摘要
第一部分大鼠原位左肺移植模型的建立和改进
【目的】建立并改进大鼠原位左肺移植动物模型
【方法】清洁级健康雄性Sprague-Dawley(SD)大鼠100只,体重200-250g。气管插管呼吸机支持下,经肺动脉灌洗LPD液后获取左肺,套管完成后保存于LPD液中备用。受体的手术采取不断肌肉的侧胸切口,肺门结构的游离采取钝性分离。肺门三结构的吻合采用套管法套入。
【结果】所有的供肺套管均顺利完成,移植手术成功45例,失败5例,成功率90%。供肺获取及套管时间25±2min,吻合时间30±4min。失败的原因:3例肺静脉套管失败,1例肺动脉套管失败,另一例为术中大出血休克死亡。
【结论】成功建立并改进了大鼠原位左肺移植的动物模型。该动物模型容易复制,成功率高,有进一步推广的价值。
第二部分供肺保存过程中细胞死亡类型的动态变化
【目的】研究供肺保存过程中细胞死亡类型的动态变化
【方法】清洁级健康雄性SD大鼠42只,体重200-250g。分为7组,每组6只。气管插管呼吸机支持下,经肺动脉灌洗LPD液后整体获取心肺。将心肺整体取出后立即置于4℃新鲜制备的LPD液中保存一定的时间(0,2,4,6,12,18,24小时)。在不同的供肺保存时间点上,经主肺动脉向供肺内灌注台盼蓝进行染色并固定。蜡块上同时进行TUNEL和Hoechst染色。在荧光显微镜下观察,严格按照盲法由两位研究者对死亡细胞进行分类计数。
【结果】随着供肺保存时间的延长,细胞逐渐出现死亡。在早期,细胞死亡以凋亡为主,在供肺保存12小时左右细胞的凋亡达到最高峰,占所有细胞的9.17±2.17%。此后细胞凋亡逐渐下降,在供肺保存18小时组基本消失,整个变化呈现钟形曲线;而细胞的坏死始于供肺保存4小时组,以后逐渐上升,至供肺保存24小时组达到最高峰33.0±2.87%,整个变化呈现向上的抛物线型。
【结论】在供肺的保存过程中,肺实质细胞的死亡在供肺保存的早期以凋亡为主,后期以坏死为主。细胞的坏死是随着供肺保存时间的延长而逐渐增加的。
第三部分供肺保存及移植肺TLR4信号系统的激活
【目的】通过对供肺保存和肺移植两阶段的Toll-like receptor 4(TLR4)及其下游炎症因子的表达的检测,探讨TLR4与肺移植后缺血再灌注损伤的关系。
【方法】清洁级健康雄性SD大鼠42只,体重200-250g。分为7组,每组6只。供肺的获取和保存同前一部分。将心肺整体取出后立即置于4℃的新鲜制备的LPD溶液(100ml LPD液中加入0.7ml 1%碳酸氢钠溶液将其PH值调整到7.40)中保存。在不同的供肺保存时间点上(0、2、6、8、12、18、24h),Western blot方法检测供肺的TLR4蛋白表达水平,RT-PCR方法检测其下游因子TNF-α和iNOS的表达水平。另取SD大鼠42只,体重250-300g。分为7组,每组6只。供肺的获取和保存同前一部分。在不同的供肺保存时间(0、2、6、8、12h)后,在改良的模型上行左肺原位移植术。移植术后30 min,Western blot方法检测供肺的TLR4蛋白表达水平,RT-PCR方法检测其下游因子TNF-α和iNOS的表达水平。对供肺保存过程中的细胞坏死的变化和YLR4的表达做相关性分析。
【结果】供肺保存过程中TLR4表达随着保存时间的推移逐渐的增加,在供肺保存24小时达到最高.其动态变化和细胞坏死规律一致。供肺保存过程中的细胞坏死和TLR4的表达密切的相关(r=0.874,P＜0.01)。不同保存时间点的供肺炎症因子(TNF-α和iNOS)的表达逐渐升高,与其相应的TLR4表达水平呈现正相关(TNF-α,r=0.779,P＜0.01;iNOS,r=0.769,P＜0.0 1)。肺移植后TLR4及其炎症因子TNF-α、iNOS的表达与相应保存时间的供肺相比显著增加,其表达量随着供肺保存时间的延长而逐渐增加。肺移植后30 min的TLR4表达水平与炎症因子TNF-α和iNOS的表达密切相关(TNF-α:r=0.617,P＜0.05;iNOS;r=0.460,P＜0.05)。
【结论】大鼠供肺的保存过程中存在TLR4及其下游炎症因子的的活化,呈时间依赖性,其变化趋势与供肺保存过程中发生的细胞坏死的情况相一致。肺移植后,移植肺TLR4及其下游炎症因子的表达与肺保存期相比显著增加,且与供肺保存时间的长短密切相关。
第四部分PDTC干预对移植肺TLR4信号系统表达和肺功能的影响
【目的】通过吡咯二硫氨基甲酸酯(Pyrrolidine dithiocarbamate,PDTC)阻断TLR4的下游炎症因子的释放,从分子水平、组织水平和移植肺功能评估等方面来考察TLR4信号系统的阻断在大鼠的缺血再灌注损伤中是否具有保护作用。
【方法】SD雄性大鼠30对,体重在200-300g之间。分为两组,对照组和PDTC干预组。每个组又按供肺的保存时间分为0h、2h、6h、8h和12h共5个亚组。每个亚组行六次成功的肺移植实验,收集实验标本。对照组供肺保存于LPD液中,实验组的保存液中加入PDTC(Sigma公司,40mmol/L)。移植术后30 min,Western blot方法检测两组供肺的TLR4蛋白和P50表达水平,EMSA方法检测NF-κB入核的变化。RT-PCR方法检测其下游因子TNF-α和iNOS的表达水平。同时比较两组气道压力、血气分析氧和指数、湿干重比、HE染色、透射电镜等肺功能指标。
【结果】PDTC干预组和对照组相比,TLR4和P50的表达统计学检验均无显著差异(TLR4-p=0.66 1,p＞0.05;P50:p=0.276,p＞0.05)。PDTC干预组和对照组相比,NF-κB入核明显的减少,其差别统计检验有显著意义(P=0.000,P＜0.01);,TNF-α和iNOS表达统计学检验有显著差异(P=0.000,P值均＜0.01)。PDTC干预组与对照组相比较,移植肺的NF-κB入核减少和TLR4下游的炎症因子TNF-α、iNOS表达上的变化密切相关(TNF-α:r=0.636,P＜0.01;iNOS:r=0.633,P＜0.01)。观察移植肺功能,PDTC干预组和对照组相比,肺移植后30 min气道峰压差(P=0.038,P＜0.05)、血气分析氧合指数(P=0.042,P＜0.05)、湿干重比(P=0.001,P＜0.05)、移植肺的肺损伤评分(P=0.017,P＜0.05),统计学检验均有显著差异。
【结论】PDTC预处理不能降低移植肺的TLR4的表达,但是可以阻断TLR4信号系统下游炎症因子的释放。其机制可能通过阻断NF-κB的入核来实现的,而非下调NF-κB的表达量。细胞因子水平的下降与术后的移植肺功能的改善明显相关。因此,PDTC预处理能明显改善移植肺的功能。
PART 1 Establishment and refining of rat' left orthotopic lung transplantation model.
Objective To build up and refine rat left lung transplantation model
Method 100 male Sprague-Dawley rats,weight ranging from 200 to 250 grams,were selected.After general anesthesia with intubation,donor left lungs were harvested individually after perfusing with LPD solution through left main pulmonary artery, then immersed into prepared fresh LPD solution.Muscle-sparing incision was adopted in Recipient lung operations.Blunt dissection was carried out during dissect hilum of left lung.Cuff techniques were introduced in anastomosis of bronchus, pulmonary artery,and pulmonary vein.
Results All cuffs of the donor lungs were accomplished successfully.In all the 50 transplantations,we yielded a 90 percent success rate.Time for harvesting and cuff techniques was 25±2min,and anastomosis time was 30±4min.The reseasons of 5 failed cases were as follows:disrupture of pulmonary vein in 3,disrupture of pulmonary artety in 1,and massive bleeding in 1.
Conclusion Rat lung transplantation model was successfully built up and refined on some techniques.The animal model could be duplicated easily and the success rate was high.
PART 2 Dynamic change of the type of cell death during donor lung preservation
Objective To investigate the dynamic change of cell death during donor lung preservation
Method Forty-two male Sprague-Dawley rats,weight ranging from 250 to 300 grams, were randomized into seven groups.After en bloc harvesting,the donor lung and heart was immersed into fresh prepared LPD solution.At different preservation time points(0,2,4,6,12,18,24hours),the donor lung was perfused with 20 ml 500mM trypan blue,20ml 0.9%saline and 10ml 4%paraformaldehyde through the main pulmonary artery.TUNEL and Hoechst staining were made on the section cuttings. Pathological studies were performed under light microscope.Varies of death cells during preservation were counted under fluorescence microscope by two individual researchers
Results Death cells increased gradually,along with the time of donor lung preservation.At the early stage of preservation,apoptosis was the major type of cell death,the peak of apoptosis appeared in the 12 hours' group,which occupied 9.17±2.17%of the whole.After that point,apoptosis decreased gradually until it almost diminished in 18 hours' group.While necrosis started at 4 hours after preservation, and increased gradually with the peak at 24 hours' group,which occupied 33.0±2.87 %of the whole.
Conclusion During donor lung preservation,apoptosis of the parenchyma cells can be observed in majority at early stage,while necrosis at the late stage.Cell necrosis increased gradually along with the preservation time.
PART 3 Activation of TLR4 signal system during donor lung preservation and after transplantation
Objective Based on the research of the expressions of TLR4 and its downstream inflammation factors during donor lung preservation and after transplantation,the possible mechanism of TLR4 signal system activation correlated with ischemia and reperfusion injury during rat lung transplantation was investigated.
Method Forty-two male Sprague-Dawley rats were randomized into seven groups. The donor lung was harvested and preserved at the same manner.TLR4 expression on donor lungs were detected by western blot at different preserve time(0,2,6,8,12,18, 24hours),and downstream inflammation factors TNF—αand iNOS were detected by RT-PCR.Another thirty male Sprague-Dawley rats were randomized into another five groups for transplantation.Orthopedic left lung transplantation were performed by using the donor lungs at different preserve time(0,2,6,8,12hours).30 minutes after lung transplantations,TLR4 expression in donor lungs were detected by western blot, and downstream inflammation factors TNF—αand iNOS by RT-PCR.
Results Expression of TLR4 increased along with the time of lung preservation,the peak was appeared in 24 hours' group.The dynamic change was similar to the cell necrosis.The correlation between them showed significantly difference(r=0.874, P＜0.01).The same phenomenon can be seen in the relationship of inflammation factors (TNF—αand iNOS) with TLR4(TNF-α,r=0.779,P＜0.01;iNOS,r=0.769,P＜0.01). After transplantation,the expressions of TLR4 and inflammation factors(TNF—αand iNOS) were significantly increased,compared with those at the correspondent time point of preservation.30 minutes after transplantation,the relationship of TLR4 expression with TNF—αand showed significant difference(TNF—α:r=0.617, P＜0.05:iNOS:r=0.460,P＜0.05).
Conclusion Activation of TLR4 and its downstream inflammation factors and increasement along with the preserve time can be revealed during donor lung preservation.This phenomenon was parallel with cell necrosis at the same time.The expression of TLR4 and inflammatory factors were increased 30 minutes after transplantation and yielded positive relation with the time of donor lung preservation.
PART 4 Influence of PDTC on TLR4 signal system and function of the transplanted lung
Objective Based on the former research,Pyrrolidine dithiocarbamate(PDTC ) which is the specific blocker of NF-κB were added into preservation solution to observe the possible protective effect of transplanted lung.
Method sixty male Sprague-Dawley rats,weigh ranging between 200-300g were randomized into two groups(PDTC group and control group).Each group contained 5 sub-groups according to its preservation time(0,2,6,8,12 hours).Six success lung transplantations were carried out in every subgroup.PDTC were added in the reseach group(Sigma,40mmol/L).30 minutes after the lung transplantations,the expression of TLR4 and P50 were detected by western blot,DNA binding activity of NF-κB by EMSA.Expression of iNOS and TNF—αwere detected by RT-PCR.In the aspect of lung function,airway pressure,oxygen indexes,wet/dry ratio,manifestation of lung injury on HE staining and transmission electron microscopy were compared between the two groups.
Results No significant difference in expression of TLR4 and P50 were yielded between the PDCT group and control group(TLR4:p=0.661,p＞0.05;P50:p=0.276, p＞0.05).DNA binding activity of NF-κB of PDTC group reduced dramatically, compared with corresponding control groups(p=0.000,p＜0.01 ) as well as the expression of TNF—α(p=0.000,p＜0.01 ) and iNOS(p=0.000,p＜0.01 ) decreased dramatically,compared with corresponding control groups.In PDTC and control groups,Correlation analysis between decreasing DNA binding activity of NF-κB and down regulation of TLR4 signal downstream inflammation factors TNF—αand iNOS showed significant difference(TNF—α:r=0.636,P＜0.01;iNOS:r=0.633, P＜0.01 ) ).As to lung function after transplantation,in PDTC groups,the peak air pressure 30 minutes after lung transplantation deceased(p=0.038,p＜0.05),the oxygen indexes increased(P=0.042,P＜0.05),wet/dry ratio deceased(P=0.001, P＜0.05),as well as lung injury assessment(P=0.017,P＜0.05),compared with corresponding control groups.
Conclusion PDTC pretreatment of donor lung could not block the expression of TLR4 and P50,but it could down regulate the TLR4 downstream inflammation factors dramatically.The process was approached by decreasing the DNA binding activity of NF-κB,not by down regulate NF-κB expression.The blockage was almost completely.The declined expression of inflammation factors was close correlation with the improvement of transplant lung function.In a word,PDTC pretreatment could improve the function of transplanted rat lung.
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