【作者】 陈舒晨；

【导师】 陈良万； 康明强；

【作者基本信息】 福建医科大学 ， 外科学， 2009， 博士

【摘要】 第一部分改良三袖套法建立大鼠左肺原位移植动物模型目的采用改良袖套法技术建立大鼠同种异体左肺移植模型。方法对供肺获取、套管制作、自体肺切除、血管和气管袖套套接技术进行改进。30例清洁级SD大鼠左供肺获取后置于40C的LPDG液保存3小时,随后行肉眼直视下同种异体左肺移植术,再灌注4h后阻断右肺门,分别测定阻断前后气道压力、动脉血气变化、肺湿/干重比率及光镜下肺组织结构变化。评价该模型复制移植物缺血再灌注损伤的能力。结果同种异体左肺移植模型在肉眼直视下单人完成,供受体动静脉和支气管套管吻合时间分别为:肺动脉(5±1)min、肺静脉(9±4)min、支气管(2±0.5)min。总手术时间(59±10)min。手术成功率90%(27/30),术后生存率100%。实验结果显示该模型成功复制出同种异体肺移植缺血再灌注损伤的变化。结论本模型符合大鼠肺移植模型建立的优点,全部操作在肉眼直视下单人完成;供肺的获取较为快速,使用的套管更简单,套管技术趋于简化,肺移植时血管套接技术成功率高,整体手术时间操作缩短。该模型复制出移植肺缺血再灌注损伤的变化,是适合肺移植缺血再灌注损伤研究的理想模型。第二部分骨髓间充质干细胞的分离、培养、鉴定、标记及其对大鼠移植肺缺血再灌注损伤的保护作用目的分离、培养、鉴定和标记骨髓间充质干细胞(BMSCs),观察BMSCs对大鼠左肺移植物缺血再灌注损伤的保护作用并探讨其作用机制。方法以SD大鼠为供受体,采用改良袖套法技术建立同种异体左肺移植模型。设立假手术组(A组)、I/R组(B组)和BMSCs组(C组),每组n=7,离体供肺静脉40C的LPDG液逆行灌注,灌注后行左肺原位移植,移植肺再灌注4h后,阻断右肺门,测量气道压力,动脉血PaO2、PaCO2。随后处死大鼠,分别测定:肺W/D比率、肺组织中SOD活性、MDA含量、MPO活性;光镜观察移植物病理组织学变化;RT-PCR和Western-blot了解旁分泌因子VEGF的表达;ELISA检测受鼠血浆中TNF-a和IL-10的变化;免疫组化检测TNF-a和bcl-2表达情况;TUNEL法检测移植肺细胞凋亡水平。结果与假手术组相比,I/R组气道压力升高、PaO2下降、肺W/D比率上升,SOD活性降低,MDA含量和MPO活性升高(P<0.01),表明I/R组相对于假手术组确实造成了明显的缺血再灌注损伤;与I/R组相比,BMSCs组气道压力降低、PaO2上升、肺W/D比率下降,SOD活性增高,MDA含量和MPO活性下降(P<0.01);与I/R组相比,BMSCs组:血浆TNF-a水平降低,IL-10水平升高;病理检查肺泡间质水肿明显减轻,炎症细胞浸润减少,肺泡腔内水肿液和红细胞少见;免疫组化TNF-a表达减弱、bcl-2表达增强(P<0.01);TUNEL法检测移植肺细胞凋亡减少(P<0.01)。基因和蛋白水平检测显示BMSCs组肺组织旁分泌因子VEGF较假手术组、I/R组明显升高(P<0.01)。结论(1)氧化损伤等指标检测表明I/R组确实复制了移植肺的IRI;BMSCs移植能减轻移植肺的缺血-再灌注损伤。(2)BMSCs通过其抗氧化、抗炎、抗凋亡和旁分泌效应等抗损伤、促组织修复等多种途径明显减轻移植肺缺血再灌注损伤,提高肺移植术后早期肺功能。因此BMSCs移植可成为预防和减轻肺移植术后缺血再灌注损伤的一种有益尝试。第三部分缺血后适应基础上骨髓间充质干细胞移植对大鼠移植肺缺血再灌注损伤的保护作用目的观察缺血后适应(I-postC)基础上BMSCs移植对大鼠同种异体左肺移植物缺血再灌注损伤的保护作用并探讨其作用机制。方法以SD大鼠为供受体,采用改良三袖套法技术建立同种异体左肺移植模型。设立I/R组(A组), I-postC组(B组),BMSCs组(C组)和I-postC + BMSCs组(D组),每组n=14,离体供肺静脉40C的LPDG液逆行灌注,灌注后行左肺原位移植,移植肺再灌注4h和72h后,分别随机抽取大鼠7只阻断右肺门,测量气道压力,动脉血PaO2、PaCO2。随后处死大鼠测定:肺W/D比率、肺组织中SOD活性、MDA含量、MPO活性;光镜观察移植物病理组织学变化;RT-PCR和Western-blot了解旁分泌因子VEGF的表达;ELISA检测受鼠血浆中TNF-a、IL-10、G-CSF和GM-CSF的变化;免疫组化检测TNF-a和bcl-2表达情况;TUNEL法检测移植肺细胞凋亡水平。免疫荧光法检测肺组织中BMSCs的定位情况。结果基因和蛋白水平检测显示单纯BMSCs组、I-postC+BMSCs组肺组织旁分泌因子VEGF表达较I/R组、I-postC组明显升高(P<0.01); G-CSF,GM-CSF(72h)明显升高(P<0.01)。与I/R组比较,I-postC组、BMSCs组、I-postC+BMSCs组:TNF-a明显降低,IL-10明显升高(P<0.01);气道压力降低、PaO2上升、肺W/D下降、SOD活性增高、MDA含量和MPO活性下降(P<0.01);bcl-2表达增强,TUNEL法检测移植肺细胞凋亡减少(P<0.01)。与I/R组、I-postC组、BMSCs组相比,光镜检查I-postC+BMSCs组肺泡间质水肿减轻最显著,炎性细胞浸润、肺泡腔内水肿和渗出改变减轻最明显。免疫荧光法检测72h取的肺组织,BMSCs在肺组织中定位,主要分布在肺细支气管壁周围。结论在I-postC的基础上,再行BMSCs移植,减轻移植肺缺血再灌注损伤的程度最显著。I-postC基础上BMSCs移植减轻移植肺IRI、提高肺移植术后早期肺功能的机制与其抗氧化、抗炎、抗凋亡、旁分泌效应和干细胞归巢等密切相关,并可能与I-postC先减轻移植肺IRI,改善BMSCs存活的微环境,提高BMSCs迁移进入肺组织的“质”和“量”有关。因此I-postC基础上BMSCs移植可成为预防和减轻同种异体肺移植物IRI的一种全新的策略。更多还原

【Abstract】 Part1.The Improvements in Establishment of Rat Orthotopic Left Lung Transplantation Model.Objective To establish rat orthotopic left lung allograft transplantation model by improved techniques. Methods This study improved the traditional cuff technique in many aspects, including graft retrieval, cuff self-making, recipient pneumonoresection,“muff-like”vessel and trachea anastomosis techniques. In a group with 30 pairs of clean-grade SD rats, the left lung allograft was stored at 4 oC LPDG solution for 3 h, and then the left lung was transplanted into the recipient rat without microscope. When the transplanted lung had been reperfused for 4 h. Airway pressure, blood gas analysis were detected before and after the right hilus of lung was block up, then left lung was removed, and W/D, histological changes under microscopy were determined. The reproducible ability of this model about ischemic- reperfusion injury of donor lung was evaluated. Results 30 pairs of rats receiving transplantation were performed the anastomosis of pulmonary artery, pulmonary vein and bronchus by single person without microscope. The time consuming was (5±1)min, (9±4)min(,2±0.5)minutes respectively. The total operational time consuming was (59±10)min. The operation successful rate was 90%, survival rate arrived 100%. The experimental outcome demonstrated that the model could duplicated the change of the ischemia-reperfusion injury. Conclusion The merit of this model corresponds with the construction of rat lung transplantation model. All the manipulations were performed by single person without microscope. The harvesting of donor lung was so fast that ischemic time was nearly 0 min. The cuff structure and the anastomosis technique were simpler than before. The improved“muff-like”technique increased the successful rate and shortened the total operational time consuming. This model duplicated the changes of ischemia reperfusion injury on lung allograft, and was demonstrated to be an ideal and suitable model for some researches just like IRI of donor lung .Part2.BMSCs Were Isolated, Cultivated, Identified and Marked,and Its Protective Effects on Ischemia-reperfusion Injury of Pulmonary Allograft in RatsObjective Bone marrow stromal stem cells were isolated, cultivated, identified and marked,and its protective effects on ischemia-reperfusion injury of pulmonary allograft in rats and explore the mechanisms. Methods A left lung transplantation formwork was constructed by using SD rat both donor and recipient by improved muff-like technique. Three groups of rats were examined. The donors in group 1 were sham operated,the donors in group 2 sufferd from ischemia-reperfusion injury,the donors in group 3 were received BMSCs .Each group have seven recipient. The donor lungs were all ex vivo reperfused by 4oC LPDG through pulmonary vein . After the left lung allograft was reperfused for 4 hours, we blocked the hilum of right lung, then measured airway pressure and made arterial blood gas analysis to measure PaO2 and PaCO2 .At last, rat was executed. Lung allograft tissue was harvested to be detected. W/D ratio, superoxide dismutase(SOD) activity, malondialdehyde (MDA) contents and myeloperoxidase(MPO) activity were detected. Graft pathologic histology was examined under light microscope. The message of expression of VEGF in donor lung tissue was measured by Real-time PCR and Western-blot. The content of TNF- a and IL-10 were quantified by ELISA. The expression of TNF-a、and bcl-2 were detected by immunohistochemical staining. Apoptotic cell death was determined by TUNEL. Results Compared with the sham operation group , the airway pressure, PaO2 ,the activity of SOD decreased and W/D ratios, content of MDA, activity of MPO increased(P<0.01)in the ischemia-reperfusion group,which demonstrated ischemia-reperfusion injury do occurred in the ischemia-reperfusion group. Compared with the ischemia-reperfusion group, the airway pressure, PaO2 ,the activity of SOD increased and W/D ratios, content of MDA, activity of MPO decreased(P<0.01)in the ischemia-reperfusion group. In the group of BMSCs, tissue edema, interstitial inflammation and exudation alleviated under light microscope, lower expression of TNF- a and higher expression of IL-10 were detected by ELISA(P<0.01), lower expression of TNF- a and higher expression of bcl-2 were detected by immunohistochemistry(P<0.01),Less apoptotic cells were found by TUNEL(P<0.01). Paracrine secretion cytokine of VEGF in the group of BMSCs was obviously higher than the sham operation group and the ischemia-reperfusion group (P<0.01) . Conclusion (1)Compared with the sham operation group , ischemia-reperfusion injury do occurred in the ischemia-reperfusion group.The transplantations of BMSCs reduced ischemic-reperfusion injury of lung allograft. (2) BMSCs reduced ischemic-reperfusion injury of lung allograft through means of anti-oxidative, anti-inflammatory and anti- apoptosis, paracrine secretion effect.So the transplantations of BMSCs may become a beneficial try which can prevent and reduce the IRI of lung allografts.Part3.Protective Effects Brought by Transplantations of Bone Marrow Stromal Stem Cells Based on Ischemic Postconditioning upon Ischemia- reperfusion Injury of Pulmonary Allograft in RatsObjective To investigate protective effects brought by transplantations of bone marrow stromal stem cells based on ischemic postconditioning upon ischemia-reperfusion injury of pulmonary allograft in rats and explore the mechanisms. Methods A left lung transplantation formwork was constructed by using SD rat both donor and recipient with improved muff-like techniques. Methods A left lung transplantation formwork was constructed by using SD rat both donor and recipient with improved muff-like techniques. The experiment was divided into four groups: In group A,open pulmonary blood flow directly to replicate ischemia-reperfusion injury . In group B, the donor lung suffered ischemic postconditioning before IRI. In group C, administration of BMSCs after IRI. In group D, administration of BMSCs after ischemic postconditioning. Each group have fourteen recipient. Then the left lung orthotopic transplantation was performed. After the left lung allograft was reperfused for 4 hours and 72 hours, extract seven rats randomly .,we blocked the hilum of right lung, then measured airway pressure and made arterial blood gas analysis to measure PaO2 and PaCO2 . At last, rat was executed. Lung allograft tissue was harvested to be detected W/D ratio, superoxide dismutase (SOD) activity, malondialdehyde (MDA) contents and myeloperoxidase(MPO) activity. Graft pathologic histology was examined under light microscope. The message of expression of VEGF in donor lung tissue was measured by Real-time PCR and Western-blot. The content of TNF- a , IL-10, G-CSF and GM-CSF were quantified by ELISA. The expression of TNF-a、and bcl-2 were detected by immunohistochemical staining. Apoptotic cell death was determined by TUNEL. Localization of BMSCs was detected through immumofluorescence method. Results Compared with group A and B, paracrine secretion cytokine of VEGF which detected by Real-time PCR and Western-blot was obviously increased in group C and D,so the G-CSF,GM-CSF detected by ELISA. Compared with group A , the airway pressure, PaO2 ,the activity of SOD decreased and W/D ratios, content of MDA, activity of MPO increased(P<0.01)in group B,C and D.TNF- a decreased ,IL-10 increased(P<0.01). Lower expression of TNF- a and higher expression of bcl-2 were detected by immunohistochemistry(P<0.01),Less apoptotic cells were found by TUNEL(P<0.01). In group D, tissue edema, interstitial inflammation and exudation alleviated significantly under light microscope. Through immunofluorescence method,we detected BMSCs were located in lung tissue after 72 hours. Conclusion Protective effects brought by transplantations of bone marrow stromal stem cells based on ischemic postconditioning upon ischemia-reperfusion injury of pulmonary allograft in rats were significant.Its mechanisms may through means of anti-oxidative, anti-inflammatory and anti- apoptosis, paracrine secretion effect and homing of BMSCs.Reduce ischemic-reperfusion injury of lung allograft by ischemic postconditioning firstly,and then administration of BMSCs may improve the microenvironment of which BMSCs survived. This study demonstrated that the transplantations of bone marrow stromal stem cells based on ischemic postconditioning upon ischemia-reperfusion injury of pulmonary allograft in rats may become a new strategy, which can reduce the IRI of lung allografts.更多还原