【作者】 刘书盈；

【导师】 钱桂生；

【作者基本信息】 第三军医大学 ， 内科学， 2007， 博士

【摘要】 全氟化碳(PFC)作为良好的呼吸气体输送剂用于急性肺损伤/急性呼吸窘迫综合征(ALI/ARDS)的液体通气和雾化/汽化吸入治疗取得了较好的疗效。研究发现其具有广泛的生物学效应,如表面活性物质样作用和非特异性抗炎作用等,但这些都是肺内局部应用或体外实验研究,全身应用可能对ALI/ARDS的防治具有更为重要的作用。因此,我们通过内毒素急性肺损伤大鼠模型,分三部分探讨全身应用PFC对急性肺损伤的作用。目的:探讨经腹腔注射PFC原液(PFO)和静脉注射PFCE(PFTE、PFT)全身应用PFC的可行性;观察腹腔注射PFO和静脉注射PFT对内毒素急性肺损伤的防治作用和抗炎作用并探讨其相关机制。方法:1.腹腔注射PFO(15ml/Kg)按24h、48h、72h和1个月时间点分4组,前三个时间点各设一个生理盐水对照组;静脉注射自制乳剂PFTE(10ml/Kg)按1h、2h、4h、6h、24h、48h、72h和10天时间点分8组,设1组生理盐水对照组。所有动物分别于相应时间点处死,测定动脉血气分析、PFO吸收率、血液PFO和PFTE浓度、肝肾功能及肺脏、肝脏和肾脏病理学检查。2.腹腔注射PFO对内毒素肺损伤防治作用和抗炎作用研究各分两个正常对照组(NC和PFO)和两个实验组(LPS和LPS+PFO),实验前48小时,PFO组和LPS+PFO组腹腔注射PFO 15 ml/kg,实验时,实验组静脉注射LPS 7 mg/kg,其它分组均注射生理盐水。各组分别于2 h、4 h、6 h时间点处死动物。防治作用研究测定LI、LPI和PaO2及肺脏病理学检查。抗炎作用研究测定血液和BALF中WBC和PMN、肺组织中PMN、血清和BALF中的IL-1β和TNFα;肺组织免疫组化检测E-选择素、ICAM-1和NF-κB表达。3.静脉注射PFT对内毒素肺损伤防治作用和抗炎作用研究各分两个正常对照组(NC和PFT)和两个实验组(LPS和LPS+PFT),PFT组和LPS+PFT组静脉注射PFT10ml/kg,5分钟后,实验组静脉注射LPS7mg/kg,其它分组均注射生理盐水。各组分别于2h、4h、6h时间点处死动物。防治作用研究测定W/D、LPI、PaO2及肺脏病理学检查。抗炎作用研究测定血液和BALF中WBC和PMN、肺组织中PMN、血清和BALF中的IL-1β和TNFα;肺组织免疫组化检测E-选择素、ICAM-1和NF-κB表达;流式细胞仪测定血液PMN表面CD11b表达。结果:1.腹腔注射PFO后,72小时吸收率达0.93±0.03,血液浓度在48小时达高峰(1.67±0.30 ug/ml),1月后测不出。对肝肾功能无明显影响;静脉注射PFTE后,血液浓度1小时最高,达3229.34±466.01ug/ml,但下降很快,至6小时下降至349.9±88.61ug/ml,第10天已测不出。PaO2较对照组明显升高(p<0.01),但下降较快,6小时已与对照组差异无统计学意义(p>0.05)。ALT和AST明显升高(p<0.01),10天后才降至与对照组差异无统计学意义(p>0.05)。2.腹腔注射PFO防治作用研究结果显示:LPS组在4h和6h时间点各有1只动物死亡,PaO2较正常对照组明显下降(p<0.01)。LPS+PFO组仅在6h有1只动物死亡,与LPS组相比,PaO2有所升高,但仅在6h差异有统计学意义(p<0.05),LI和LPI降低,但仅在4h和6h时间点才有统计学意义(p<0.05和0.01),肺组织除水肿和炎性细胞侵润有所较轻外,其它无明显改变。腹腔注射PFO抗炎作用研究结果显示:与正常对照组比较,LPS组动物BALF中WBC、PMN和肺组织PMN及血液WBC(2h)明显升高(p<0.01),血液PMN和WBC(4h和6h)降低(p<0.01);LPS+PFO组与LPS组相比,BALF中WBC、PMN和肺组织PMN及血液中WBC(2h)均降低(p<0.01),血液中PMN和WBC(4h和6h)均升高(p<0.01),且与正常对照组差异无统计学意义(p>0.05);血清和BALF中IL-1β和TNFα:LPS组较正常对照组明显升高(p<0.01或<0.05),LPS+PFO组较LPS组明显降低(p<0.01),但仍高于正常对照组(p<0.01或0.05),血清TNFα降低仅在6h具有统计学意义(p<0.05)。免疫组化检测E-选择素、ICAM-1和NF-κb表达(IOD)显示:LPS组较正常对照组明显升高(p<0.01),LPS+PFO组较LPS组明显降低(p<0.01),但仍高于正常对照组(p<0.01或0.05)。3.静脉注射PFT防治作用研究结果显示:LPS组和LPS+PFT组各在6h时间点有1只动物死亡。PaO2比较:LPS组较NC组明显下降(p<0.01),LPS+PFT组较LPS组明显升高(p<0.01),在2h甚至高于NC组(p<0.05),至6h尽管仍高于LPS组(p<0.01),但低于NC组(p<0.01),PFT组在各时间点均高于其它三组(p<0.01);W/D和LPI:LPS组明显高于NC组(p<0.01),LPS+PFT组较LPS组明显降低,但在4h和6h时间点才有统计学意义(p<0.05或0.01)。肺组织病理学检查显示:LPS+PFT组较LPS组的炎性细胞侵润和水肿减轻,可见明显肺泡扩张,并见泡沫样PFC微粒。静脉注射PFT抗炎作用研究结果显示:与正常对照组比较,LPS组BALF中WBC、PMN和肺组织PMN及血液WBC(2h)明显升高(p<0.01),而血液PMN和WBC(6h)降低(p均<0.01);LPS+PFO组与LPS组比较:BALF中WBC、PMN和肺组织PMN及血液WBC(2h)均明显降低(p<0.01);血液PMN和WBC(6h)均升高(p<0.01);血清和BALF中IL-1β和TNFα:LPS组较NC组均升高(p<0.01),LPS+PFT组较LPS组均降低(p<0.05或0.01);血液中PMN表面CD11b表达(MIF):LPS组明显高于NC组(p<0.01),LPS+PFT组明显低于LPS组(p<0.01);免疫组化检测E-选择素、ICAM-1和NF-κb表达(IOD):LPS组明显高于NC组(p<0.01),LPS+PFT组明显低于LPS组(p<0.01),但仍高于正常对照组(p<0.01或0.05)。结论:1.通过腹腔注射PFC原液(PFO)和静脉注射PFTE(或PFT)途径全身应用PFC能够到达一定的血液浓度和维持一定的时间,PFO对肝肾功能无明显影响,除PFTE有一过性肝功能损伤外,无其它不良反应,安全性好,且PFTE可明显提高PaO2,说明此两种全身应用途径可行。2.腹腔预注射PFO和静脉预注射PFT可明显提高PaO2,减轻内毒素肺损伤导致的肺水肿,降低肺泡通透性,PFT亦可改善肺病理性损伤,提示全身应用PFO和PFT对内毒素急性肺损伤具有一定的防治作用。3.腹腔注射PFO和静脉注射PFT可明显抑制PMN肺侵润、降低促炎细胞因子IL-1β和TNFα的产生,具有明显的非特异性抗炎作用,其机制可能与抑制E-选择素、ICAM-1和NF-κb以及PMN表面CD11b表达有关。更多还原

【Abstract】 Perfluorocarbons (PFCs) have been used in ALI/ARDS treatment by liquid ventilation and aerosol or vapor inhalation and revealed its extensive biological effects such as surfactant-like and nonspecific antiinflammatory effects. These suggest that systemic administration may have therapeutic effects on ALI/ARDS. In this study, with animal model of LPS-induced lung injury, we made researchs on the effects of systemic PFC on acute lung injury in three parts:Objectives1.To explore the feasibility of systemic application through peritoneum and vein;2.To investigate the therapeutic and antiinflammatory effects of PFC (PFO) injected intraperitoneally and PFCE (PFTE or PFT) injected intravenously on acute lung injury.Methods1. In the exploration of feasibility, Wistar rats grouped by timepoint in four treatment groups were intraperitoneally injected with PFO (15ml/Kg) and in eight treatment groups were intravenously administrated with PFTE (10ml/Kg); control groups with normal saline. The animals were exsanguinated and sampled for measurment of arterial blood gas analysis, liver and kidney function, blood concentrations of PFC and absorption rate of PFO. The tissue of lung, liver and kidney were histologically assessed.2. In the research on therapeutic and antiinflammatory effects of PFO, the animals were divided into two treatment groups(LPS and LPS+PFO) and two control groups(NC and PFO). The rats were intraperitoneally injected with PFO (15ml/Kg) in PFO group and LPS+PFO group, with normal saline in others at 48 hours before experiment; received LPS (7mg/Kg) to induced lung injury in treatment groups and normal saline in controls at experiment hour. At 2h, 4h and 6h timepoints, the animals were exsanguinated and sampled for measurements of PaO2, lung index (LI), lung permeability index(LPI) and lung histologic examination in therapeutic effect study. The animals were sampled for counts of WBC and PMN in blood and BALF, and PMN in lung tissue, measurements of IL-1βand TNFαin serum and BALF, the expression(IOD) of E-selectin, ICAM-1 and NF-κB measured immunohistochemically in antiinflammatory study.3. In the research on therapeutic effects and antiinflammation of PFT, the animals divided into two treatment groups(LPS and LPS+PFT) and two control groups(NC and PFT)were injected via penile vein with PFT (10ml/Kg) in PFT group and LPS+PFT group, with normal saline in others. Five minutes late, the animals received LPS (7mg/Kg) to induced lung injury in treatment groups and normal saline in controls. At 2h, 4h and 6h timepoints, the rats were exsanguinated and sampled for measurements of PaO2, the ratio of wet and dry weight of lung (W/D), lung permeability index(LPI) and lung histologic examination in therapeutic effect study. The animals were sampled for counts of WBC and PMN in blood and BALF, PMN in lung tissue, measurements of IL-1βand TNFαin serum and BALF, the expression (MFI) of CD11b on PMN measured with Flow cytometry and the expression (IOD) of E-selectin, ICAM-1 and NF-κB measured immunohistochemically in antiinflammatory study.Results1. The absorption rate of PFO intraperitoneally administrated was to 0.93±0.03 at 72h. The blood concentration of PFO reached highest (1.67±0.30 ug/ml) at 48h and cannot detected 1 month late. There were no effects on the liver and kidney function. After PFTE intravenously administrated, its blood concentration was highest (3229.34±466.01ug/ml) at 1h and rapidly reduced to 349.9±88.61ug/ml at 6h and cannot detected 10 days late. The PaO2 significantly increased (p<0.01), but rapidly decreased to the level of no significant difference (p>0.05) compared with control group at 6h timepoint. The levels of ALT and AST also significantly increased (p<0.01) and decreased to the level of control group (p>0.05) at tenth day.2. The results of therapeutic effects of PFO showed that the LPS significantly reduced PaO2 compared with NC, PFO increased PaO2 compared with LPS but significantly only at 6h timepoint (p<0.05). LI and LPI were significantly reduced in LPS+PFO group compared with LPS group at 4h and 6h timepoints (p<0.05 or 0.01). The edema and infiltration of leukocytes in lung tissue slightly reduced in PFO group. The results of antiinflammation of PFO showed that LPS significantly increased the counts of WBC and PMN in BALF and PMN in lung tissue at all timepoints and WBC in blood at 2h timepoint, decreased these of WBC in blood at 6h timepoint and PMN in blood at three timepoints compared with NC (p<0.01). The PFO significantly inhabited the effects of LPS (p<0.01). The levels of IL-1βand TNFαin serum and BALF markedly increased in LPS group than in normal controls (p<0.01or 0.05) and significantly decreased in LPS+PFO group, when compared with LPS group (p<0.01) except in 2h and 4h timepoint for TNFαin serum. The expression(IOD) of E-selectin, ICAM-1 and NF-κb significantly reduced in LPS+PFO group than in LPS group(p<0.01).3. The results of therapeutic effects of PFT showed that PaO2 significantly decreased in LPS group than in controls (P<0.01) and increased in PFT and LPS+PFT groups compared with LPS group (p<0.01). W/D and LPI were significantly lower in LPS+PFT group than in LPS group at 4h and 6h timepoints (p<0.05 and 0.01).The edema and infiltration of leukocytes in lung tissue markedly reduced by PFT. Alveolar inflation and foam granules of PFC were found histologically in PFT and LPS+PFT groups.The results of antiinflammation of PFT showed that LPS significantly increased the counts of WBC and PMN in BALF and PMN in lung tissue at all timepoints and WBC in blood at 2h timepoint, decreased these of WBC in blood at 6h timepoint and PMN in blood at three timepoints compared with controls(p<0.01). The PFT significantly inhabited these effects of LPS (p<0.01). The levels of IL-1βand TNFαin serum and BALF markedly increased in LPS group than controls (p<0.01) and decreased in LPS+PFT group compared with LPS group (p<0.01 or 0.05). The expression(MFI) of CD11b on PMN and the expression (IOD) of E-selectin, ICAM-1, NF-κb significantly reduced in LPS+PFT group than in LPS group(p<0.01).Conclusions1. The systemic application of PFC through peritoneum(PFO) and vein(PFTE) can reach certain blood concentrations of PFC and maintain some hours. PFO has no toxicity on liver and kidney function. Thus, the way of systemic application of PFC is safe and feasible.2. PFO administrated intraperitoneally and PFT injected intravenously increase PaO2, decrease lung edema, permeability. Furthermore, PFT may improve the pathological changes of lung. These suggest that systemic application of PFO and PFT may have therapeutic effects on LPS-induced acute lung injury.3. Systemic PFO and PFT inhabit the lung infiltration of PMN, reduce the release of proinflammatory cytokine. These suggest that systemic PFO and PFT may have significant effects of nonspecific antiinflammation on LPS-induced aute lung injury, which may be correlated with the prohibition of the expession of E-selectin, ICAM-1, NF-kb and CD11b.更多还原