eNO在肝移植手术中监测缺血再灌注损伤的应用

目的通过监测肝移植术中呼出气一氧化氮(eNO)的变化,来研究eNO与肝移植手术中缺血/再灌注损伤的关系。方法收集2015-09/2017-02哈尔滨医科大学附属第一临床医学院10例于静吸复合全身麻醉下行原位肝移植术患者。分别于预计无肝期前10 min(T0)、预计新肝期前10 min(T1)、新肝期开始即刻(T2)、新肝期10 min(T3)、新肝期20 min(T4)、新肝期30 min(T5)、手术结束前(T6)采集患者的呼出气和动脉血,分析eNO、乳酸(Lac)。结果所有患者顺利完成手术,术中血流动力学变化明显。T0-T6,eNO先上升后下降,T2与T0相比,eNO显著升高且达到最高值(20.20±5.55 vs 14.40±3.86),T5恢复至T0水平;T0-T6,Lac持续升高;eNO与Lac无相关性。结论肝移植患者进入新肝期eNO显著升高,后又逐渐下降至无肝前期水平,其机制可能与新肝期缺血/再灌注损伤有关。eNO可以作为一个监测肝移植手术中缺血/再灌注损伤的新指标。     

急性心肌梗死患者经皮冠状动脉介入治疗前后血清可溶性Fas、可溶性Fas配体和内皮素-1检测的临床意义

目的 探讨急性心肌梗死患者经皮冠状动脉介入(PCI)治疗前后血清可溶性Fas(sFas)、可溶性Fas配体(sFasL)和内皮素-1(ET-1)水平的变化及意义.方法 应用放射免疫法和酶联免疫法对40例急性心肌梗死患者(急性心肌梗死组)进行了PCI治疗前后血清sFas、sFasL和ET-1的检测,并与40名正常健康人(正常对照组)作比较.结果 在PCI治疗前,急性心肌梗死组血清sFas、sFasL和ET-1水平均高于正常对照组(P均＜0.01),治疗后2周,与正常对照组比较差异均无统计学意义(P均＞0.05),血浆sFas水平与sFasL水平呈正相关(r=0.5398,P＜0.01);血浆sFas水平与ET-1水平呈正相关(r=0.5282,P＜0.01);血浆sFasL水平与ET-1水平也呈正相关(r=0.5484,P＜0.01).结论 检测急性心肌梗死患者血清sFas、sFasL和ET-1水平的变化对了解病情、观察预后均有重要的临床价值.     

急性心肌梗死患者经皮冠状动脉介入治疗前后血清可溶性Fas、可溶性Fas配体和内皮素-1检测的临床意义

目的 探讨急性心肌梗死患者经皮冠状动脉介入(PCI)治疗前后血清可溶性Fas(sFas)、可溶性Fas配体(sFasL)和内皮素-1(ET-1)水平的变化及意义.方法 应用放射免疫法和酶联免疫法对40例急性心肌梗死患者(急性心肌梗死组)进行了PCI治疗前后血清sFas、sFasL和ET-1的检测,并与40名正常健康人(正常对照组)作比较.结果 在PCI治疗前,急性心肌梗死组血清sFas、sFasL和ET-1水平均高于正常对照组(P均＜0.01),治疗后2周,与正常对照组比较差异均无统计学意义(P均＞0.05),血浆sFas水平与sFasL水平呈正相关(r=0.5398,P＜0.01);血浆sFas水平与ET-1水平呈正相关(r=0.5282,P＜0.01);血浆sFasL水平与ET-1水平也呈正相关(r=0.5484,P＜0.01).结论 检测急性心肌梗死患者血清sFas、sFasL和ET-1水平的变化对了解病情、观察预后均有重要的临床价值.     

肝移植围手术期血流动力学及血浆一氧化氮和内皮素含量的变化

目的探讨一氧化氮(NO)和内皮素(ET)对肝硬化患者肝移植围手术期体、肺循环的影响。方法24例终末期肝硬化患者接受改良背驮式肝移植术,术中持续监测心率(HR)、心排血量(CO)、平均动脉压(MABP)、平均肺动脉压(MPAP)、中心静脉压(CVP)、肺动脉楔压(PAWP)、心排血指数(CI)、体循环阻力指数(SVRI)和肺循环阻力指数(PVRI)。分别于麻醉后术前、无肝前10min、无肝30min、新肝30min和术毕5个时间点采集中心静脉血,用硝酸还原法和放射免疫法分别测定血浆NO和ET1水平。结果1MABP在下腔静脉和门静脉阻断及开放后短期内有一过性下降〔分别由(81±11)mmHg(1mmHg=0.133kPa)降至(79±9)mmHg,再降至(57±19)mmHg,P均<0.05〕,应用血管活性药物后,可基本维持稳定。2CVP、MPAP和PAWP在无肝期均显著降低(P均<0.05);而在新肝期显著增高并维持高于术前水平。3CI在无肝期显著降低(P<0.05),新肝10min后显著增高(P<0.05)。4SVRI和PVRI在无肝期均显著增高(P均<0.05);血管开放后新肝15min内SVRI和PVRI高于术前水平,新肝30min后SVRI显著低于术前水平。5与术前值比较:阻断后,血浆NO水平明显降低(P<0.05),新肝期和术毕均升高(P均<0.05);在无肝30min、新肝30min血浆ET1水平均升高(P均<0.05)。结论肝硬化患者肝移植围手术期血流动力学变化显著,新肝期易发生轻度肺高压。新肝期NO和ET增高,其临床意义有待进一步研究。     

改良背驮式肝移植围术期血酸碱和电解质变化

目的:探讨改良背驮式肝移植围术期血酸碱、电解质变化及处理。方法:39例病人采用静吸复合全麻,术中监测血流动力学、呼吸功能、血气、生化、凝血功能、血糖、体温和尿量等。麻醉后、无肝期控制血K 在3.5～4.0mmol/L,无肝前期控制输液量,无肝期严格控制输入总量,并给予血管活性药物维持循环功能稳定。分别于麻醉前,麻醉后30min,无肝期前15min,无肝期15、30min,新肝期5、15、30min及术毕抽取动脉血进行血气分析、检测生化和凝血功能等。结果:麻醉后30min、无肝期前15minpH、BE值和HCO3与麻醉前-比较无明显变化,但无肝期随门、腔静脉血流阻断时间延长,pH、BE值和HCO3下降,至新肝期5min达高峰。-血K 麻醉后30min、无肝期均较麻醉前低,新肝期血流开放后有一过性升高,但仍维持在正常范围内。结论:肝移植无肝期,尤其在无肝后期维持血K 在可耐受的较低水平,对减轻新肝期门、腔静脉开放后血K 过高,预防肝再灌注综合征发生,维持循环功能稳定是有利的。     

甘露醇于肝移植中的应用研究

目的观察甘露醇于肝移植无肝期及再灌注期对患者血流动力学及尿量的影响。方法肝移植患者30例,ASAⅡ-Ⅲ级,分为2组。实验组（n=15）：于无肝期内快速输注甘露醇250mL,进入再灌注期后缓慢静滴甘露醇直至手术结束。对照组（n=15）：根据手术需要及时补充晶体和胶体液。2组患者均于必要时输浓缩红细胞和血浆。分别于门脉开放前20、10min,门脉开放时、开放后10、20、30min观察患者的中心静脉压（CVP）、肺动脉楔压（PAWP）、平均动脉压（MAP）、肺动脉压（PAPm）;以及原肝期、无肝期、再灌注期的尿量。结果与实验组比较,对照组在原肝期和再灌注前20minCVP、PAWP、MAP、PAPm无统计学差异,对照组于再灌注前10min及其门脉开放后各时点CVP、PAWP、MAP、PAPm明显下降（P〈0．05）。实验组无肝期和再灌注期的尿量明显高于对照组（P〈0．05）。结论对于心功能正常的患者,甘露醇完全可以应用于肝移植手术,并且对血流动力学的稳定和肾功能的保护及再灌注期酸碱平衡和电解质的恢复有积极的作用。     

乙型肝炎患者血清中sFas和sFasL检测临床意义

目的探讨可溶性Fas(sFas)和可溶性FasL(sFasL)在乙型肝炎患者血清中水平及临床意义.方法采用ELISA双抗体夹心法检测139例乙型肝炎和30名健康体检者血清中sFas和sFasL的浓度.结果重型乙型肝炎、肝硬变、慢性活动性乙型肝炎患者血清中sFas和sFasL的水平,与健康对照组之间存在显著差异(P＜0.05);血清中sFas和sFasL比值,除急性肝炎与对照组无差异外,其余各组均呈明显下降趋势.结论乙型肝炎患者血清中sFas、sFasL水平与病情严重程度呈正相关,而二者的比值则与病情严重呈负相关.     

丙泊酚麻醉对围手术期肝缺血再灌注损伤患者血浆一氧化氮/内皮素-1和炎症细胞因子的影响

目的:观察围手术期应用丙泊酚麻醉对肝缺血再灌注损伤患者血浆一氧化氮/内皮素-1(NO/ET-1)和炎症细胞因子的影响.方法:选择择期行肝叶切除的手术患者60例,分成对照组和丙泊酚组.对照组采用咪唑安定、芬太尼、阿曲库胺麻醉诱导;丙泊酚组采用丙泊酚2mg/kg静注,余同对照组.诱导后两组均静注芬太尼、阿曲库胺间断维持麻醉,丙泊酚组加用丙泊酚4～6 mg/(kg·h)微泵持续静脉输注至关腹,对照组以同样方法输注等量生理盐水.两组患者分别在术前(T1)、肝门阻断末(T2)、术后第1天(T3)和术后第3天(T4)抽取静脉血,采用ELISA法检测血浆NO、ET-1、白介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)含量,放射免疫法检测血浆白介素-8(IL-8)含量.结果:两组患者术前血浆NO、ET-1、IL-6、IL-8和TNF-α含量无统计学差异(P＜0.05).肝门阻断末和术后第1天两组患者血浆NO含量均较杏前明显下降,血浆ET-1、IL-6、IL-8和TNF-α含量均明显增加,但丙泊酚组下降或增加幅度均低于对照纽,且术后第3天两组患者血浆NO、ET-1、IL-6、IL-8和TNF-α含量均恢复至术前水平.结论:肝缺血再灌注损伤可引起血浆NO/ET-1的比例失衡和促炎症细胞因子释放,从而引起血管内皮细胞功能紊乱,产生或加重炎症反应.围手术期应用丙泊酚麻醉对肝缺血再灌注损伤有保护作用,其作用可能通过调节NO/ET-1比例失衡和抑制促炎症细胞因子释放,保护血管内皮细胞和减轻炎症反应而实现.     

基质金属蛋白酶-2与体外循环下心肌再灌注损伤的研究

目的揭示基质金属蛋白酶-2（MMP-2）在体外循环（CPB）围手术期的释放规律，并探讨基质金属蛋白酶抑制剂（TIMP）——强力霉素在CPB围手术期对心肌的保护作用。方法将20例风心、二尖瓣狭窄患者随机分成Ⅰ组（n=10）和Ⅱ组（n=10），Ⅱ组患者术前1d始口服强力霉素100mg、2次／d，至术后3d，其他处理两组无差别，均在CPB下行二尖瓣置换术。两组分别在手术前1d（T1）、CPB前1min（T2）、主动脉阻断（ACC）前1min（T3）、主动脉开放后1min（T4）、主动脉开放后15min（T5）、及术后3d（T6）共6个时间点采集静脉回流血，测定血浆中MMP-2的浓度。结果Ⅰ组患者血浆中MMP-2浓度在T4时间点达峰值，后逐渐下降，但术后3d仍高于术前；而Ⅱ组患者血浆中MMP-2浓度水平在T1时间点和Ⅰ组患者无明显差异，余各时间点均低于Ⅰ组患者，且无明显峰值出现。结论MMP-2在围手术期对CPB下心肌再灌注损伤诊断和评估预后有重要作用；强力霉素则在围手术期对心肌保护有重要的临床价值。     

同种异体肾移植排斥患者血清sFas和sFasL水平的临床观察

目的观察肾同种移植急性排斥患者血清可溶性Fas(sFas)和sFas配体(sFasL)的水平及临床意义.方法采用酶联免疫吸附试验(ELISA)分别对健康对照组及实验组透析前后sFas、sFasL进行检测.结果对照组sFas为(256.8±72.0)ng/L,sFasL为(227.9±65.9)ng/L;实验组透析前后sFas分别为(1 225.7±467.6)ng/L、(1 225.8±464.0)ng/L,sFasL分别为(227.9±147.2)ng/L、(226.9±109.6)ng/L.实验组与对照组的sFas比较差异有显著性(P＜0.01),而sFasL比较差异无显著性(P＞0.05).结论 sFas在排异的病理反应过程中参与了细胞凋亡的抑制,透析并不能改善Fas-FasL介导的细胞凋亡.     

Fas and Fas Ligand Interactions Suppress Melanoma Lung Metastasis

Apoptosis induced by Fas (CD95) ligation is frequently lost during tumor progression; however, there is no direct evidence to support an association of Fas loss-of-function with metastatic tumor behavior. To determine whether Fas loss-of-function is critical for acquisition of the metastatic phenotype, we have compared the ability of Fas-sensitive K1735 murine melanomas to form spontaneous lung metastases in wild-type and Fas ligand–deficient mice. Fas-sensitive melanoma clones are highly tumorigenic but rarely metastatic in wild-type syngeneic mice. However, in Fas ligand–deficient mice, both the incidence and number of metastases are increased. These findings provide the first evidence that Fas–Fas ligand interactions can suppress metastasis and that tumor Fas loss-of-function may be causally linked to metastatic progression.     

Roles of Fas and Fas ligand during mammary gland remodeling

Mammary involution is associated with degeneration of the alveolar structure and programmed cell death of mammary epithelial cells. In this study, we evaluated the expression of Fas and Fas ligand (FasL) in the mammary gland tissue and their possible role in the induction of apoptosis of mammary cells. FasL-positive cells were observed in normal mammary epithelium from pregnant and lactating mice, but not in nonpregnant/virgin mouse mammary tissue. Fas expression was observed in epithelial and stromal cells in nonpregnant mice but was absent during pregnancy. At day 1 after weaning, high levels of both Fas and FasL proteins and caspase 3 were observed and coincided with the appearance of apoptotic cells in ducts and glands. During the same period, no apoptotic cells were found in the Fas-deficient (MRL/lpr) and FasL-deficient (C3H/gld) mice. Increase in Fas and FasL protein was demonstrated in human (MCF10A) and mouse (HC-11) mammary epithelial cells after incubation in hormone-deprived media, before apoptosis was detected. These results suggest that the Fas-FasL interaction plays an important role in the normal remodeling of mammary tissue. Furthermore, this autocrine induction of apoptosis may prevent accumulation of cells with mutations and subsequent neoplastic development. Failure of the Fas/FasL signal could contribute to tumor development.     

Functional expression of Fas and Fas ligand on human colonic intraepithelial T lymphocytes

The expression of Fas, a cell surface receptor directly responsible for triggering cell death by apoptosis, and its ligand (FasL) was investigated on both human colonic intraepithelial T lymphocytes (IELs) and peripheral blood mononuclear lymphocytes (PBMLs). FACS analysis indicated that IELs have increased expression of Fas compared with PBMLs, together with the progress activation marker, CD45RO. A discrete fraction of freshly isolated IELs also constitutively expressed FasL, perhaps as a result of recent in vivo activation. Using monoclonal antibody APO2.7, which detects mitochondrial 7A6 antigen specifically expressed by cells undergoing apoptosis, we further investigated the apoptosis-inducing effect of anti-Fas monoclonal antibody (CH11) on both IELs and PBMLs. FACS analysis revealed that CH11 increased the percentage of apoptotic cells, in IELs but not in PBMLs. Culture with anti-FasL monoclonal antibody (4H9) significantly recovered cell viability in IELs, but not in PBMLs. These results indicate that IELs constitutively express both Fas and FasL and that Fas crosslinking generates signals resulting in apoptosis, outlining a potential mechanism involved in intestinal tolerance.     

Soluble Fas and soluble Fas ligand levels in patients with acute hepatic failure

PURPOSE: The Fas ligand (FasL)/Fas system is an apoptosis induction system that plays an important role in homeostasis and biophylaxis. We measured tumor necrosis factor alpha (TNF-alpha), soluble FasL (sFasL), and soluble Fas (sFas) in patients with acute hepatic failure to determine the relation between such failure and apoptosis. MATERIALS AND METHODS: We assayed 21 blood samples from patients with acute hepatic failure and 8 from patients with sepsis but without acute hepatic failure. Serum TNF-alpha, sFas, and sFasL levels were determined by enzyme-linked immunosorbent assay. RESULTS: sFasL levels were significantly higher in the patients with acute hepatic failure than in the patients with sepsis (0.68 +/- 0.42 ng/mL vs. 0 ng/mL, P =.0001). No significant differences were observed in sFas levels between the two groups. A significant correlation was observed between TNF-alpha and sFas levels (r = 0.657, P =.0008); a negative correlation was observed between TNF-alpha and sFasL levels (r = 0.454, P =.038). CONCLUSIONS: Our results suggest that pathologic aggravation of acute hepatic failure are related to changes in the FasL/Fas system and that TNF-alpha and sFasL, in particular, may play hepatoprotective roles.     

Spontaneous Development of Plasmacytoid Tumors in Mice with Defective Fas–Fas Ligand Interactions

B cell malignancies arise with increased frequency in aging individuals and in patients with genetic or acquired immunodeficiency (e.g., AIDS) or autoimmune diseases. The mechanisms of lymphomagenesis in these individuals are poorly understood. In this report we investigated the possibility that mutations at the Fas (lpr) and Fasl (gld) loci, which prevent Fas-mediated apoptosis and cause an early onset benign lymphoid hyperplasia and autoimmunity, also predispose mice to malignant lymphomas later in life. Up to 6 mo of age, hyperplasia in lpr and gld mice results from the predominant accumulation of polyclonal T cell subsets and smaller numbers of polyclonal B cells and plasma cells. Here, we examined C3H-lpr, C3H-gld, and BALB-gld mice 6–15 mo of age for the emergence of clonal T and B cell populations and found that a significant proportion of aging mice exclusively developed B cell malignancies with many of the hallmarks of immunodeficiency-associated B lymphomas. By 1 yr of age, ∼60% of BALB-gld and 30% of C3H-gld mice had monoclonal B cell populations that grew and metastasized in scid recipients but in most cases were rejected by immunocompetent mice. The tumors developed in a milieu greatly enriched for plasma cells, CD23⁻ B cells and immunodeficient memory T cells and variably depleted of B220⁺ DN T cells. Growth factor–independent cell lines were established from five of the tumors. The majority of the tumors were CD23⁻ and IgH isotype switched and a high proportion was CD5⁺ and dull Mac-1⁺. Considering their Ig secretion and morphology in vivo, most tumors were classified as malignant plasmacytoid lymphomas. The delayed development of the gld tumors indicated that genetic defects in addition to the Fas/Fasl mutations were necessary for malignant transformation. Interestingly, none of the tumors showed changes in the genomic organization of c-Myc but many had one or more somatically-acquired MuLV proviral integrations that were transmitted in scid passages and cell lines. Therefore, insertional mutagenesis may be a mechanism for transformation in gld B cells. Our panel of in vivo passaged and in vitro adapted gld lymphomas will be a valuable tool for the future identification of genetic abnormalities associated with B cell transformation in aging and autoimmune mice.     

转染mFas配体的COS-7细胞诱导Fas+淋巴瘤细胞系(Yac-1)凋亡

本研究探讨通过Fas配体(Fas ligand,FasL)-Fas途径进行免疫治疗淋巴瘤的可能性。常规转化pBillneo-mFasL至大肠杆菌DH5α,经扩增、质粒抽提和纯化后,进行限制性内切酶酶切、mFasL基因PCR及其产物DNA测序,以鉴定pBillneo-mFasL内mFasL基因一级结构及插入方向;用脂质体法转染 pBillneo-mFasL至猴肾COS-7细胞,G418选择培养后,Western印迹分析外源性mFasL cDNA基因表达水平,将高表达mFasL的COS-7细胞与Fas~+小鼠淋巴瘤细胞系Yac-1以不同比例混合培养,5小时后收集悬浮的Yac-1细胞,用膜联蛋白(annexin)V/PI标记后,借助FCM观察细胞调亡率。结果表明,质粒pBillneo-mFasL的EcoRI酶切获得920 bp和7227 bp产物,Hind Ⅲ酶切获得1293 bp和6807 bp产物,初步证实插入mFasL cDNA片段与理论预计大小一致,并系正向插入;PCR扩增出自起始密码子(ATG)至终止密码子(TAA)后+36 bp的mFasL cDNA全长890 bp序列,DNA测序结果与基因库已知序列完全一致;pBillneo-mFasL转染COS-7细胞,并经G418选择培养后,Western印迹检测到明显mFasL蛋白质表达;当用这种高表达mFasL蛋白质的COS 7细胞与Fas~+ Yac-1细胞以1:1,5:1和10:1混合培养5小时后,用annexin V/PI标记悬浮Yac-1,结果后者调亡率分别为(22±4.8)％,(32.18±7.8)％和(51.8±5.4)％,与     

Antigen presenting cells expressing Fas ligand down-modulate chronic inflammatory disease in Fas ligand-deficient mice

We assessed the effect of modified antigen presenting cells (APCs) expressing high levels of Fas ligand (APC-FasL) on post-viral chronic inflammatory disease. FasL-deficient B6-gld/gld mice infected with murine cytomegalovirus (MCMV) cleared the virus from their lungs, kidneys, and livers within 2 weeks of infection. However, inflammation persisted in these organs for more than 8 weeks, with a chronically increased T-cell response to MCMV-infected APCs and production of autoantibodies. Administration of APC-AdFasL at 4 weeks suppressed this inflammation and diminished the T-cell response and autoantibody production. APC-AdFasL that had been transfected with ultraviolet-irradiated MCMV were more effective than uninfected APC-AdFasL in ameliorating the chronic inflammation. APC-AdFasL migrated preferentially to the spleen, where they triggered apoptosis of lymphocytes in the marginal zone of the spleen. These results confirm that Fas-mediated apoptosis is not required for clearance of virus, but is required for down-modulation of the virally induced chronic inflammatory response. This organwide effect of APC-AdFasL appears to be mediated by elimination of activated T lymphocytes in the spleen before their emigration to the target organs.