【作者】 刘凯；

【导师】 吴树明；

【作者基本信息】 山东大学 ， 心血管外科， 2011， 博士

【摘要】 研究背景：高动力性肺动脉高压是左向右分流型先天性心脏病的常见并发症,严重影响患者外科手术治疗时机,手术成功率及手术后的生存质量。有关肺动脉高压形成的机理和治疗研究已经取得了很大进展,现已明确肺动脉高压是以肺血管阻力进行性升高和肺血管阻塞性病变为特征的恶性肺血管病,特征性的病理改变为肺小动脉中膜增生肥厚,最终导致肺小动脉的闭塞,肺血管床减少,使增高的肺动脉压力更高,肺功能严重恶化。左向右分流型先天性心脏病引起的肺动脉高压,在经过手术矫治心内畸形后,部分病例可以缓解,但是肺血管的阻塞性病变一旦发生,却是手术不能恢复的。单纯扩血管治疗肺动脉高压,在一定程度上降低了肺动脉压力,改善了病人的生活质量,但对于肺血管的破坏性病变其疗效有限。近年来,治疗性血管新生成为医学领域的研究热点,干细胞的应用更是组织工程的重点研究对象。脂肪组织来源的间充质干细胞是一种来源于脂肪可以向中胚层多种细胞诱导分化的多能干细胞,因其取材简单,创伤小,少量脂肪组织即可获取大量细胞,体外培养方便,扩增容易而倍受研究者的关注。而且体外研究指出ADSCs(Adipose Derived Stromal Cells,ADSCs)可以分泌大量的促血管新生的细胞因子如肝细胞生长因子(Hepatocyte Growth Factor, HGF),血管内皮生长因子(Vascular Endothelial Growth Factor, VEGF)等。HGF是一种高效的血管生成因子,对多种组织器官的损伤起到修复作用,以往的多项研究将ADSCs用于缺血性疾病取得了满意的结果,更有研究将该种细胞用于肺损伤模型,如肺气肿的动物模型,依靠ADSCs的多向分化功能和/或其分泌的HGF的作用增加肺组织的血管及腺泡的生成和修复,从而改善了肺组织的血流灌注和换气功能。因此我们设想：在肺动脉高压的情况下利用自体ADSCs增加肺组织的血管生成,增加肺组织的灌注,对进行性恶化的肺血管进行修复,改善肺功能。从而为这种破坏性疾病寻求一种有效的治疗方式,目前,将脂肪间充质干细胞用于肺动脉高压的研究尚未见报道。目的：建立大鼠的动力性肺动脉高压模型,体外分离培养大鼠自体ADSCs并进行有效标记,经静脉途径移植细胞到肺组织,观察大鼠肺动脉压力变化及肺血管的病理变化,分析ADSCs移植后与肺动脉压力变化之间的关系,同时检测肺组织HGF的表达情况,为ADSCs移植后对肺动脉高压的影响寻求可能的解释。方法：1.以大鼠为研究对象,行大鼠颈动脉-颈静脉套管法分流手术,分别在手术后4周,8周,12周,通过心脏血管超声进行无创检查,检测分流血管是否通畅,测量大鼠的肺动脉瓣环内径和主动脉瓣环内径,肺动脉血流频谱测量计算肺动脉血流加速时间,心脏超声探测右心室前壁和左心室后壁厚度,计算厚度指数,并行心尖四腔超声观察右心室的变化,有创检查包括经右心导管及开胸测量大鼠的肺动脉压力。动物处死后肺组织行病理形态学分析,计算各组动物肺小血管的肌化血管的百分比,血管壁厚度指数和相对血管面积指数,从病理学角度观察肺血管的病变,确定是否成功建立肺动脉高压模型。2.取大鼠腹股沟脂肪组织,剪碎后用胶原酶消化,反复过滤离心获取ADSCs,体外培养观察细胞形态,流式细胞仪鉴定不同代细胞的免疫表型,并用不同条件的培养基诱导细胞向脂肪细胞,骨细胞及内皮细胞分化,通过油红染色,碱性磷酸酶染色及Ⅷ因子免疫荧光鉴定其多向分化功能。不同代数的细胞传代时进行台盼兰染色测定细胞活力,MTT法测定体外培养的不同代数细胞的生长曲线,计算细胞倍增时间。酶联免疫吸附法测定常氧和低氧条件下体外培养的大鼠ADSCs分泌的HGF含量,CM-DiL标记细胞并传代观察其标记效率,同时对标记后的细胞进行MTT活力检测以揭示该染料对细胞活力的影响。3.将大鼠颈动静脉分流手术后12周处死的动物模型资料作为基线水平,另有造模12周的动物分为空白对照组,细胞移植组,DMEM组作为阳性对照组,仅作血管分离的假手术组作为阴性对照组。细胞移植组经非手术侧颈静脉给予含5×107个自体ADSCs的培养基,DMEM组仅给予不含细胞的培养基注射,空白对照组为造模12周后的动物不给予任何处理。细胞移植2周后,行超声多普勒检查,右心导管肺动脉测压。处死动物后行肺组织病理检查,冰冻组织切片行免疫荧光检查,观察移植到体内的ADSCs在肺组织的分布,并鉴定其在体内是否继续分泌HGF。免疫组织化学检测ADSCs移植后的HGF含量,Ⅷ因子染色进行肺小血管计数,观察细胞移植后对肺血管数量的影响。Western Blot及RT-PCR测定各组动物肺组织HGF含量及细胞移植后eNOS的表达变化。结果：1.分流手术中因麻醉过量或出血死亡10只,死亡率为13.4%(10/60),手术后因套管脱落及其他原因死亡4只,手术后死亡率为8%(4/50),超声及解剖发现分流手术后存活的46只大鼠有10只动物血管桥阻塞,2只血管桥套管不明原因丢失,从分流组排除,分流通畅率73.9%(34/46)。大鼠颈动静脉分流12周后,超声显示肺动脉瓣环内径(3.52±0.27mm)明显大于主动脉瓣环内径(2.99±0.32mm,P<0.05)。肺动脉血流频谱显示肺动脉血流加速时间(75.31±22.12 ms)较正常对照组(135.14±26.42ms)及分流4周(119.38±12.81 ms),8周时(106.56±31.45 ms)缩短,右心室游离壁与左心室后壁的厚度比值计算分析显示分流12周后(63.02±14.36%)比正常对照组(41.08±4.54%)及4周(40.46±13.41%),8周时(42.24±5.87%)明显增大,P<0.05,心尖四腔图提示分流12周后右心室扩大,收缩期室间隔左向偏移；以上结果间接反映了手术后肺动脉压力的升高。右心导管及开胸测压显示分流组动物的肺动脉收缩压(37.69±7.81 mmHg)较正常对照组(16.59±4.51mmHg)升高,P<0.05。病理学检查发现分流12周后,肺组织小动脉管壁增厚,管腔狭窄,肺小动脉的管壁厚度指数(34.25±9.11%),及管壁面积指数(69.72±13.19%)均比正常对照组(分别为16.71±4.99%,29.05±9.79%)明显增高,P<0.05。2.大鼠腹股沟脂肪组织经过胶原酶消化后,72小时细胞贴壁,成纤维细胞样生长,原代细胞分离种植后7-10天融合至80%左右可以传代,传代后细胞生长旺盛,约3-5天即可再次传代。流式细胞仪测定细胞表面免疫表型,原代细胞CD29, CD105, SCa-1阳性率较高,CD31阳性率稍高,CD45基本阴性,传代2次后细胞CD31基本阴性,其余表型变化不大。油红染色鉴定成脂诱导7天后的细胞可见细胞浆内脂滴染成红色,碱性磷酸酶染色鉴定成骨诱导14天的ADSCs,细胞变长,聚集,细胞浆可见棕红色的钙化沉积颗粒,Ⅷ因子染色显示细胞浆内呈阳性表达。MTT测定细胞的生长曲线显示不同代的细胞生长旺盛,贴壁24-60小时达到倍增时间,72小时后到达平台期。细胞传代时台盼兰染色测定细胞传代15次以内的活力均在90%以上。酶联免疫吸附法测定细胞上清HGF的含量显示：低氧条件下培养的细胞其上清液中HGF含量明显高于常氧条件下培养的细胞,而且细胞代数越低,细胞上清液中HGF的含量越高。CM-DiL标记细胞后进行荧光观察显示细胞标记率高达99%以上,而且进行多次传代后荧光无明显淬灭。标记后的P2代细胞MTT活性检测发现与正常未标记的细胞生长曲线相似,没有明显差异。3.大鼠自体ADSCs移植后2周,超声检查发现肺动脉血流加速时间比造模后未行任何治疗的空白对照组延长(129.58±35.14毫秒VS 80.49±21.29毫秒,P<0.05),心尖四腔图显示右心室室腔减小,收缩期室间隔的左向偏移消失,右心室前壁与左心室后壁的厚度比值较空白对照组明显减小(42.63±8.71% VS 59.39±7.12%,P<0.05)。右心导管及开胸测量肺动脉压力显示细胞移植组的肺动脉压力(19.83±2.32mmHg)较空白对照组的肺动脉压力(35.82±5.09 mmHg)降低,P<0.05。荧光显微镜下观察细胞移植后的肺组织冰冻切片显示移植的细胞大部分聚集在肺血管周围,但是移植的细胞没有形成明显的环状血管样结构。免疫荧光化学分析显示移植到体内的ADSCs仍然表达HGF,免疫组织化学检测表明细胞移植组的肺组织HGF表达高于空白对照组及DMEM组,RT-PCR及Western Blot也提示细胞移植后增加了肺组织HGF的mRNA及蛋白表达。同时RT-PCR及Western Blot检测eNOS的表达显示在细胞移植后eNOS的mRNA及蛋白表达与HGF的变化相一致。Ⅷ因子进行的血管染色分析显示细胞移植后肺小血管明显增多,单位面积内的血管数量大于空白对照组及DMEM组。结论：1.大鼠颈动脉-颈静脉套管法分流手术12周后,血管通路保持通畅可以形成三尖瓣前型动力性肺动脉高压。手术后12周肺血管的变化符合肺动脉高压的病理特征。2.从大鼠腹股沟脂肪组织可以成功分离获取ADSCs。该细胞在形状,表型特征及多向分化的性能方面符合干细胞的标准。细胞在体外培养环境下生长旺盛,多次传代后细胞活力不减。低氧条件下培养的ADSCs较常氧条件下培养的细胞分泌更多的HGF。CM-DiL标记ADSCs效率高,多次传代后荧光不减,可以作为细胞移植示踪的良好标记物。3.ADSCs移植减轻了肺动脉高压动物模型的肺动脉压力,逆转了恶化的血液动力学,减轻了肺小血管的病理改变,改善了肺换气功能。移植的细胞可能通过增加肺组织HGF的分泌,促进肺小血管新生,同时增加了eNOS的含量,减轻了肺组织的血管病变。更多还原

【Abstract】 Background:Hyperkinetic pulmonary arterial hypertension (PAH) is the common complication of congenital heart disease (CHD) with left to right shunt. The opportunity of surgery, success of surgery and the quality of postoperative life were seriously effected by PAH. Now, studies on the mechanism and therapy of PAH have made great progress. It has been recognized that PAH was characterized by an increased resistance in pulmonary circulation and the occlusive remodeling of the pulmonary arterioles. Pathologically, hypertrophy of the media in pulmonary arterioles contributed to the occlusion of the vessels and ultimately, made the decrease of pulmonary vascular bed. Even worse, the increased pulmonary arterial pressure developed higher than before and then deteriorated the pulmonary function. In some cases, pulmonary hypertension induced by CHD could be reversed after surgical treatment. Vasodilation therapy has improved the life quality of patients with PAH. While the obstructive remodeling occurred, mere vasodilation treatment or the surgery could not reverse the pathological changes in the lung. Recent years, therapeutic angiogenesis and the tissue engineering cells for vascular regeneration have been a hot spot in the medical field. Adipose derived stromal cells (ADSCs) as a multipotential stem cell attracted the interests of researchers for its advantages such as the minor invasion and easy culture. ADSCs could secrete some cytokines such as hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) for angiogenesis. HGF was an efficient vasculogenesis factor and could restore the injured organ. Many studies about the application of ADSCs in the ischemia diseases have made success. Previous study has proved that ADSCs implantation could improve the damaged lung in animal models such as emophysematous models via participating in the regeneration of vessels and alveolus or through secreting some cytokines. The perfusion of lung was improved and subsequently the gas exchange function of lung was also restored. But, whether the transplantation of ADSCs could restore the lung of the PAH models has not been reported. So, we put forward the hypothesis that the autologous ADSCs transplantation might improve the function of lung under PAH situation.Objective:To establish a hyperkinetic PAH model, to isolate the ADSCs from rat fat, to label the ADSCs in vitro efficiently before injected, to investigate the hemodynamics and the pathological changes of pulmonary arterioles and to analyze the expression of HGF in the lung for explaining the possible reason for the changes of pulmonary arterial pressure after ADSCs tansplantation.Methods:1. Carotid artery-jugular vein shunt was performed using a cannulation style in rat. Echocardiogram was performed to measure the internal diameter of pulmonary valve and aortic valve, frequency spectrum of pulmonary flow and the thickness of right ventricular anterior wall (RVAW) and left ventricular posterior wall (LVPW) and to investigate right ventricle by apical four chamber view. Invasive examination was the right heart catheter and thoracotomy for the measurement of pulmonary arterial pressure. Pathomorphyology analysis of pulmonary arterioles included vascular wall thickness index (TI), relative vascular wall area index (AI), and the percentage of the muscularized vessels. 2. The fat was obtained form rat inguina and epididymis. A series of filtration and centrifugation was performed after the digestion of collagenase for the isolation of adipose derived stromal cells (ADSCs). Morphous, the immunophenotype, and the multi-directional differentiation of ADSCs such as adipogenic, osteogenic inductions were investigated. Growth curve of ADSCs was draw after MTT test and the doubling generation time was calculated. Enzyme linked immunosorbent assay (ELISA) was used for detecting the HGF in supernatant of ADSCs cultured under hypoxia or normaxia condition in vitro. Cells were labeled by CM-DiL and MTT test was also performed to investigate the cell vitality after labeling.3. The data of the PAH models 12 weeks after A-V shunt was regarded as the baseline data. Another PAH models were separated into blank group (without any treatment after shunt surgery), cell transplantation group (5×107 cells in 0.5 mL DMEM were injected per rat through right jugular vein), DMEM group (injection of 0.5 mL DMEM) and the sham group was taken as negative control. Two weeks after cell transplantation, Echocardiogram, right heart catheter and gas blood ananlysis were performed. Frozen slices and immunofluorescence was made for investigating the location of the transplanted cells and HGF secreted by ADSCs in vivo. Immunohistocytochemistry of HGF and VIII factor were also performed to compare the vascular number among the groups for revealing the possible mechanisms of the decreasing of pulmonary arterial pressure. Real time PCR and Western blot analysis were done to demonstrate the expressions of mRNA and protein levels of HGF and eNOS.Results:1.Ten rats died because of the overdose of anesthesia drugs or bleeding during the operation. The surgery mortality rate was 13.4%(10/60). Four rats died of the loss of cannulation or other accidents. The postoperative mortality rate was 8% (4/50). In the 46 residual living rats,12 animals were excluded from the study because occlusion of the shunt which was detected by echocardiogram. The patency rate was 73.9% (34/46). Twelve weeks after the shunt surgery, echocardiogram showed that the internal diameter of pulmonary valve (3.52±0.27mm) was much wider than the internal diameter of aortic valve (2.99±0.32mm, P<0.05) in animals underwent shunt operation. Pulmonary arterial blood frequency spectrum analysis suggested that the pulmonary arterial acceleration time (PAAT) of the shunt rats (75.31±22.12 ms) was much shorter than that in the normal rats (135.14±26.42 ms) and that in the 4th week (119.38±12.81 ms) and 8th week (106.56±31.45 ms) rats after operation. Twelve weeks after shunt operation, the rate of RVAW to LVPW increased (63.02±14.36%) compared with that in the normal control animals (41.08±4.54%) or that in the 4th week (40.46±13.41%) and 8th week (42.24±5.87%) animals. The enlargement of right ventricle was shown by apical four chamber view in the animals after 12 weeks shunt and the interventricular septum shifting toward left side during systolic period was also displayed by echocardiogram. Pulmonary arterial pressure was higher in the shunt rats (37.69±7.81 mmHg)than that in the normal animals (16.59±4.51mmHg, P<0.05) which was confirmed by the right heart catheter and the thoracotomy for the pressure measurement. Pathological examination for the pulmonary arterioles showed that the TI and AI were both increased in the shunt animals (34.25±9.11% and 69.72±13.19%, respectively) than that in the normal rats (16.71±4.99% and 29.05±9.79%, respectively, P<0.05)2. ADSCs from the rat’s adipose tissue exhibited a fibroblast-like morphology, had the ability to self-renew and adhere to plastic 72 hours after the planting, and extensively expanded in culture without loss of differentiation potential. These cells could be induced into mature adipocytes, which was confirmed by microscopic observation of intracellular lipid droplets after Oil Red O staining. ADSCs also differentiated into osteoblasts, which was evaluated by alkaline phosphatase staining. Immunocytochemistry revealed that these cells showed a positive signal for factor-Ⅷin vitro after the endothelial induction. Fluorescence-activated cell sorter analysis showed that the cultured ADSCs were positive for stem cell such as CD29, CD105 and SCa-1. The percentage of CD31 positive cells was a little higher in the primary cells and decreased after twice passages. MTT examination showed that the ADSCs have high vitality and the doubling generation time was between 24-60 hours since the planting. During the passage, trypan blue stain showed that the rate of living cells maintained over 90% within the passage 15. ELISA results demonstrated that the HGF content in supernatant of the cells cultured under hypoxia condition was much higher than that of the cells cultured under normoxia condition. Furthermore, the younger the cell is, the more the HGF was detected in the supernatant. The labeling rate of CM-DiL in ADSCs was more than 99% and the fluorescence did not quench even after several passages. In addition, the MTT test for the labeled cells with CM-DiL showed that no difference in the growth curve and the doubling generation time compared with the normal cells.3. Two weeks after the ADSCs transplantation, the PAAT was extended than that in the blank control group (129.58±35.14 milliseconds VS 80.49±21.29milliseconds, P<0.05). The decreased size of right ventricle and the disappearance of interventricular septum shift were displayed in the apical four chamber view by echocardiogram and the rate of RVAW/LVPW was also significantly lower compared with that in the blank control group (42.63±8.71% VS 59.39±7.12%, P<0.05) Pulmonary arterial pressure in cell transplantation group (19.83±2.32mmHg) was decreased than that in the blank control group (35.82±5.09 mmHg, P<0.05). The transplanted ADSCs located around the vessels in lung which was observed under fluorescence microscope. Immunofluorescence for the HGF analysis indicated that the transplanted cells secreted HGF in vivo. Furthermore, the cells transplantation augments the HGF expression in lung which was confirmed by RT-PCR and Western blot analysis. In addition, the expression of eNOS in lung was also increased after cell transplantation which was in accordance with the increasing of the HGF. Finally, VIII factor immunohistochemistry stain showed that the vessels in certain area of the lung were much more than that in the blank control group.Conclusion:1. Carotid artery-Jugular vein shunt in rat could establish a hyperkinetic pulmonary arterial hypertension model after 12 weeks if the patent was patent. The pathological alteration conforms to the pre-tricuspid valve hyperkinetic pulmonary hypertension.2. ADSCs could be obtained from rat fat. ADSCs were in conformity with the standard of stem cells on the appearance, immunophenotype and the multidirectional differentiation. ADSCs were of high vitality even in high passage cells. ADSCs cultured under hypoxia condition secreted more HGF than that in cells cultured under normoxia condition. CM-DiL was a good tracking probe for the cell transplantation.3. Autologous ADSCs transplantation ameliorated pulmonary hypertension induced by shunt flow, reversed the deteriorative hemodynamics caused by PAH, and decreased the pathological process of the pulmonary arterioles, therefore, improved the gas exchange function of lung. All the results mentioned above might due to the HGF secretion of ADSCs and subsequently the increased HGF improved the angiogenesis and the eNOS expression in the lung tissue.更多还原