【作者】 宋小莲；

【导师】 白冲；

【作者基本信息】 第二军医大学 ， 内科学， 2009， 博士

【摘要】 慢性阻塞性肺病(COPD)是人类的常见病,是世界范围内第四位的死亡原因。近年来,随着环境污染等因素的加重,其发病率有逐年增高的趋势。COPD以不完全可逆的气流受限为特征,病情呈进行性发展,其发病机制复杂,病理学改变则累及肺脏的多级结构,包括中央及周围的气道、肺实质乃至肺血管。目前认为,COPD的病理损伤属于不可逆性病变,依靠机体自身的能力无法达到组织的完全修复。因此,迫切需要寻找到一种参与肺部组织结构的修复和重建的有效方法。研究认为,骨髓中的造血干细胞(hematopoieticstemcells,HSC)和间充质干细胞(mesenchymal stem cells,MSC)都具有多向分化的潜能,其中MSC由于取材方便、来源丰富、容易培养、增殖力旺盛而日益成为干细胞研究的热点。近年来大量研究显示,间充质干细胞在治疗诸如糖尿病、心肌梗塞、移植物抗宿主反应等多种疾病方面具有广阔的临床应用前景。之前有研究已证实,骨髓MSC可以在体内、体外分化为肺实质细胞,在炎症等因素的趋化下,外源的骨髓MSC有向肺损伤部位汇集的现象。外源MSC可能通过两方面参与肺的损伤修复:1.在局部微环境作用下诱导分化为肺泡上皮和支气管上皮细胞等结构细胞,参与组织的修复过程;2.通过免疫调节作用,为损伤修复提供的有利环境。但目前而言,应用MSC干预治疗COPD的相关研究十分少见,现有的关于肺部疾病干细胞治疗的有限相关研究,主要集中在急性肺实质损伤和肺纤维化方面。而且,对于不同移植条件对干细胞治疗效果也没有相应的对比研究。因此,本课题经过严格的对比和系统的观察,研究观察了移植的骨髓MSC在COPD动物模型肺局部的定植存活情况,了解MSC移植的治疗效果。本研究包括三部分内容,分述如下。一、大鼠骨髓间充质干细胞的体外培养和生物学特性目的:分离和培养大鼠MSC(rMSC),研究其生物学特征,为动物实验做准备。方法:取6-8周龄正常雄性Wistar大鼠的股骨和肱骨,无菌状态下冲洗骨髓腔,收集骨髓细胞悬液,接种于塑料培养皿,使用10%FBS/DMEM-LG的培养基,在37℃、5%CO2和100%饱和湿度条件下进行培养,每3-4d按照1:3进行常规方法传代;倒置显微镜下观察细胞形态学特点;MTT法绘制细胞生长曲线;流式细胞仪检测细胞表面标记和细胞周期;定向诱导rMSC在体外向成骨细胞和脂肪细胞分化;裸鼠皮下注射rMSC检测其成瘤性。结果:体外培养的rMSC大部分呈梭形的成纤维细胞样形态,细胞呈辐射状排列。在体外观察传代20代,细胞形态和生长速度无明显改变;生长汇合率达30%-40%和90%时的P5代rMSC,G0/G1期细胞的百分比分别为53.07%和85.24%,S+G2+M期细胞的百分比分别为46.93%和14.76%;P5代细胞高表达CD29、CD44和Sca-1,而不表达造血干细胞的标记CD34、CD117和CD45,不表达Flk-1、MHC-II;在体外诱导培养下,rMSC可向成骨细胞和脂肪细胞分化,具有多向分化潜能;裸鼠背部种植rMSC,3个月未见成瘤现象。结论:全骨髓贴壁法可成功分离和培养出大鼠骨髓间充质干细胞,分离所得的细胞性状稳定、状态良好,形态学和表型符合间充质干细胞特点,具有多向分化潜能且无明显成瘤性。二、COPD模型的构建和鉴定目的:研究建立合理的慢性阻塞性肺病(COPD)大鼠模型的有效方法。方法:8周龄健康Wistar大鼠15只,随机分入健康对照组(C组)和两个COPD模型组。用两次气管内注入脂多糖(LPS 200μg/次)加熏香烟1个月的复合刺激法(A组)和单纯熏烟法(B组)分别建立大鼠COPD模型,然后行病理学检测造模效果,包括平均内衬间隔(MLI)和平均肺泡数(MAN)的测定;观察实验动物一般情况;检测动物的外周血常规和支气管肺泡灌洗液(BALF)细胞计数与分类。结果:两COPD模型组大鼠纳差消瘦,伴有间歇咳嗽和气促。病理学检查显示两模型组均具有慢性支气管炎和肺气肿的典型病变。两模型组外周血和BALF中的白细胞总数及中性粒细胞百分比均较健康对照组显著增高(P<0.01);两模型组MLI较对照组有显著性增高,而MAN较对照组显著性下降(P<0.01);但A、B两组间无显著差异(P>0.05)。病理学切片上,A组比B组各级支气管及肺组织的炎症浸润更明显,上皮细胞损伤和腺体增生明显;而B组更突出表现为肺泡的过度扩大融合。结论:用单纯熏烟法和两次气管内注入脂多糖(LPS 200μg/次)加熏香烟的方法,均可成功制备大鼠COPD模型,其病理生理改变与人类COPD类似,后者比前者更符合COPD自然发病过程。三、MSC在COPD大鼠模型肺部的定植分化与治疗作用目的:观察rMSC移植在COPD动物肺部的定植和分化及其治疗作用。方法:选健康雌性Wistar大鼠36只,随机分为5组:rMSC小剂量/单次尾静脉移植组(A组,12只,COPD大鼠,尾静脉输注rMSC 1×106/1ml);rMSC大剂量/两次尾静脉移植组(B组,12只,COPD大鼠,尾静脉输注rMSC 1×106/1ml×2次);rMSC对照组(C组,4只,正常大鼠,处理同A组);COPD模型组(D组,4只,尾静脉输注生理盐水1ml);健康对照组(E组,4只)。体外培养扩增雄性Wistar大鼠来源的rMSC,以CM-Dil标记细胞后将其经尾静脉注入用熏烟加LPS气管注入法制作的同系雌性COPD大鼠模型体内,在注射后的1d、7d、15d、30d分别处死动物,取新鲜肺组织冰冻切片荧光显微镜下观察Dil阳性细胞定植、分布和存活时间;原位杂交法检测肺组织中外源细胞的定位、定植效率,以及形态学观察和在肺外脏器的分布;CM-Dil联合免疫组化法检测rMSC在肺组织的转分化情况;观察肺组织病理学变化以及血常规、肺泡灌洗液(BALF)细胞计数与分类;使用双抗夹心ELISA方法检测COPD大鼠BALF和外周血中IL-10、TNF-α和G-CSF的表达情况。实验数据采用SSPS13.0软件进行统计学分析,检验水平为α=0.05。结果:(1)荧光显微镜观察发现,A、B两组在进行rMSC移植后,肺组织内有较多的CM-Dil阳性细胞(红色荧光)进入,主要分布于肺间质、肺泡和气道壁,并可在肺局部存留30d以上;而C组肺组织内偶见少量荧光阳性细胞;未经rMSC移植的D、E组肺组织内未见阳性细胞。(2)原位杂交结果与CM-Dil检测结果类似,并可见原位杂交阳性的细胞光镜下呈现II型肺泡上皮和气道粘膜上皮形态,血管平滑肌细胞也可见少量阳性细胞;心脏、骨骼肌和肝脏等肺外组织未见或极少见外源细胞;A、B两组肺组织内的外源细胞定植效率无明显差异。(3)CM-Dil染色联合免疫组化检测提示,部分CM-Dil阳性细胞同时有免疫组化检测SPC或CC16表达阳性,提示外源细胞部分转分化为II型肺泡上皮和气道粘膜上皮。(4)A、B组外周血和BALF中的白细胞总数及中性粒细胞分类较D组有显著降低(P<0.01),但仍高于C和E组(P<0.01),提示rMSC移植后COPD炎症水平有所下调。(5)A、B组外周血和BALF中的IL-10较D组有显著性升高(P<0.01),但低于C和E两组(P<0.01);TNF-α和G-CSF低于D组(P<0.01),但高于C和E两组(P<0.01)。(6)HE染色显示,A、B组肺气肿和气道病变较D组明显减轻,但与C、E组比较,COPD病变仍较明显;说明MSC使大鼠COPD病理改变得到一定程度的改善。结论:经尾静脉注射的rMSC能在大鼠COPD模型的肺组织内定植并长期存留,改善COPD肺部的病理学损伤,调节局部及全身的炎症因子水平,且无明显致瘤性和成纤维化倾向;在一定范围内,这种治疗效果无明显量效关系。更多还原

【Abstract】 Chronic obstructive pulmonary disease(COPD)is one of the most common disease and the 4th leading cause of death in the world. The morbidity has been rising even in recent years due to several factors such as environmental pollution. COPD is a disease state characterized by airflow limitation that is not fully reversible. The airflow limitation is usually progressive. The pathogenesis of COPD is complicated and its pathobiology involve s multilevel structures in the lung, including central and peripheral airways, lung parenchyma,even blood vessel, which is regarded as inreversible. The injure of COPD can not be fully repaired by itself or drugs. Thus, we need badly to explore a new way of structural reparation and reestablishment of the lung.Recently, many studies have identified that bone marrow derived mesenchymal stem cells (MSC) and hematopoieticstemcells (HSCs) have the potency of multi-directional differentiation. Because of the abundant resource, convenient isolation, easy cultivation and vigorous proliferative ability, MSC has become increasingly common in stem cell research. By far, the MSC transplantation was widely used on the treatment study of different diseases and presented as a promising clinical application to them, including diabetes mellitus, myocardial infarction, graft-versus-host disease, and so on.Bone marrow derived MSC can differentiate into some lung parenchymal cells in vivo and in vitro, as confirmed by several studies, which observed that MSC could be guided to location of lungs by inflammation and injury. Exogenous MSC might repair the lesions in two ways: induction and cell differentiation into alveolar epithelium or tracheal epithelium under the local microenvironment, and immunological regulation of MSC for the advantageous repairing conditions.Nevertheless, studies on the application of MSC in COPD is few. Most researchs focused on the use of MSC in the acute pulmonary parenchyma injury or pulmonary fibrosis. Moreover, therapeutic efficacy of different stem cells transplantation program also need to be studied. This research was designed to explore the field planting and survivorship of exogenous bone marrow derived MSC in the lungs of the COPD model rats after transplantation, understanding the therapeutic efficacy of MSC on COPD models via systematical observation.The experiment consists of three parts described as follows:1. Isolation, culture in vitro and study of biological characteristic of the rat bone marrow derived mesenchymal stem cells.Objective To isolate the bone marrow derived mesenchymal stem cells and culture them in vivro. To study the biological characteristics of rMSC.Methods and materials Under aseptic condition, bone marrow cells were collected by flushing the femurs and tibias from 6-8-week-old healthy male Wistar rats with PBS. All the mononuclear cells from bone marrow were plated in the plastic culture dish. The cells was maintained in a incubator with humidified atmosphere of 95% air and 5% CO2 at 37℃, using DMEM-LG medium supplemented with 10% fetal bovine serum (FBS), and was splitted as 1:3 every 3 or 4 days. The morphological features were observed under the invert microscope; Cell growth curve was determined by MTT method; cell cycle and phenotype were taken by flow cytometry. Moreover, their abilities to differentiate along osteoplastic and adipocytic pathways were also investigated. Tumor genesis were observed in BALB/c-nu/nu nude mice which received rMSC subcutaneously.Results The cells cultured in vitro showed spindle-shaped appearance like fibroblast, spreading radiatly. Up to 20 passages of rMSC were observed and no visible morphologic or growth velocity change was detected. Cell cycle ananlyses revealed that 53.07% and 85.24% of the cells at G0/G1 phase, 46.93% and 14.76% at S+G2+M phase when they attached to 30%-40% and 90% confluences, respectively. Flow cytometric analysisshowed that the rMSC were high positive for CD29、CD44 and Sca-1 and negative for CD34、CD117,CD45, Flk-1 and MHC-II. The rMSC could be differentiated into adipocyte and osteoblast cells in vitro. No tumor formation were observed in nude mice during those 3 months.Conclusions The rMSC can be isolated and cultured by all bone marrow cells adherence successfully. The cell population we harvested expressed morphology and phenotype of mesenchymal stem cells, significant renewal capacity, biological stabilities, multi-potentiality and free from tumorgenesis in vitro.2. Establishment and assessment of the rat COPD model. Objective To explore an effective and reasonable way of establishing a rat model of chronic obstructive pulmonary disease(COPD).Methods and materials A total of 15 8-week-old healthy Wistar rats were divided into 2 model groups and the control group(Group C) randomly. The rat COPD models were established by two ways, intratracheal instillation of lipopolysaccharide (LPS) twice + exposure to cigarette smoke for 1 month(Group A), and cigarette smoke inhalation for 80 days only(Group B). The pathologic characteristics of animal models, including the mean lining interval(MLI) and the mean alveoli number(MAN), were determinated to assess the model quality. The general state of health of those rats were observed. Total cell counts and different cell percentage of bronchoalveolar lavage fluid(BALF) were determined. The comparison between the two model groups were also carried out.Results In the two model groups, the rats presented cough or breathlessness periodically, and amplification of weight were reduced than that of control group(P<0.01). Rats in Group A and B shared specific pathological features in tracheobronchial and lung tissues with that of human chronic bronchitis and obstructive emphysema. Significant increase in total white blood cells and neutrophils in BAIF and peripheral blood was found in Group A and B (P< 0.01) than those of Group C. MLI in Group A and B was significantly higher in comparison with Group C, while MAN was reversely much lower( both P<0.01). But there was no statistical difference between Group A and B on those measurements. The histology of Group A showed more often inflammatory cells infiltrating in the bronchial and lung tissue and the secretion of airway than Group B. The latter was charactered on the ruptured and enlarged alveoli.Conclusions By intratracheal instillation of LPS twice in addition to exposure to cigarette smoke and by cigarette inhalation only for more time both successfully established the rat COPD models, which shared many characteristics of human COPD including the pathological and pathophysiological feature.3. Implantation and differentiation of rMSC in the lungs of COPD model rats and its therapeutical effect.Objective To observe the implantation and differentiation of rMSC in the lungs of COPD rats, to study the therapeutical effect of rMSC.Methods and materials The rMSC from the bone marrow of male rats were cultured in vitro and labeled with CM-Dil before implantation into rats. The recipient Wistar rats, totally 36 female rats, were divided randomly into five groups: low-dosed/one-timed transplantation group (Group A, n=12, COPD model rats, intravenous infusion of 1×106/1ml CM-Dil-labeled rMSC after model establishment); large-dosed/twice transplantation group (GroupB, n=12, COPD model rats, intravenous infusion of 1×106/1ml CM-Dil-labeled rMSC twice in the same day after model establishment); rMSC control group (Group C, n=4, the same amounts of rMSC as Group A were injected into the normal female rats); COPD model rats group (Group D, n=4, COPD model rats, intravenous infusion of 1ml NS after model establishment) and normal control group (Group E, n=4). The COPD models were established by smoke inhalation + LPS perfused intratracheally. The rats in Group A and B were sacrificed at d1, d7, d15 and d30 after the intravenous infusion of rMSC. The frozen sections of fresh lung tissues samples were viewed under a fluorescence microscope to observe the implantation, distribution and survivorship of the CM-Dil positive cells. Hybridization in situ(ISH) and histopathological analysis were carried out to detect the localization, implantation efficiency and morphology of foreign cells in the lungs and other organs. CM-Dil label combined immunohistochemistry was used to identify the transdifferentiation of rMSC in the lung. Peripheral blood(PB)and bronchoalveolar lavage fluid(BALF)were harvested for cell counting and classification and the measurement of IL-10, TNF-αand G-CSF levels by ELISA. Statistical comparison were tested with single-factor variance analyses. The reported P value was 2-sided(α=0.05). Calculations were performed using the software SSPS13.0.Results (1)Many CM-Dil positive cells(red fluorescence)were observed in the lung tissues of Group A and B, mainly located in the interstitium, pulmonary alveoli and airway walls, even 30 days after the intravenous infusion of rMSC. Only a few CM-Dil positive cells were found in Group C, and none was detected in Group D and E,which were infused NS instead of rMSC. (2) The results of ISH were similar as those above. Moreover, some ISH positive cells presented with the morphology of type II alveolar epithelium, bronchial epithelium or vascular smooth muscle cell. None or few foreign cell was detected in other organs like the heart, the skeletal muscle and the liver. The implantation efficiency of rMSC in Group B were close to that in Group A. (3) Some CM-Dil positive cells also showed immunohistochemical positivity of SPC(molecular marker for type II alveolar epithelium) or CC16(molecular marker for Clara cell) in the same time. (4)The total white blood cells and neutrophil percentage in the PB and BALF of Group A and B were significantly lower, comparing to those of Group D(P<0.01),but still higher than those of Group Cand E(P<0.01). (5)The IL-10 level of the PB and BALF in Group A and B were significantly higher than that of Group D, while the level of TNF-αand G-CSF were higher(all P<0.01), even the IL-10 level of Group A and B were still much lower than those of Group C and E, and the TNF-α, G-CSF levels were higher reversely (all P<0.01). (6)HE-stained sections revealed obviously pathologic improvement in Group A and B in comparison with Group D, which implied the therapeutic effect of rMSC.Conclusions The rMSC infused from caudal vein can implant into the lungs of COPD model rats and be permanent planting there, improving the pathology of the recipient rats and regulating the local and systemic inflammatory factor level. The therapeutic efficacy of rMSC transplantation showed dose-independent to some extent.更多还原