摘要
第一部分:猪自体肺移植模型的建立及不同灌注液对移植肺损伤的影响
目的:
建立猪自体肺叶移植的实验动物模型;观察不同的肺灌注方法对猪自体肺叶移植术后肺组织光镜及超微结构、氧自由基水平、气体交换能力变化的影响,为临床开展肺叶移植术提供参考依据。
方法:
本地健康杂种猪18只,体重49.7±3.0kg,雌雄不限,随机分为对照组(A组)、常温肝素灌注组(B组)及低温EC液灌注组(C组)。各组均在全肺切除后立即行自体肺叶移植(左侧全肺切除+左肺下叶自体肺叶移植),对照组不予任何处理,常温肝素灌注组常温下予肝素液灌注供肺,低温EC液组予4℃Euro-Collins液灌注。分别于移植前、移植后0.5h、1h、2h、4h检测移植肺的肺静脉血氧分压(PvO2)、二氧化碳分压(PvCO2)、肺动态顺应性(Cdyn)、平均动脉压(mPAP),移植肺组织中的氧自由基髓过氧化物酶(MPO)活性及丙二醛(MDA)含量;移植前、移植后0.5h、1h、2h、4h取移植肺组织,观察移植肺组织的湿干重比(W/D)、光镜及电镜结构变化。
结果:
1、移植前,移植后0.5h、1h、2h,三组移植肺的肺静脉血PvO2、PvCO2及动态肺顺应性均无显著性差异(P>0.05);随着再灌注时间的延长,三组的PvO2及Cdyn下降,而PvCO2上升;与术前相比,对照组移植后2h的PvO2差异有显著性(P<0.05),移植后4h的PvO2、PvCO2及Cdyn差异均有显著性(P<0.05);与对照组相比,常温肝素组及低温EC液灌注组移植后4h的PvO2、PvCO2及Cdyn差异有显著性(P<0.05)。
2、移植前,移植后1h、2h、4h,三组移植肺的mPAP没有差异;与术前相比,对照组术后0.5h的mPAP有显著性差异(P<0.05);与对照组相比,常温肝素组及低温EC液组术后0.5h的mPAP差异有差异性(P<0.05)。
3、移植前,移植后0.5h三组的肺组织中的MDA及MPO的含量没有差异(P>0.05);与术前相比,对照组术后1h的MPO有显著性差异(p>0.05),而对照组术后2h及4h、常温肝素组及低温EC液组术后4h肺组织中的MPO及MDA有非常显著差异(P<0.01);与对照组相比,常温肝素组及低温EC液组术后4h的MPO及MDA差异显著(P<0.05)。
4、移植前,三组肺组织湿干重比、病理光镜评分、光镜及电镜结构均无显著差异;与术前相比,对照组术后1h,常温肝素组及低温EC液组术后2h的肺湿干重比有显著差异(P<0.05),对照组术后2h、4h,常温肝素组及低温EC液组术后4h的湿干重比有非常显著差异(P<0.01);与对照组相比,常温肝素组及低温EC液组术后4h的肺干湿重比、病理光镜评分、光镜及电镜结构有显著差异(P<0.05)。
结论:
1、猪自体肺叶移植在技术上是可行的,猪自体肺叶移植实验具有较好的稳定性和可重复性,是评价肺叶移植后移植肺功能及各项指标较为理想的动物模型。
2、无论是常温肝素液还是低温EC液,对供肺叶的灌注均能较好地保持移植肺的组织结构,减轻肺的缺血再灌注损伤,保护移植肺的气体交换功能。
3、常温肝素灌注后的供肺叶能满足自体肺叶移植的要求。
第二部分:猪自体肺叶移植热缺血时限的实验研究
目的:
观察不同的肺热缺血时间对猪自体肺叶移植术后肺组织结构、氧自由基水平及气体交换能力变化的影响,明确肺耐受热缺血的时限,初步探讨其可能机理,为临床开展肺叶移植术提供参考依据。
方法:
本地健康杂种猪36只,体重49.5±2.3kg,雌雄不限,随机分为常温肝素对照组(B组)、常温肝素热缺血60分钟组(B1组)、常温肝素热缺血120分钟组(B2组)、低温EC液对照组(C组)、低温EC液热缺血60分钟组(C1组)及低温EC液热缺血120分钟组(C2组),其中B组及C组为第一部分所用动物。各组均行自体肺叶移植(左侧全肺切除+左肺下叶自体肺叶移植),对照组肺切除后立即予常温肝素或4℃E uro-Collins液灌注供肺,热缺血60分钟组则在缺血60分钟后予常温肝素或4℃E uro-Collins液灌注供肺,热缺血120分钟组则在缺血120分钟后予常温肝素或4℃E uro-Collins液灌注供肺。分别于移植前,移植后0.5h、1h、2h、4h检测移植肺的肺静脉血氧分压(PvO2)、二氧化碳分压(PvCO2)、肺动态顺应性(Cdyn)、平均动脉压(mPAP),移植肺组织中的氧自由基髓过氧化物酶(MPO)活性及丙二醛(MDA)含量;移植前,移植后0.5h、1h、2h、4h取移植肺组织,观察移植肺组织的湿干重比(W/D),移植前及移植后4h移植肺叶组织光镜及电镜结构变化;测定移植后4h肺静脉血浆肿瘤坏死因子(TNF-α)及基质金属蛋白酶(MMP)-9浓度及移植肺组织中MMP-9含量。
结果:
1、移植前,移植后0.5h、1h、2h,六组移植肺的肺静脉血PvO2、PvCO2及动脉肺顺应性均无显著性差异(P>0.05);随着再灌注时间的延长,各组的PvO2及Cdyn下降,而PvCO2上升;与低温EC液缺血120分钟及常温肝素对照组相比,常温肝素120分钟组移植后4h的PvO2、PvCO2及Cdyn差异有显著性(P<0.05)。
2、移植前,移植后1h、2h、4h,各组移植肺的mPAP没有差异;与低温EC液缺血120分钟及常温肝素对照组相比,常温肝素120分钟术后0.5h的mPAP有显著性差异(P<0.05)。
3、移植前,移植后0.5h各组的肺组织中的MDA及MPO的含量没有差异(P>0.05);与低温EC液缺血120分钟及常温肝素对照组相比,常温肝素120分钟组术后2h的MPO有显著性差异(p>0.05),术后4h肺组织中的MPO及MDA有非常显著差异(P<0.01)。
4、移植前,六组肺组织湿干重比、病理光镜评分、光镜及电镜结构均无显著差异;与低温EC液缺血120分钟及常温肝素对照组相比,常温肝素120分钟术后2h、4h的湿干重比有非常显著差异(P<0.01),病理光镜评分、光镜及电镜结构有显著差异(P<0.05)。
5、移植后4h,与常温肝素对照组及低温EC缺血120分钟相比,常温肝素缺血120分钟组的血浆TNF-α、MMP-9浓度有非常显著差异(P<0.01),肺组织MMP-9阳性评分也有显著差异(P<0.05)。
结论:
1.移植肺叶的组织结构破坏、缺血再灌注损伤及气体交换能力下降程度随热缺血时间延长而加重。
2.至少在60分钟的热缺血时间内,单纯应用常温肝素液灌注猪的肺叶能满足移植需要;而在120分钟内,应用低温EC液灌注猪的肺叶也能满足移植需要。
3.相对于常温肝素灌注,应用低温EC液灌注移植肺叶效果更好的原因可能是低温EC液灌注更能抑制血浆TNF-α、MMP-9及肺组织MMP-9浓度的升高。
第三部分:自体肺叶移植术治疗上叶中心型肺癌的临床研究
目的:
本研究旨在探讨临床应用自体肺叶移植技术根治切除上叶中央型肺的可行性及其益处,比较同期全肺切除术、支气管肺动脉双袖状切除及自体肺移植术的围术期死亡率(30天以内)、并发症发生率、生存时间及肺功能、生活质量的差异,并为自体肺移植术进一步临床应用提供更多经验及依据。
方法:
自2000年8月至2006年8月,自体肺移植病人共7例。5例作双袖状左上叶联合肺叶切除,2例右上中叶联合肺叶切除,因主支气管或肺动脉切除过长,吻合张力过大,遂切断下肺静脉,肺短时间离体后作下叶移植,将下肺静脉移植在上肺静脉残端。选择2000年8月至2006年8月在我科行支气管肺动脉双袖状切除的病人41例,全肺切除术的非小细胞肺癌(NCLC)的病人194例。比较三组病人主要的手术并发症,如支气管胸膜瘘,围术期并发症,围术期死亡率,术后肺功能、长期生活质量以及长期生存时间。
结果:
随访至2006年8月,7例自体肺移植病人中5例已无瘤存活2-73个月,生活质量良好, 1例因术后2天肺静脉血栓行重植肺切除,术后15月死于肺癌复发合并呼吸衰竭;另1例术后31个月死于肺癌脑转移。自体肺移植组病人预计中位生存时间为71个月,未发生吻合口瘘,无手术死亡。双袖状切除组病人支气管胸膜瘘的发生率为2.4%(41例中1例),无围术期死亡,预计中位生存时间为36个月。全肺切除组病人支气管胸膜瘘的发生率为3.6%(194例中7例),而围术期死亡率为2.6%(194例中5例),预计中位生存时间为30个月。与全肺切除组相比,自体肺移植组和双袖状切除组病人的术后肺功能及生活质量存在明显差异(P<0.05);与双袖状切除组相比,自体肺移植组病人的术后肺功能及生活质量无明显差异。
结论:
对心肺功能不能耐受全肺切除的III期上叶中心型肺癌病人,自体肺移植术是一种可供选择的、能保全肺组织的肺癌根治术式。自体肺移植术在技术条件成熟时,效果与袖状切除手术相似,比全肺切除术能更好的保护患者的肺功能,提高患者的生活质量,有利于降低患者的围术期死亡率及并发症率,以及延长患者生存时间。
Part I: Establishment of a porcine model of lobar lung auto-transplantation and the effects of different pulmonary infusion on the transplanted lung injury
Objective:
To duplicate the swine model of left single lung auto-transplantation; to compare the difference of pulmonary function and other parameters within different pulmonary artery perfusion in this model.
Methods:
Eighteen healthy local pigs were used as experimental animals, the body weight was 49.7±3.0 kg. The animals were divided into three groups randomly (n = 6 in each group); control group (group A), received left pneumonectomy and lobar lung auto-transplantation (left lower lobe) without pulmonary artery perfusion; Normothermic heparin group (group B), received left pneumonectomy and lobar lung auto-transplantation (left lower lobe) and pulmonary artery infused with heparin in normal temperature; Cold EC group (group C), received left pneumonectomy and lobar lung auto-transplantation (left lower lobe) and with cold Euro-Collins pulmonary artery perfusion. The blood sample and lung tissues were collected at baseline, 0.5, 1, 2, and 4 hours after lobar lung auto-transplantation respectively. The indices were determined as follows: the oxygen pressure of pulmonary vein blood (PvO2), the carbon dioxide pressure of pulmonary vein blood (PvCO2), dynamic lung compliance (Cdyn) and mean pulmonary artery pressure (mPAP) of the transplanted lobar lung, the malondialdchyde (MDA) and myeloperoxidase (MPO) content of the transplanted lung tissue, and the lung water content of the transplanted lung tissue. The lung tissue wet/dry ratio (W/D) was analyzed simultaneously. At 4 h after lung (or lobar lung) auto-transplantation, the pulmonary grafts were sampled for histological examination and ultra-structure observation under the electronic microscopes.
Results:
1、There were no statistical significance of the PvO2, PvCO2 and Cdyn levels within the three groups at the time-points of the baseline, 0.5, 1, or 2 hours after transplantation. The levels of PvO2 and Cdyn decreased after reperfusion while PvCO2 increased in all groups. Compared with the baseline, PvO2 levels at time-point of 2 and 4 hours post-operatively and PvCO2 levels at time-point of 4 hours post-operatively, Cdyn levels at time-point of 4 hours post-operatively were significantly different in group A (P<0.05). Compared to group A, 4 hours post-operative PvO2, PvCO2 and Cdyn levels have significant difference in group B and C (P<0.05).
2、At the time-points of the baseline, 1, 2, and 4 hours after auto-transplantation, mPAP levels have no difference within the three groups. It increased immediately after surgery and decreased thereafter. At 0.5 hour after transplantation, the mPAP level was significantly elevated in group A compared with the baseline (P<0.05). However, the levels in groups B and C were markedly retarded compared with group A (P<0.05).
3、At the time-points of baseline, 0.5, 1, and 2 hours after transplantation, there were no statistical significance in all groups with respect to MDA and MPO contents, which elevated immediately after transplantation in all groups. Compared with the baseline, MDA concentrations at 2 and 4 hours post-operatively and MPO contents at 1, 2, and 4 hours post-operatively were significantly increased in group A. Compared to the baseline, 4 hours post-operative MDA and MPO contents were significantly elevated?? in group B and C (P<0.05). Compared with group A, MDA and MPO levels at 4 hours after operation were significantly decreased in group B and C (P<0.05).
4、The lung tissue W/D ratio, ultra-structure and structure within the three groups at the baseline were comparable. The W/D ratio elevated immediately after reperfusion in all groups. When compared with the baseline, W/D ratio were remarkably raised at 1, 2, and 4 hours after transplantation in group A (P<0.05) and at 2 and 4 hours after transplantation in group B and C (P<0.01). Moreover, as compared with group A, both normothermic heparin and cold EC perfusion decrease the W/D ratio (P<0.05), as well as retarding the injury under electronic and light microscopy in group B and C at 4 hours after transplantation.
Conclusions:
1. It is practicable of the establishment of porcine model of lobar lung auto-transplantation. This model has satisfactory stability and repeatability. Consequently, it is an ideal experimental animal model for the evaluation of the pulmonary function and other parameters after lobar lung transplantation.
2. Pulmonary artery perfusion either with normothermic heparin or cold Euro-Collins solution can preserve the transplanted lobar lung structure, reduce the ischemia-reperfusion injury, and maintain the gas exchange function.
3. Pulmonary artery infusion with normothermic heparin meets the requirement of lobar lung auto-transplantation.
Part II: Warm ischemic time limit of porcine lobar lung auto-transplantation
Objective:
To observe the change of pulmonary function and other parameters such as structure and oxyradical levels after porcine lobar lung auto-transplantation between different pig left pulmonary ischemic periods; to determine the largest warm ischemic time limit of lobar lung transplantation and explore the possible involved mechanisms.
Methods:
hirty-six healthy local pigs were used as experimental animals, and the body weight was 49.5±2.2 kg. The animals were divided into six groups randomly (n=6 in each group); Normothermic heparin without ischemia group (group B), received left pneumonectomy and lobar lung auto-transplantation (left lower lobe) without additional pulmonary circulation occlusion and pulmonary artery infused with heparin in normal temperature; Normothermic heparin with 60 min ischemic period group (group B1), received left pneumonectomy and lobar lung auto-transplantation (left lower lobe), pulmonary artery infused with heparin in normal temperature and additional 60 min pulmonary circulation occlusion; Normothermic heparin with 120 min ischemic period group (group B2), received left pneumonectomy and lobar lung auto-transplantation (left lower lobe), pulmonary artery infused with heparin in normal temperature and additional 120 min pulmonary circulation occlusion; Low temperature EC without ischemia group (group C), received left pneumonectomy and lobar lung auto-transplantation (left lower lobe) without additional pulmonary circulation occlusion and with cold Euro-Collins pulmonary artery perfusion. Low temperature EC with 60 min ischemic period group (group C1, ), received left pneumonectomy and lobar lung auto-transplantation (left lower lobe) with cold Euro-Collins pulmonary artery perfusion and 60 min pulmonary circulation occlusion; Low temperature EC with 120 min ischemic period group (group C2), received left pneumonectomy and lobar lung auto-transplantation (left lower lobe) with cold Euro-Collins pulmonary artery perfusion and 120 min pulmonary circulation occlusion. The blood sample and lung tissues were collected at baseline, 0.5, 1, 2, 4 hours after lobar lung auto-transplantation. The indices were determined as follows: the oxygen pressure of pulmonary vein blood (PvO2), the carbon dioxide pressure of pulmonary vein blood (PvCO2), dynamic lung compliance (Cdyn) and mean pulmonary artery pressure (mPAP) of the transplanted lobar lung, the malondialdchyde (MDA) and myeloperoxidase (MPO) content of the transplanted lung tissue, and the lung water content of the transplanted lung tissue were detected. The lung tissue wet/dry ratio (W/D) was analyzed simultaneously. At 4 hours after lung (or lobar lung) auto-transplantation, the transplanted lung tissue was sampled for histological examination and ultra-structure observation under the electronic microscopes. Additionally, we measured the serum levels of tumor necrosis factor-alpha (TNF-α) and matrix metalloproteinase-9 (MMP-9) and lung tissue matrix metalloproteinase concentration at 4 hours after the operation.
Results:
1、There were no difference of the PvO2, PvCO2 and Cdyn levels within the six groups at the time-points of the baseline, 0.5, 1 and 2 hours after transplantation. The levels of PvO2 and Cdyn decreased after reperfusion while PvCO2 increased in all groups. Compared with no additional ischemia group, PvO2 level at 2 and 4 hours post-operatively, PvCO2 level and Cdyn level at 4 hours post-operatively were significantly different in group B2 (P<0.05). Compared to the level of group C2, 4 h post-operative PvO2, PvCO2 and Cdyn levels have significant difference in group B2 (P<0.05).
2、At the time-point of the baseline, and 1 h, 2 h, 4 h after auto-transplantation, there was no difference of the mPAP level within the six group. It increased immediately after surgery and decreased thereafter. Compared to the levels of no additional ischemia group, 0.5 h post-operative mPAP level was significantly elevated in group B2 (P<0.05). At time-point of 0.5 h following operation, mPAP level have significant increased in group B2 comparing the level of group C2 (P<0.05).
3、MDA and MPO contents was no difference within the six groups at the time-point of the baseline and 0.5 h after auto-transplantation, which rise immediately after transplantation in all groups. Compared to the levels of no additional ischemia group, 4 h post-operative MDA and MPO levels were significantly increasing in group B2. At time-point of 4 h post-operatively, MDA and MPO contents were significant high in group B2 compared with the levels of group C2 (P<0.05).
4、The lung tissue W/D ratio, ultra-structure and structure within the three groups at the baseline were comparable. The W/D ratio elevated immediately after reperfusion in all groups. Compared to the levels of no additional ischemia group, W/D ratio at the time-point of 1 h, 2 h, and 4 h post-operatively were significantly increased in group B2 (P<0.05). At the time-point of 2 h and 4 h after surgery, W/D ratio were significant different in group B2 compared to the levels of group C2 (P<0.05). Compared to the level of group A, 4 h post-operative W/D ratios have significant difference in group B and C (P<0.05). The 4 h post-operative ultra-structure and light microscope score were significantly different in group B2 compared with no additional ischemia group.
5、Compared with the levels of no additional ischemia group, serum TNF-αand MMP-9 levels and MMP-9 positive scores were significantly elevated in group B2 (P<0.01). Serum TNF-αand MMP-9 levels and MMP-9 positive scores were significantly different in group B2 compared to the levels of group C2 (P<0.01).
Conclusions:
1、Additional ischemia period before lobar auto-transplantation either with normothermic heparin or cold Euro-Collins solution will damage the transplanted lobar lung structure, induce the ischemia-reperfusion injury, increase the oxyradical level, and impair the gas exchange function.
2、At least less than 60 min additional ischemic period, pulmonary artery infusion with normothermic heparin meets the requirement of porcine lobar lung auto-transplantation. If the additional ischemia period more than 60 min but less than 120 min, pulmonary artery infusion with cold Euro-Collins is recommended.
3、Compared with pulmonary infusion with the normothermic heparin, pulmonary artery infusion with cold Euro-Collins might be better, and the involved mechanism is possibly associated with suppressing the elevation of serum TNF-αand MMP-9 and tissue MMP-9 more effectively.
Part III: Lung auto-transplantation technique in the treatment for central lung cancer of upper lobe
Objective:
To assess the feasibility and benefit of lung auto-transplantation technique in the treatment for central lung cancer of upper lobe which could not tolerate pneumotomy. To compare the perioperative mortality (within 30 days), major complication morbidity, survival time, pulmonary function and life quality within the patients who underwent pneumonectomy, sleeve lobectomy and auto-transplantation. To obtain more evidence and experience for the clinical application of the lung auto-transplantation technique.
Method:
Seven patients underwent double - sleeve right upper and middle bilobectomy from Aug 2000 to Aug 2006. Because the length of resected bronchus or pulmonary artery was too long to perform a tension - free anastomoses, we had to transect and transplant the lower lobar vein to proximal stump of the upper lobar vein. Meanwhile, 41 patients underwent sleeve lobectomy and 194 patients underwent pneumonectomy. To compare the peri-operative mortality (within 30 days), major complication morbidity (i.e. anastomotic fistulas), survival time, pulmonary function and life quality between the patients who underwent pneumonectomy, sleeve lobectomy and auto-transplantation.
Result:
Until Aug. 2006, in the auto-transplantation group, five patients had been alive tumor free for 2-73 months with good quality of life, and the estimated median live time was 24 months. One patient was performed resection of the replanted lung because of pulmonary vein thrombus on 2nd day after transplantation, and died of respiratory failure caused by pulmonary relapse on 15th month postoperatively. Another patient died of brain metastases 31 months postoperatively. The predicted median survival time of all patients was 71 months. The incidence of bronchial anastomotic stoma fistulas after pneumonectomy and sleeve lobectomy was 3.6% (7 of 194 patients) and 2.4% (1 of 41 patients) respectively. The incidence of early mortality was 2.6% (5 of 194 patients) in pneumonectomy group. The estimated median live time after pneumonectomy was 30 months and after sleeve lobectomy, 36 months. The pulmonary function and life quality was significantly different within groups (p<0.05).
Conclusion:
Lung auto-transplantation is an alternative and beneficial technique with pulmonary preservation for patient with stage III central lung cancer of upper lobe when his pulmonary reserve is too low to afford pneumonectomy. Compare with people undergoing pneunonectomy, patients undergoing lobar auto-transplantation have better pulmonary function and life quality, lower peri-operative morbidity and mortality, and longer survival time.
引文
1 Reece TB, Ellman PI, Maxey TS, et al. Adenosine A2A receptor activation reduces inflammation and preserves pulmonary function in an in vivo model of lung transplantation. J Thorac Cardiovasc Surg JT-The Journal of thoracic and cardiovascular surgery, 2005,129(5):1137-43.
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