人工气腹对新生猪循环、呼吸及腹膜形态和免疫功能影响的实验研究
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摘要
腹腔镜手术越来越多的应用于小儿外科疾病的诊断及治疗,特别是在新生儿疾病领域的应用。除仪器设备及手术方法不断的革新以外,手术术式越来越复杂,手术时间越来越长,术前合并有其它合并症的患儿也越来越多,二氧化碳(CO_2)气腹对这一群体的影响还未充分了解。CO_2气腹对机体呼吸、循环及代谢等方面的影响促使人们对最佳充填气体的探求一直未有间断。氧化亚氮(N_2O)以其与CO_2相近的理化性质能否在腹腔镜技术中应用成为又一关注的焦点。大量临床实践证明氧化亚氮气体应用的安全性,其在酸碱及血流动力学、术后疼痛等方面优于CO_2及其他气体,应用于孕妇、心肺功能不全等病人时,具有明显的优越性。随着腹腔镜技术的成熟,其在小儿肿瘤疾病中的应用更是日益广泛。然而,CO_2作为气腹的充填介质对恶性肿瘤的细胞行为及对抗肿瘤细胞的潜在影响仍是目前讨论的焦点问题之一。有学者认为,这一影响与CO_2气体造成的腹膜损伤有关。
因此,本实验通过人工CO_2气腹与N_2O气腹的建立,了解CO_2气腹和N_2O气腹对新生猪心肺功能的影响,对比不同气体类型对机体影响的优劣,以求更加深入的了解腹腔镜手术在新生儿应用中产生的问题;通过观察不同气体暴露下的腹膜超微结构的改变,CO_2暴露对间皮细胞代谢及形态的影响,神经母细胞瘤细胞跨间皮细胞迁移实验,讨论CO_2气腹对腹腔免疫的影响及腹腔镜术后高肿瘤转移率的潜在机制,试图发现新生儿及肿瘤病人腹腔镜手术时的最佳膨腹气体。
第一部分二氧化碳和氧化亚氮气腹对新生猪循环、呼吸功能影响的实验研究
目的建立新生猪气腹及急性心功能不全模型,观察人工CO_2及N_2O气腹时新生猪血流动力学及呼吸指标的变化规律,比较新生儿腹腔镜手术的膨腹气体的优劣。
方法乳猪35头,年龄7-14d,体重2-4kg。分为5组,对照组(CON)7头,CO_2气腹组(CO_2)7头,N_2O气腹组(N_2O)7头,心功能不全CO_2气腹组(CO_2-HF)7头,心功能不全N_2O气腹组(N_2O-HF)7头。CON、CO_2及N_2O组进行2h气腹,监测时间点为麻醉稳定后气腹开始前5min,气腹开始后15min、30min、60min、120min,关腹后60min;CO_2-HF及N_2O-HF组进行4h气腹,监测时间点为麻醉稳定后气腹开始前5min,气腹开始后15min、60min、120min、180min、240min,关腹后30min。监测指标为:心率(HR)、每搏输出量指数(SVI)、心输出量指数(CI)、平均动脉压(MAP)、中心静脉压(CVP)、平均肺动脉压(MPAP)、系统血管阻力指数(SVRI)、左室内压变化速率(dP/dtmax)、动脉血二氧化碳分压(PaCO_2)、动脉血氧分压(PaO_2)、动脉血氧饱和度(SaO_2)、血碳酸氢根离子浓度(HCO_3~-)及血PH值(PH)。
结果CON组血流动力学及呼吸参数基本维持恒定。CO_2组HR加快(P＜0.05):MPAP及SVRI增加(P＜0.001,P＜0.01);SVI下降(P＜0.05),CI基本维持恒定。与CON组相比,N_2O组HR减慢(P＜0.001);CVP、MPAP及SVRI增加(P＜0.05,P＜0.001,P＜0.001);dP/dtmax、MAP、SVI及CI下降(P＜0.001,P＜0.001,P＜0.01,P＜0.01)。CO_2-HF组及N_2O-HF组,与成模前相比,成模时HR降低(P＜0.01,P＜0.01),SVI降低(P＜0.05,P＜0.01),CI降低(P＜0.001,P＜0.001),MPAP升高(P＜0.05,P＜0.05),SVRI增加(P＜0.01,P＜0.05),dP/dtmax、MAP及CVP无明显改变;随后两组HR无明显改变,两组CI继续下降(P＜0.05,P＜0.05),两组CVP升高(P＜0.001,P＜0.05),两组MPAP升高(P＜0.01,P＜0.05);CO_2-HF组MAP下降(P＜0.001),SVI继续下降(P＜0.05),SVRI继续增加(P＜0.001),dP/dtmax降低(P＜0.05)。
CO_2组PaCO_2升高(P＜0.001),伴有PH下降(PH＜0.001)。N_2O组PaCO_2升高(P＜0.01)。CO_2-HF及N_2O-HF组,与成模前相比,成模时PH均下降(P＜0.01,P＜0.001),PaCO_2、PaO_2、SaO_2及HCO_3~-均无明显改变;随后两组PaCO_2升高(P＜0.05,P＜0.001).PaO_2(P＜0.05,P＜0.001)及SaO_2下降(P＜0.05,P＜0.01),HCO_3~-浓度下降(P＜0.01,P＜0.01),血PH继续下降(P＜0.05,P＜0.001)。
结论健康新生猪在严密监测下可以耐受短时间CO_2气腹且较N_2O气腹具有优越性。心功能不全新生猪进行长时间气体充填时CO_2气腹对心功能影响明显,导致心肌收缩力明显下降及系统血管阻力明显增加;长时间N_2O气腹对心功能不全新生猪无进一步心肌损害作用。
第二部分二氧化碳对腹膜形态和免疫功能影响的实验研究
目的间皮细胞是腹腔一个重要的细胞群,外科创伤对这一细胞群影响的研究甚少。在体实验研究提示,CO_2气腹后,腹膜结构出现改变。近期尚有文献报道CO_2气腹增加小鼠神经母细胞瘤模型的系统转移率。本实验研究不同气体条件对腹膜间皮细胞形态及分泌功能的影响,探讨CO_2气腹对腹腔肿瘤细胞影响的潜在机制。
方法经0.125%胰酶预处理后,通过腹腔灌洗分离培养C57/BL6小鼠腹膜间皮细胞。间皮细胞纯化通过细胞形态学及免疫组化方法(小鼠间皮细胞角蛋白特异性抗体AE1/AE3)鉴定。MTT实验确定细胞活力。脂多糖(LPS)(10μg/ml)或肿瘤坏死因子-α(TNF-α)(10ng/ml)刺激间皮细胞,于CO_2暴露期间及随后不同时间点使用酶联免疫吸附试验(ELISA)检测间皮细胞粒细胞集落刺激因子(G-CSF)、白细胞介素-6(IL-6)、巨噬细胞集落刺激因子(M-CSF)、单核细胞趋化蛋白-1(MCP-1)及正常T细胞表达和分泌因子(RANTES)产量。使用Transwell系统培养连续间皮细胞层,分别暴露于100%CO_2及5%CO_22h,经荧光染色后的小鼠神经母细胞瘤细胞(Neuro2a)加入至Transwell系统上室,细胞迁移数目经多功能检测仪进行测定。不同气体条件下腹膜及分离培养的间皮细胞行电镜观察。
结果100%CO_2暴露后,LPS及TNF-α刺激组间皮细胞活力均下降,TNF-α刺激组间皮细胞活力下降时间持久,但两组24h后细胞活力均有所恢复;LPS及TNF-α刺激后间皮细胞释放一定数量的IL-6及G-CSF(P＜0.05),M-CSF、MCP-1及RANTES产量变化不明显。100%CO_2可阻断炎症细胞因子G-CSF及IL-6的产生(P＜0.05),但暴露结束后可部分恢复。M-CSF的产量受100%CO_2影响不显著。迁移实验表明,100%CO_2暴露后间皮细胞屏障功能下降,Neuro2a迁移数目增加(P＜0.05)。扫描电镜提示,离体实验观察100%CO_2破坏间皮细胞微绒毛,在体实验腹膜电镜结果提示压力12mmHg100%CO_2气腹维持4h导致腹膜间皮细胞层破坏,基底膜暴露,仅存细胞骨架;相同条件的100%N_2O气腹使间皮细胞间隙增大,部分区域基底膜暴露。
结论100%CO_2能暂时性抑制腹膜间皮细胞的线粒体活动。经刺激后,腹膜间皮细胞能释放一定数量的细胞因子。暴露于100%CO_2可以对这一炎性反应产生暂时的阻断作用。100%CO_2使在体腹膜及离体间皮细胞超微结构发生明显改变,同时利于神经母细胞瘤细胞迁移。因此,小鼠模型中发现的CO_2气腹后神经母细胞瘤转移增加可能与间皮细胞屏障削弱有关。
Recently, more and more diagnosis and treatment of disease in childhood can be solved by using laparoscopy, especially applying in the field of neonatal diseases. In addition to innovation of surgical instruments and operative method, surgical procedure is more complex, operative time is longer. And also there are an increasing number of children with preoperative complications. The influence of CO_2 pneumoperitoneum of this group has not yet fully understood. Effect of CO_2 pneumoperitoneum on respiratory, circulatory and metabolic area arouses people to search for the best filling gas constantly. Because of similar physical and chemical properties of N_2O to CO_2, N_2O becomes another focal point. A large number of clinical practices have shown the safety of N_2O applications. It is better than CO_2 in acid-base and blood flow dynamics, postoperative pain and so on. It has obvious advantages when appling in pregnant woman, heart and lung dysfunction patients. With the maturity of laparoscopy, it is gradually applied in pediatric oncology. However, it is still discussion intensely that whether CO_2 as a filling medium effect malignant tumor cells behavior and has potential impact to anti-tumor cells. Some scholars believe that this effect is caused by peritoneal damage because of applying CO_2 pneumoperitoneum.
Therefore, this experiment established artificial CO_2 and N_2O pneumoperitoneum, in order to understand the influence of CO_2 and N_2O pneumoperitoneum on cardio-pulmonary function in piglets, compare advantages and shortcomings of different gas types, look into issues arising in neonates by using laparoscopy. By observation changes of peritoneal morphology under different gas exposition, investigation influence of CO_2 on metabolic and morphology changes in mesothelial cells and on transmigration of neuroblastoma cells across mesothelial cells, in order to discuss influence of CO_2 on immune of peritoneal cavity and explaining underlying mechanism of high rates tumor metastasis after lapascopy. Thus it can provide clue to discover the best filling gas when apply laparoscopy in neonates and tumor patients.
PART 1
A Research on The Influrence of Carbon Dioxide and Nitrous Oxide Pneumoperitoneum on The Circulatory, Respiratoryof Piglets
Objective This experiment established pneumoperitoneum model and acute cardiac insufficiency model in piglets. It studies changes of hemodynamics and resiperitory system of artificial pneumoperitoneum with CO_2 and N_2O in piglets and in order to contrast superior and inferior of the filling gas for laparoscopy in neonate.
Methods Thirty five piglets (2-4 kilogram in weight, 7-14 days of age) were equally divided into five groups: control group (CON), CO_2 group (CO_2), N_2O group (N_2O), cardiac failure & CO_2 group (CO_2-HF) and cardiac failure & N_2O group (N_2O-HF). It was going on 2h pneumoperitoneum in CON, CO_2, N_2O groups and 4h pneumoperitoneum in CO_2-HF, N_2O-HF groups. Investigation time points were 5min after anaesthesia and before pneumoperitoneum started, 15min, 30min, 60min, 120min and 60min after abdominal closure in CON, CO_2 and N_2O groups. Investigation time points were 5min after anaesthesia and before pneumoperitoneum started, 15min, 60min, 120min, 180min, 240min and 30min after abdominal closure in CO_2-HF and N_2O-HF groups. Investigation index were heart rete, stroke volume index, cardiac index, mean arterial pressure, central venous pressure, mean pulmonary arterial pressure, systemic vascular resistance index, dP/dtmax, partial pressure of carbon dioxide in arterial blood, partial pressure of oxygen in arterial blood, arterial oxygen saturation, concentration of HCO_3~- in arterial blood and blood PH value.
Results Hemodynamics and parameters of respiratory were nearly constant in CON group. In CO_2 group, HR increased (P<0. 05), MPAP and SVRI increased (P<0. 001, P<0. 01), SVI decreased (P<0. 05), CI was stable. In N_2O group, HR decreased (P<0.001); CVP, MPAP and SVRI increased (P<0. 05, P<0. 001, P<0. 001); dP/dtmax, MAP, SVI and CI decreased (P<0.001, P<0. 001, P<0. 01, P<0.01). In CO_2-HF and N_2O-HF groups, compared to before modeling, when model was established, HR decreased (P<0. 01, P<0.01), SVI decreased (P<0. 05, P<0.01), CI decreased (P<0.001, P<0.001), MPAP increased (P<0.05, P <0. 05), SVRI increased (P<0. 01, P<0. 05), dP/dtmax, MAP and CVP were stable. Afterwards, HR were stable in both groups, CI decreased further (P<0. 05, P<0. 05) in both groups, CVP increased (P<0. 001, P<0. 05) in both groups, MPAP increased (P<0.01, P<0.05) in both groups. In CO_2-HF group, MAP decreased (P<0.001), SVI decreased further (P<0.05), SVRI increased further (P<0. 001), dP/dtmax decreased (P<0. 05).
In CO_2 group, PaCO_2 increased (P<0. 001), accompanyed with PH value decreased (PH<0. 001). In N_2O group, PaCO_2 increased (P<0. 01). In CO_2-HF and N_2O-HF groups, compare to before modeling, when model was established, PH value decreased (P<0.01, P<0.001), PaCO_2, PaO_2, SaO_2 and HCO_3~- were stable. Afterwards, PaCO_2 increased (P<0. 05, P<0.001) in both groups, PaO_2 (P< 0.05, P<0. 001) and SaO_2 decreased (P<0. 05, P<0. 01), HC0_3~- decreased (P< 0.01, P<0. 01), PH value decreased (P<0. 05, P<0. 001).
Conclusion The experimental results suggested that healthy piglets can tolerate CO_2 pneumoperitoneum and CO_2 pneumoperitoneum was superior to N_20 pneumoperitoneum. Cardiac contractility decreased and systemic vascular resistance index increased obviously when applying CO_2 pneumoperitoneum in cardiac insufficient piglets for long duration. Long duration of N_20 pneumoperitoneum has no further myocardial damage to cardiac insufficient piglets.
PART 2
A Research on The Influence of Carbon dioxide on Morphology of Peritoneum and Immunologic function
Objective
The response of mesothelial cells, one of the dominant cell population in the peritoneal cavity, to surgical trauma is poorly defined. Nonetheless, electron microscopic studies have shown significant morphologic changes of these cells after CO_2 pneumoperitoneum in vivo. It is recently shown that CO_2 pneumoperitoneum increases systemic metastasis of peritoneal neuroblastoma cells in a murine model. Thus, this experiment was tested the effection under different gas condition to the morphology and the inflammatory response of peritoneal mesothelial cells and discussed the underlying mechanism of the effection of CO_2 pneumoperitoneum to tumor cells in peritoneal cavity in vitro.
Methods
Purification of primary murine peritoneal mesothelial cells was achieved by sequential peritoneal lavage after 0. 125% trypsin pretreatment. Purity of the mesothelial cell culture was confirmed by cell morphology and immunohistochemical staining for specific cytokeratine (AE1/AE3). In all experiments vitality of the cells was confirmed by MTT assay. Cells were stimulated by LPS (10μg/ml) or murine TNF-α(10ng/ml) and the release of G-CSF, IL-6, M-CSF, MCP-1 and RANTES during and following CO_2 incubation were measured by ELISA. In an additional set of experiments monolayers of mesothelial cells were established on transwell systems. Following incubation with 100%CO_2 or 5%CO_2 for 2h, fluorescent stained Neuro2a cells were added to the upper chamber and their migration into the lower chamber was measured by multi-detection reader. Peritoneum and isolated mesothelial cells were detected by scanning electron microscopy.
Results MTT conversion was decreased during the CO_2 incubation both in LPS and TNF-αstimulation groups. This inhibition effect was longer in TNF-αgroup than in LPS stimulation group. After 24h, their mitochondrial activity recovered. Mesothelial cells stimulated with LPS or TNF-αreleased significant amounts of IL-6 and G-CSF (P<0. 05). The production of M-CSF, MCP-1 and RANTES can not be stimulated obviously. The release of G-CSF and IL-6 was completely blocked during the incubation with 100%CO_2 (P<0. 05), but directly after the incubation the inflammatory capacity recoverd. M-CSF can not be effected by CO_2 obviously. Migration studies showed that the barrier function of the mesothelial monolayer decreased. A significantly increased migration of neuroblastoma cells was identified after 100%CO_2 exposure (P<0. 05). Scanning electron microscopy investigation suggested 100%CO_2 exposition was associated with a significant destruction of the microvilli formation of isolated mesothelial cells in vitro. In vivo, it also suggested 12mmHg 100%CO_2 pneumoperitoneum destroyed mesothelial cells layer of peritoneum when lasting for 4h, basal lamina was exposed and cytoskeleton was existed merely. In contrast, 100%N_2O pneumoperitoneum leaded to increase of intercellular gaps and basal lamina was exposed in part areas under same pressure and duration.
Conclusions
100%CO_2 suppressed mitochondrial activity of mesothelial cells temporarily. Peritoneal mesothelial cells released significant amounts of cytokines following stimulation. This inflammatory response was blocked during the incubation with 100%C0_2, but there appeared to be no lasting effect. Moreover the peritoneal mesothelial cells lose their typical cell morphology by 100%CO_2 exposition, which is accompanied by facilitated migration of neuroblastoma cells. Thus, the increased Neuroblastoma metastasis observed after CO_2 pneumoperitoneum in mice might be related to an impaired mesothelial barrier function.
因此,本实验通过人工CO_2气腹与N_2O气腹的建立,了解CO_2气腹和N_2O气腹对新生猪心肺功能的影响,对比不同气体类型对机体影响的优劣,以求更加深入的了解腹腔镜手术在新生儿应用中产生的问题;通过观察不同气体暴露下的腹膜超微结构的改变,CO_2暴露对间皮细胞代谢及形态的影响,神经母细胞瘤细胞跨间皮细胞迁移实验,讨论CO_2气腹对腹腔免疫的影响及腹腔镜术后高肿瘤转移率的潜在机制,试图发现新生儿及肿瘤病人腹腔镜手术时的最佳膨腹气体。
第一部分二氧化碳和氧化亚氮气腹对新生猪循环、呼吸功能影响的实验研究
目的建立新生猪气腹及急性心功能不全模型,观察人工CO_2及N_2O气腹时新生猪血流动力学及呼吸指标的变化规律,比较新生儿腹腔镜手术的膨腹气体的优劣。
方法乳猪35头,年龄7-14d,体重2-4kg。分为5组,对照组(CON)7头,CO_2气腹组(CO_2)7头,N_2O气腹组(N_2O)7头,心功能不全CO_2气腹组(CO_2-HF)7头,心功能不全N_2O气腹组(N_2O-HF)7头。CON、CO_2及N_2O组进行2h气腹,监测时间点为麻醉稳定后气腹开始前5min,气腹开始后15min、30min、60min、120min,关腹后60min;CO_2-HF及N_2O-HF组进行4h气腹,监测时间点为麻醉稳定后气腹开始前5min,气腹开始后15min、60min、120min、180min、240min,关腹后30min。监测指标为:心率(HR)、每搏输出量指数(SVI)、心输出量指数(CI)、平均动脉压(MAP)、中心静脉压(CVP)、平均肺动脉压(MPAP)、系统血管阻力指数(SVRI)、左室内压变化速率(dP/dtmax)、动脉血二氧化碳分压(PaCO_2)、动脉血氧分压(PaO_2)、动脉血氧饱和度(SaO_2)、血碳酸氢根离子浓度(HCO_3~-)及血PH值(PH)。
结果CON组血流动力学及呼吸参数基本维持恒定。CO_2组HR加快(P＜0.05):MPAP及SVRI增加(P＜0.001,P＜0.01);SVI下降(P＜0.05),CI基本维持恒定。与CON组相比,N_2O组HR减慢(P＜0.001);CVP、MPAP及SVRI增加(P＜0.05,P＜0.001,P＜0.001);dP/dtmax、MAP、SVI及CI下降(P＜0.001,P＜0.001,P＜0.01,P＜0.01)。CO_2-HF组及N_2O-HF组,与成模前相比,成模时HR降低(P＜0.01,P＜0.01),SVI降低(P＜0.05,P＜0.01),CI降低(P＜0.001,P＜0.001),MPAP升高(P＜0.05,P＜0.05),SVRI增加(P＜0.01,P＜0.05),dP/dtmax、MAP及CVP无明显改变;随后两组HR无明显改变,两组CI继续下降(P＜0.05,P＜0.05),两组CVP升高(P＜0.001,P＜0.05),两组MPAP升高(P＜0.01,P＜0.05);CO_2-HF组MAP下降(P＜0.001),SVI继续下降(P＜0.05),SVRI继续增加(P＜0.001),dP/dtmax降低(P＜0.05)。
CO_2组PaCO_2升高(P＜0.001),伴有PH下降(PH＜0.001)。N_2O组PaCO_2升高(P＜0.01)。CO_2-HF及N_2O-HF组,与成模前相比,成模时PH均下降(P＜0.01,P＜0.001),PaCO_2、PaO_2、SaO_2及HCO_3~-均无明显改变;随后两组PaCO_2升高(P＜0.05,P＜0.001).PaO_2(P＜0.05,P＜0.001)及SaO_2下降(P＜0.05,P＜0.01),HCO_3~-浓度下降(P＜0.01,P＜0.01),血PH继续下降(P＜0.05,P＜0.001)。
结论健康新生猪在严密监测下可以耐受短时间CO_2气腹且较N_2O气腹具有优越性。心功能不全新生猪进行长时间气体充填时CO_2气腹对心功能影响明显,导致心肌收缩力明显下降及系统血管阻力明显增加;长时间N_2O气腹对心功能不全新生猪无进一步心肌损害作用。
第二部分二氧化碳对腹膜形态和免疫功能影响的实验研究
目的间皮细胞是腹腔一个重要的细胞群,外科创伤对这一细胞群影响的研究甚少。在体实验研究提示,CO_2气腹后,腹膜结构出现改变。近期尚有文献报道CO_2气腹增加小鼠神经母细胞瘤模型的系统转移率。本实验研究不同气体条件对腹膜间皮细胞形态及分泌功能的影响,探讨CO_2气腹对腹腔肿瘤细胞影响的潜在机制。
方法经0.125%胰酶预处理后,通过腹腔灌洗分离培养C57/BL6小鼠腹膜间皮细胞。间皮细胞纯化通过细胞形态学及免疫组化方法(小鼠间皮细胞角蛋白特异性抗体AE1/AE3)鉴定。MTT实验确定细胞活力。脂多糖(LPS)(10μg/ml)或肿瘤坏死因子-α(TNF-α)(10ng/ml)刺激间皮细胞,于CO_2暴露期间及随后不同时间点使用酶联免疫吸附试验(ELISA)检测间皮细胞粒细胞集落刺激因子(G-CSF)、白细胞介素-6(IL-6)、巨噬细胞集落刺激因子(M-CSF)、单核细胞趋化蛋白-1(MCP-1)及正常T细胞表达和分泌因子(RANTES)产量。使用Transwell系统培养连续间皮细胞层,分别暴露于100%CO_2及5%CO_22h,经荧光染色后的小鼠神经母细胞瘤细胞(Neuro2a)加入至Transwell系统上室,细胞迁移数目经多功能检测仪进行测定。不同气体条件下腹膜及分离培养的间皮细胞行电镜观察。
结果100%CO_2暴露后,LPS及TNF-α刺激组间皮细胞活力均下降,TNF-α刺激组间皮细胞活力下降时间持久,但两组24h后细胞活力均有所恢复;LPS及TNF-α刺激后间皮细胞释放一定数量的IL-6及G-CSF(P＜0.05),M-CSF、MCP-1及RANTES产量变化不明显。100%CO_2可阻断炎症细胞因子G-CSF及IL-6的产生(P＜0.05),但暴露结束后可部分恢复。M-CSF的产量受100%CO_2影响不显著。迁移实验表明,100%CO_2暴露后间皮细胞屏障功能下降,Neuro2a迁移数目增加(P＜0.05)。扫描电镜提示,离体实验观察100%CO_2破坏间皮细胞微绒毛,在体实验腹膜电镜结果提示压力12mmHg100%CO_2气腹维持4h导致腹膜间皮细胞层破坏,基底膜暴露,仅存细胞骨架;相同条件的100%N_2O气腹使间皮细胞间隙增大,部分区域基底膜暴露。
结论100%CO_2能暂时性抑制腹膜间皮细胞的线粒体活动。经刺激后,腹膜间皮细胞能释放一定数量的细胞因子。暴露于100%CO_2可以对这一炎性反应产生暂时的阻断作用。100%CO_2使在体腹膜及离体间皮细胞超微结构发生明显改变,同时利于神经母细胞瘤细胞迁移。因此,小鼠模型中发现的CO_2气腹后神经母细胞瘤转移增加可能与间皮细胞屏障削弱有关。
Recently, more and more diagnosis and treatment of disease in childhood can be solved by using laparoscopy, especially applying in the field of neonatal diseases. In addition to innovation of surgical instruments and operative method, surgical procedure is more complex, operative time is longer. And also there are an increasing number of children with preoperative complications. The influence of CO_2 pneumoperitoneum of this group has not yet fully understood. Effect of CO_2 pneumoperitoneum on respiratory, circulatory and metabolic area arouses people to search for the best filling gas constantly. Because of similar physical and chemical properties of N_2O to CO_2, N_2O becomes another focal point. A large number of clinical practices have shown the safety of N_2O applications. It is better than CO_2 in acid-base and blood flow dynamics, postoperative pain and so on. It has obvious advantages when appling in pregnant woman, heart and lung dysfunction patients. With the maturity of laparoscopy, it is gradually applied in pediatric oncology. However, it is still discussion intensely that whether CO_2 as a filling medium effect malignant tumor cells behavior and has potential impact to anti-tumor cells. Some scholars believe that this effect is caused by peritoneal damage because of applying CO_2 pneumoperitoneum.
Therefore, this experiment established artificial CO_2 and N_2O pneumoperitoneum, in order to understand the influence of CO_2 and N_2O pneumoperitoneum on cardio-pulmonary function in piglets, compare advantages and shortcomings of different gas types, look into issues arising in neonates by using laparoscopy. By observation changes of peritoneal morphology under different gas exposition, investigation influence of CO_2 on metabolic and morphology changes in mesothelial cells and on transmigration of neuroblastoma cells across mesothelial cells, in order to discuss influence of CO_2 on immune of peritoneal cavity and explaining underlying mechanism of high rates tumor metastasis after lapascopy. Thus it can provide clue to discover the best filling gas when apply laparoscopy in neonates and tumor patients.
PART 1
A Research on The Influrence of Carbon Dioxide and Nitrous Oxide Pneumoperitoneum on The Circulatory, Respiratoryof Piglets
Objective This experiment established pneumoperitoneum model and acute cardiac insufficiency model in piglets. It studies changes of hemodynamics and resiperitory system of artificial pneumoperitoneum with CO_2 and N_2O in piglets and in order to contrast superior and inferior of the filling gas for laparoscopy in neonate.
Methods Thirty five piglets (2-4 kilogram in weight, 7-14 days of age) were equally divided into five groups: control group (CON), CO_2 group (CO_2), N_2O group (N_2O), cardiac failure & CO_2 group (CO_2-HF) and cardiac failure & N_2O group (N_2O-HF). It was going on 2h pneumoperitoneum in CON, CO_2, N_2O groups and 4h pneumoperitoneum in CO_2-HF, N_2O-HF groups. Investigation time points were 5min after anaesthesia and before pneumoperitoneum started, 15min, 30min, 60min, 120min and 60min after abdominal closure in CON, CO_2 and N_2O groups. Investigation time points were 5min after anaesthesia and before pneumoperitoneum started, 15min, 60min, 120min, 180min, 240min and 30min after abdominal closure in CO_2-HF and N_2O-HF groups. Investigation index were heart rete, stroke volume index, cardiac index, mean arterial pressure, central venous pressure, mean pulmonary arterial pressure, systemic vascular resistance index, dP/dtmax, partial pressure of carbon dioxide in arterial blood, partial pressure of oxygen in arterial blood, arterial oxygen saturation, concentration of HCO_3~- in arterial blood and blood PH value.
Results Hemodynamics and parameters of respiratory were nearly constant in CON group. In CO_2 group, HR increased (P<0. 05), MPAP and SVRI increased (P<0. 001, P<0. 01), SVI decreased (P<0. 05), CI was stable. In N_2O group, HR decreased (P<0.001); CVP, MPAP and SVRI increased (P<0. 05, P<0. 001, P<0. 001); dP/dtmax, MAP, SVI and CI decreased (P<0.001, P<0. 001, P<0. 01, P<0.01). In CO_2-HF and N_2O-HF groups, compared to before modeling, when model was established, HR decreased (P<0. 01, P<0.01), SVI decreased (P<0. 05, P<0.01), CI decreased (P<0.001, P<0.001), MPAP increased (P<0.05, P <0. 05), SVRI increased (P<0. 01, P<0. 05), dP/dtmax, MAP and CVP were stable. Afterwards, HR were stable in both groups, CI decreased further (P<0. 05, P<0. 05) in both groups, CVP increased (P<0. 001, P<0. 05) in both groups, MPAP increased (P<0.01, P<0.05) in both groups. In CO_2-HF group, MAP decreased (P<0.001), SVI decreased further (P<0.05), SVRI increased further (P<0. 001), dP/dtmax decreased (P<0. 05).
In CO_2 group, PaCO_2 increased (P<0. 001), accompanyed with PH value decreased (PH<0. 001). In N_2O group, PaCO_2 increased (P<0. 01). In CO_2-HF and N_2O-HF groups, compare to before modeling, when model was established, PH value decreased (P<0.01, P<0.001), PaCO_2, PaO_2, SaO_2 and HCO_3~- were stable. Afterwards, PaCO_2 increased (P<0. 05, P<0.001) in both groups, PaO_2 (P< 0.05, P<0. 001) and SaO_2 decreased (P<0. 05, P<0. 01), HC0_3~- decreased (P< 0.01, P<0. 01), PH value decreased (P<0. 05, P<0. 001).
Conclusion The experimental results suggested that healthy piglets can tolerate CO_2 pneumoperitoneum and CO_2 pneumoperitoneum was superior to N_20 pneumoperitoneum. Cardiac contractility decreased and systemic vascular resistance index increased obviously when applying CO_2 pneumoperitoneum in cardiac insufficient piglets for long duration. Long duration of N_20 pneumoperitoneum has no further myocardial damage to cardiac insufficient piglets.
PART 2
A Research on The Influence of Carbon dioxide on Morphology of Peritoneum and Immunologic function
Objective
The response of mesothelial cells, one of the dominant cell population in the peritoneal cavity, to surgical trauma is poorly defined. Nonetheless, electron microscopic studies have shown significant morphologic changes of these cells after CO_2 pneumoperitoneum in vivo. It is recently shown that CO_2 pneumoperitoneum increases systemic metastasis of peritoneal neuroblastoma cells in a murine model. Thus, this experiment was tested the effection under different gas condition to the morphology and the inflammatory response of peritoneal mesothelial cells and discussed the underlying mechanism of the effection of CO_2 pneumoperitoneum to tumor cells in peritoneal cavity in vitro.
Methods
Purification of primary murine peritoneal mesothelial cells was achieved by sequential peritoneal lavage after 0. 125% trypsin pretreatment. Purity of the mesothelial cell culture was confirmed by cell morphology and immunohistochemical staining for specific cytokeratine (AE1/AE3). In all experiments vitality of the cells was confirmed by MTT assay. Cells were stimulated by LPS (10μg/ml) or murine TNF-α(10ng/ml) and the release of G-CSF, IL-6, M-CSF, MCP-1 and RANTES during and following CO_2 incubation were measured by ELISA. In an additional set of experiments monolayers of mesothelial cells were established on transwell systems. Following incubation with 100%CO_2 or 5%CO_2 for 2h, fluorescent stained Neuro2a cells were added to the upper chamber and their migration into the lower chamber was measured by multi-detection reader. Peritoneum and isolated mesothelial cells were detected by scanning electron microscopy.
Results MTT conversion was decreased during the CO_2 incubation both in LPS and TNF-αstimulation groups. This inhibition effect was longer in TNF-αgroup than in LPS stimulation group. After 24h, their mitochondrial activity recovered. Mesothelial cells stimulated with LPS or TNF-αreleased significant amounts of IL-6 and G-CSF (P<0. 05). The production of M-CSF, MCP-1 and RANTES can not be stimulated obviously. The release of G-CSF and IL-6 was completely blocked during the incubation with 100%CO_2 (P<0. 05), but directly after the incubation the inflammatory capacity recoverd. M-CSF can not be effected by CO_2 obviously. Migration studies showed that the barrier function of the mesothelial monolayer decreased. A significantly increased migration of neuroblastoma cells was identified after 100%CO_2 exposure (P<0. 05). Scanning electron microscopy investigation suggested 100%CO_2 exposition was associated with a significant destruction of the microvilli formation of isolated mesothelial cells in vitro. In vivo, it also suggested 12mmHg 100%CO_2 pneumoperitoneum destroyed mesothelial cells layer of peritoneum when lasting for 4h, basal lamina was exposed and cytoskeleton was existed merely. In contrast, 100%N_2O pneumoperitoneum leaded to increase of intercellular gaps and basal lamina was exposed in part areas under same pressure and duration.
Conclusions
100%CO_2 suppressed mitochondrial activity of mesothelial cells temporarily. Peritoneal mesothelial cells released significant amounts of cytokines following stimulation. This inflammatory response was blocked during the incubation with 100%C0_2, but there appeared to be no lasting effect. Moreover the peritoneal mesothelial cells lose their typical cell morphology by 100%CO_2 exposition, which is accompanied by facilitated migration of neuroblastoma cells. Thus, the increased Neuroblastoma metastasis observed after CO_2 pneumoperitoneum in mice might be related to an impaired mesothelial barrier function.
引文
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