意义,BMSC治疗组与PBS对照组有差异(P=0.000),差异有统计学意义。提示BMSC对高氧肺损伤有修复作用。肺组织经天狼星红染色及Masson染色结果显示,空气对照组肺部结构正常,肺泡大小正常均一,肺间质厚度适中；高氧暴露注射PBS对照组小鼠肺组织失去正常结构,肺泡腔隙扩大融合,肺间质增厚不规则,见蓝色胶原沉积,并融合成片状、束状；高氧暴露注射BMSC治疗组小鼠肺组织结构不如正常肺组织规整,有正常肺泡,肺间质较厚,有部分线条状蓝色胶原沉积,说明高氧暴露对肺组织结构的损伤是显而易见的,BMSC治疗能显著改善支气管和肺泡周围纤维组织增生的严重程度；提取3个不同处理组(即BMSC治疗组、空气对照组、PBS处理对照组)小鼠肺组织,经提取RNA——反转录—real time qPCR以及血清进行ELISA检测,结果分别显示,BMSC治疗的细胞外基质重塑和纤维化相关的基因,即TGFβ1、TIMP-1、胶原1α等的表达显著性减少：TGFβ1三组组间比较差异有统计学意义(F=53.443,P=0.000),组间多重比较,BMSC治疗组表达量接近空气对照组(P=0.388),差异无统计学意义；BMSC治疗组、PBS对照组比较有差异(P=0.000),差异有统计学意义；TIMP-1:三组组间比较差异有统计学意义(F=88.195,P=0.000),组间多重比较,空气对照组、BMSC治疗组接近(P=0.100),差异无统计学意义；BMSC治疗组、PBS对照组比较有差异(P=0.000),差异有统计学意义；胶原1α：三组组间比较差异有统计学意义(F=37.917,P=0.000),组间多重比较,空气对照组、BMSC治疗组比较有差异(P=0.000),差异有统计学意义；BMSC治疗组、PBS对照组比较有差异(P=0.000),差异有统计学意义；血清中IL-1、TNFα降低：IL-1：三组组间比较差异有统计学意义(F=29.285,P=0.000),组间多重比较,空气对照组、BMSC治疗组比较有差异(P=0.006),差异有统计学意义；BMSC治疗组、PBS对照组比较有差异(P=0.000),差异有统计学意义；TNFα:三组组间比较差异有统计学意义(F=22.768,P=0.000),组间多重比较,空气对照组、BMSC治疗组接近(P=0.661),差异无统计学意义；BMSC治疗组、PBS对照组比较有差异(P=0.000),差异有统计学意义。60%吸入氧浓度是制作BPD小鼠模型的适宜浓度；由于肺通气、换气功能障碍,不能满足新生小鼠新陈代谢需要,以及高浓度氧对细胞的毒性作用,高氧暴露小鼠在发生BPD的过程中,出现慢性呼衰、氧依赖,体格生长缓慢,反应差,活力下降的表现,证实我们的造模条件不仅能引起类似BPD的肺部损害,还能全面模拟人类BPD发病时的系统性表现。而肺部结构的改变从第2天开始出现,并渐进性发展为肺泡化受阻、纤维化形成及非正常结构的破坏,因此,我们认为,作为BPD治疗的研究工具,持续给予60%浓度氧诱导的肺损伤小鼠模型符合研究要求。全骨髓贴壁培养方法成熟、易于操作,随着培养时间的延长及换液去除悬浮细胞,可有效分离出纯化的BMSC,经细胞形态鉴定以及表面免疫标记物的检测,证实这种方法分离BMSC效果良好；原代细胞培养的过程中,随着纯化程度的提高,BMSC生长速度加快,传代后的细胞增殖较为迅速,3代BMSC诱导分化效率良好,适于体内实验。腹腔注射外源性BMSC,达到了向损伤肺组织的迁移,并可诱导SP-C表达,从而对损伤肺直接进行修复；BMSC抑制TGFβ1胶原1α、TIMP-1等表达,继而调控细胞外基质增生、纤维组织增生相关基因表达,从而抑制高氧引起的纤维组织增生,减少胶原沉积；在对小鼠血清的检测中,TNF-α、IL-1含量减少,从而证实BMSC的应用可减少炎症反应；最后,从实验效果来看,BMSC使BPD模型小鼠的存活率得到提高,因此还可能通过一些未知的途径改善BPD小鼠的预后。供体BMSC的移植方法很多,我们证实了腹腔注射能够通过细胞迁移的方式对损伤肺发挥修复作用,为BMSC未来的临床试验提供一个有意义的给药途径。更多还原

【Abstract】 Bronchopulmonary dysplasia(BPD), as known as chronic lung disease(CLD) before, is a chronic disorder characterized with chronic respiratory distress and pulmonary dysfunction. Along with there have being more and more extremely preterm neonates and very preterm neonates rescued successfully, the incidence of BPD is a growing trend, especially in the very low birth weight infants(VLBW) among which20～40%infants get the disease. BPD develops in the neonatal period and maintains very long, for the most they will be afflicted during whole life. The disease often happens following the respiratory, inhalation syndrome, neonatal pneumonia, which present tachypnea, hypoxemia, acidosis, three depressions sign, cyanosis and respiratory and therefore receive respiratory support therapy. After a long time they can not wean with oxygen and ventilator, BPD happens. The little patients grow stunt, death often follows in a few weeks while they can survive several years even until early adult, they would be cursed by again and again rehospitalization for repeatedly pulmonary infections, cardiopulmonary dysfunction and other distresses which in fine lead to death.The typical clinical pathological manifestations are like that:early stage starts from the destriction of alveolar epithelial cells and capillary endothelial cells companied with stromal and perivascular edema, the small bronchi injury, squamous Cell metaplasia, ciliated epithelial cell apoptosis and necrosis, smooth muscle hypertrophy. Pathogenesis progresses quickly followed by the invasion of inflammatory cells, macrophages, fibroblasts and collagen depositon, interstial thickening, bronchial damage so that there form local atelectasis and emphysema.BPD is a disease syndrome involving multiple factors. The development of the respiratoy system is divided into two periods included with embryonic and postnatal. At gestational age24weeks it is the small tube period, the alveoli precursor, the bronchioles, the precursor of the capillaries, mucous glands, bronchial stent structure start to form. After28weeks embryonic lung progress into the sac period involving the formation of alveoli and alveoli begins secrete pulmonary surfactant, so that real respiratory function begins to form. However, the accomplishments of the structure and function of alveolar were gotten until about8years old. Therefore, during the duration any factors that abrupt the process can cause serious damage to the structure and function development, especially in the neonatal period, all relevant factors can lead to BPD.Bone marrow-derived mesenchymal stem cells(BMSC) are from bone marrow stromal stem cells. BMSC can support the function of bone marrow-derived hematopoietic stem cells. However, the most important function of BMSC is differentiation into bone cells, cartilage cells and fat cells, even then under some inducing conditions into nerve cells, alveolar epithelial cells, cardiac muscle cells, liver cells and other organs cell. BMSC are pocessed with some immunophenotype as MHCl+、MHC2-、CD40-、CD80-and no immunogenicity. On the other hand, BMSC can activate and induce peripheral T cells and then play the roles of immune regulations. Eventually, BMSC can be obtained easily in not animal experiments also in clinical donor or self body drawn. Substituting cells cultured in vitro can keep homogenous, what’s more, under specific induced stimulation BMSC also have a characteristics of multi-directional differentiation potent. In a word, as a multiple potential stem cells, the obtain of BMSC is easy and low cost, BMSC should have broad prospects.Based on these findings, we raised mice exposed to60%oxygen from birth, the control group mice were exposed to21%oxygen (air). the mice general condition were recorded including body weight. On21days after birth the mice were sacrificed and lung tissue were obtained. In the process of feeding mice BPD model, we found even healthy mice (i.e. air control mice) grew slowly in the first3days after birth. throughout the21days-feeding period, the hyperoxia group and the air control group mice shared weight gain, but the hyperoxia group mice grew generally lower down in compared with the normal air control group, showing significant difference at21days of age. Furthermore, the hyperoxia group mice were lack of energy, exhibiting poor general condition. Through7days exposure the hyperoxia group and the air control group appeared more obvious differences, in comparison with the control mice the hyperoxia group were poorer in subcutaneous fat, the general reaction, oxygen intolerance, fur sleeky. On21day-old the hyperoxia group were poorer. There demonstrating the symptoms of poor feeding, shaggy, arch back, head, breathing audible and nasals, oxygen from toleration, shortness of breath, cyanosis, pale skin, and even convulsions, while the control group grew lively. This is more in line with human BPD infant malnutrition. With the rest of alveolarization followed by the reduce of gas exchange, the body could not meet demand for oxygen. At the same time, cytotoxic reactive oxygen species damaged the cell metabolism, leading to malnutrition and growth retardation. In compared with the air group, no significant quantitative changes happened to the hyperoxia group on2-day age, with the distal hypertrophic alveolar and interstitial tissue. On7-day age the alveolar morbidity of the hyperoxia group increased and lung parenchyma appeared focal thickening. As the disease progresses, on21-day age, the hyperoxia group showed histopathological features similar to human BPD. P7, P21, RAC were reduced by34%(P<0.01),33%(P<0.01).We analyzed the surface immunophenotype and found BMSC family were positive for CD105and CD106whereas negative for CD34and CD45. The results of co-culture of BMSC with the hyperoxia injury lung cells manifested that SP-C and proteins(specific markers for typeⅡ alveolar epithelial cells). Conversely, BMSC alone did not express those, neither co-cultured with normoxic lung cells. The results reveal injured lung could promte BMSC migration in vitro.In situ hybridization indicated the presence of Y-chromosome cells in the corner of alveolar. The results indicated intraperitoneal injection of exogenous BMSC can migrate to injury lung. Immunofluorescence verified that SP-C and BMSC are expressed in BPD mice lung. Furthermore, given BMSC, the expression were increased significantly. Treated with BMSC the survival rate of BPD mice increased. We studied RAC and found more aveolarization with injection of BMSC. Trichrome stain indicated that BMSC reduced the severity of pulmonary fibrosis around the alveoli and the airway. Real time qPCR and ELISA also revealed that the group with BMSC treatment TGF β1,TIMP-1,collagen1α expressed decreasedly significantly and the plasma concentration of IL-land TNF α decreased significantly.MiRNA regulate lung septation and play an important role on the development of BPD. Exogenous BMSC can migrate to injury lung and improve the destroyed lung structure.更多还原