肠内肠外营养对大鼠肠道损伤后屏障功能恢复的实验研究

目的 比较肠内、肠外两种营养支持途径及不同营养物质对胃肠手术后大鼠肠屏障功能的恢复情况.方法 将40只雄性SD大鼠随机分为4组:空白对照组(C组)、肠外营养组(PN组)、普通肠内营养组(EN组)、富含谷氨酰胺的肠内营养组(G-EN组).PN组、EN组、G-EN组行盲肠切除+胃造口置管手术并联合使用阿莫西林50 mg+甲硝唑20 g两次进行干预.术后第1天起各组等氮等热卡连续给予营养支持7 d,于末段回肠5 cm处取肠段1 cm,在光镜下观察肠黏膜形态;采用酶联免疫吸附实验(ELISA)比较血浆D-乳酸含量,检测肠道黏膜通透性;双抗体夹心ABC-ELISA法检测肿瘤坏死因子水平(TNF-α);免疫组化法观察肠黏膜occludin蛋白表达.结果 PN组肠黏膜明显萎缩,其绒毛高度、黏膜厚度、隐窝深度、绒毛表面积及肠黏膜occludin蛋白表达均显著低于C组、EN组、G-EN组(P<0.05),D-乳酸及TNF-α水平显著高于C组、EN组、G-EN组(P<0.05);EN组肠黏膜萎缩较明显,其肠黏膜形态及肠黏膜occludin蛋白表达均低 于C组、G-EN组(P<0.05),D-乳酸及TNF-α水平显著高于C组及G-EN组(P<0.05);G-EN组肠道形态、肠黏膜occludin蛋白表达、D-乳酸及TNF-α水平与C组无统计学差异(P>0.05).结论 谷氨酰胺强化的肠内营养在维护肠黏膜屏障功能、减轻炎症反应,增加肠上皮occludin蛋白表达等方面优于单独使用肠内营养液或肠外营养液,更有助于胃肠手术后大鼠肠屏障功能的恢复.     

核因子κB在重症急性胰腺炎大鼠肠黏膜屏障损伤中的作用机制

目的探讨核因子κB(NF-κB)在重症急性胰腺炎(SAP)大鼠肠黏膜屏障损伤中的作用机制。方法 24只Wastar大鼠按随机数字表法分为对照组、SAP组和NF-κB抑制剂组即二硫代氨基甲酸吡咯烷(PDTC)组,每组各8只。制作SAP大鼠模型,对照组仅翻动胰腺,PDTC组于建模后2 h经尾静脉注射PDTC溶液40 m L/kg。各组大鼠于建模后24 h处死,观察各组大鼠腹腔内大体改变,比较各组肠黏膜的病理改变、通透性的变化及肿瘤坏死因子α(TNF-α)、白细胞介素1(IL-1)的表达水平,应用免疫组织化学法检测大鼠肠黏膜上皮细胞紧密连接蛋白Occludin的表达。结果对照组大鼠腹腔内无明显炎症征象,肠管无水肿充血;SAP组和PDTC组大鼠均有不同程度的腹水、肠管水肿、充血,且SAP组更为严重。光学显微镜下对照组肠黏膜未见明显损伤,SAP组和PDTC组大鼠回肠可见不同程度的肠黏膜破坏,且PDTC组较轻。三组大鼠的病理评分[(3.6±1.4)、(21.3±3.7)、(33.8±4.2)分]和肠黏膜通透性[(22±6)、(188±26)、(328±35)n L·min-1·cm-1]比较,差异均有统计学意义(F=7.259、6.402,P均0.05),且SAP组肠黏膜通透性较PDTC组增加更为显著(P0.05)。三组大鼠TNF-α[(146±37)、(1 684±80)、(2 896±99)ng/L],IL-1[(53±10)、(1 756±60)、(2 893±88)ng/L]表达水平比较,差异均有统计学意义(F=5.751、7.247,P均0.05),其中SAP组和PDTC组肠道TNF-α、IL-1表达水平均较对照组显著增加(P均0.05),且SAP组大鼠TNF-α和IL-1表达水平均较PDTC组更高(P均0.05)。免疫组织化学结果显示,对照组Occludin蛋白沿肠黏膜上皮细胞分布,呈很强的棕褐色线状信号;PDTC组Occludin的定位与对照组没有明显区别,但棕褐色阳性信号有所减弱;SAP组阳性细胞明显减少。三组大鼠Occludin[(15.6±3.0)、(6.4±1.4)、(3.1±1.2)]表达水平比较,差异有统计学意义(F=5.427,P0.05)。结论 SAP大鼠肠黏膜屏障损伤可能与肠组织中NF-κB表达升高有关,从而下调肠上皮细胞紧密连接蛋白Occludin的表达,抑制NF-κB表达对SAP肠黏膜屏障损伤具有保护作用。     

慢性乙醇摄取对急性肺损伤大鼠气道上皮细胞间连接蛋白occludin和E-cadherin及屏障功能的影响

目的 观察慢性乙醇摄取及内毒素处理对大鼠气道上皮屏障功能及紧密连接(TJ)特征性蛋白occludin和黏附连接(AJ)蛋白E-cadherin的影响.方法 将40只SD大鼠随机均分为对照组、慢性乙醇摄取组(乙醇组)、内毒素处理组(LPS组)、慢性乙醇摄取合并内毒素处理组(乙醇+LPS组).利用荧光示踪剂异硫氰酸荧光素标记的右旋糖酐(FD4)测定支气管肺泡上皮的通透性;免疫荧光共聚焦显微镜下观察大鼠气道上皮occludin和E-cadherin蛋白分布及表达;蛋白质免疫印迹法(Western blotting)和逆转录-聚合酶链反应(RT-PCR)测定肺组织中occludin和E-cadherin的蛋白及mRNA表达;并观察肺组织病理学改变.结果 乙醇组及LPS组支气管肺泡上皮通透性均较对照组明显增高(P均＜0.05);乙醇+LPS组支气管肺泡上皮的通透性进一步增高(P＜0.01).occludin和E-cadherin蛋白在对照组大鼠气道上皮呈连续、均匀的胞膜及胞质中表达;在乙醇组、LPS组胞膜呈部分断裂、不连续的表达,且胞膜和胞质的表达下降;在乙醇+LPS组的表达显著下降,且胞膜表达呈明显的断裂甚至消失.Western blotting和RT-PCR显示,乙醇组和LPS组肺组织中occludin和E-cadherin的蛋白及mRNA表达均较对照组明显下降(P均＜0.05);乙醇+LPS组中蛋白及mRNA表达下降最为明显,与其余各组比较差异均有统计学意义(P均＜0.01).结论 慢性乙醇摄取通过降低TJ蛋白occludin和AJ蛋白E-cadherin的蛋白及mRNA表达水平,并干扰各蛋白在胞膜上的定位,最终导致气道上皮屏障功能受损,加重内毒素诱导的急性肺损伤.     

抑制c-Jun蛋白氨基末端激酶信号通路对内毒素血症大鼠肠屏障功能的保护作用

目的 探讨阻断细胞质内应激信号通路c-Jun蛋白氨基末端激酶(JNK)对内毒素血症大鼠肠屏障损伤的保护作用.方法 24只雄性SD大鼠,按随机数字表法分为对照组、内毒素血症模型组、JNK抑制剂组3组,每组8只.对照组仅给予生理盐水2 ml/kg+JNK抑制剂SP600125的溶剂PPCES液2.5 ml/kg;内毒素血症模型组静脉注射脂多糖( LPS) 10 mg/kg+PPCES液2.5 ml/kg;JNK抑制剂组静脉注射LPS 10 mg/kg+JNK抑制剂SP600125 10 mg/kg.记录各组大鼠活动和生存情况;并于12h后取大鼠同肠,光镜下观察肠道黏膜病理改变;同时取血,采用酶联免疫吸附试验( ELISA)测定血浆D-乳酸含量.结果 对照组大鼠活动正常,无死亡;模型组大鼠精神萎靡、活动减少,12h内死亡1只;JNK抑制剂组大鼠活动较模型组活跃,无死亡.回肠黏膜病理检查显示:与对照组相比,模型组大鼠回肠组织黏膜水肿,绒毛缩短,炎性细胞浸润;INK抑制剂组回肠组织病变较模型组减轻.模型组大鼠血浆D-乳酸含量(μg/L)较对照组显著升高(943.8±439.6比227.9±130.0,P＜0.05);JNK抑制剂能显著降低内毒素血症大鼠血浆D-乳酸含量(637.4±114.4比943.8±439.6,P＜0.05).结论 抑制细胞质内应激信号通路JNK能减轻内毒素血症大鼠肠屏障损伤.     

肝素对感染性休克犬早期肠黏膜损伤保护作用的研究

目的 通过对血浆标示物D-乳酸和I-FABP的测定及分析,验证感染性休克犬早期肠脂肪酸结合蛋白和D-乳酸表达水平发生变化以及肝素对感染性休克犬早期空肠绒毛微循环改善及肠脂肪酸结合蛋白和D-乳酸表达水平的影响.方法 通过静脉注射LPS方法建立比格犬早期感染性休克模型.将40只实验犬随机(随机数字法)分为空白对照组,LPS组,基础治疗组,肝素治疗组.术后稳定1h,即T0点,给LPS造模后1h,即T1点,之后序贯观察5h分别为T2,T3,T4,T5,T6点.在相应观察时间点(T1、T3、T6时间点)采集犬静脉血,经1 500 r/min,15 min离心后取血浆,以EP管分装后置入-80℃保存,通过ELISA法测定肠脂肪酸结合蛋白(I-FABP)和D-乳酸(D-lac).同时应用OPS观察空肠绒毛微循环的变化.结果 肝素组IFABP (21.6±2.3) pg/mL比较LPS组IFABP(100.48±8.42) pg/mL会明显下降,差异具有统计学意义(P＜0.05);同时微循环有相应的改善.基础治疗组与LPS组比较未见明显变化,P＞0.05.结论 感染性休克早期存在肠黏膜微循环障碍及肠黏膜损伤.肝素对早期感染性休克犬肠黏膜微循环障碍具有改善作用,进而保护肠黏膜屏障功能.     

髓系细胞触发受体-1对重症急性胰腺炎大鼠肠屏障功能的影响

目的 探讨髓系细胞触发受体-1（triggering receptor-1 on myeloid cells,TREM-1）的表达与重症急性胰腺炎（severe acute pancreatitis,SAP）肠屏障功能障碍的关系。方法 雄性Wistar大鼠64只,随机（随机数字法）分为假手术组(SO)和SAP组,每组32只,采用逆行胰胆管注射5％牛磺胆酸钠制备SAP大鼠模型,分别于造模后2,6,12,24h时点取血和回肠组织。改良分光光度法检测血浆D-乳酸、二胺氧化酶（diamine oxidase,DAO）和内毒素浓度。逆转录-聚合酶链反应( RT-PCR)方法检测回肠组织TREM-1、白细胞介素-1β(interleukin-1β,IL-1β)和肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）mRNA的表达水平。对数据进行单因素方差分析和spearman相关性分析,P＜0.05表示差异具有统计学意义。结果SAP组各时点血浆D-乳酸、DAO和内毒素的水平均高于假手术组(P ＜0.01,P＜0.05);SAP组各时点回肠组织TREM-1,IL-1β和TNF-α mRNA的表达水平较假手术组显著增高(P ＜0.01,P＜0.05),TREM-1 mRNA表达水平与IL-1β及TNF-α mRNA表达水平均呈正相关(r=0.956,P =0.044;r =0.986,P=0.015),IL-1β mRNA表达水平与TNF-α mRNA表达水平无明显相关性(P=0.133)。结论 SAP时,大鼠肠组织内TREM-1表达上调,促进炎症介质释放和肠黏膜损伤加重,TREM-1在SAP肠屏障功能障碍的发生发展中起重要作用。     

梗阻性黄疸对大鼠肠黏膜上皮细胞紧密连接蛋白ZO-1和肠黏膜通透性的影响

目的研究梗阻性黄疸对大鼠肠黏膜上皮细胞紧密连接蛋白ZO－1和肠黏膜通透性的影响。方法30只健康雄性Wistar大鼠随机分为3组：正常对照组、假手术组、梗阻性黄疸组（OJ组）。OJ组制成梗阻性黄疸动物模型,7d后各组同一时间点取材。检测门静脉血D-乳酸含量,免疫组化检测肠黏膜紧密连接蛋白ZO-I的分布。结果OJ组大鼠门静脉血D晋L酸水平显著高于正常对照组和假手术组（P〈0．05）,ZO-1染色变淡,分布不均匀,边缘粗糙,强阳性表达率下降。结论梗阻性黄疸时大鼠肠黏膜上皮紧密连接蛋白ZO－1分布紊乱、表达下降,肠黏膜通透性增加,肠黏膜屏障受损。     

谷氨酰胺对急性肺损伤大鼠肺泡上皮屏障功能的影响

目的:观察谷氨酰胺对急性肺损伤大鼠肺泡上皮屏障功能及紧密连接(TJ)特征性蛋白occludin和黏附连接蛋白E-cadherin的影响。方法:40只SD大鼠随机分为对照组、谷氨酰胺处理组(Gln组)、内毒素处理组(LPS组)、谷氨酰胺并内毒素处理组(Gln+LPS组)。测定支气管肺泡渗透性、运用免疫印迹测定和RT-PCR测定肺泡Ⅱ型上皮细胞中occludin和E-cadherin的蛋白及mRNA表达。结果:内毒素导致支气管肺泡上皮渗透性明显增高2倍左右(P<0.01);补充谷氨酰胺可以明显改善由内毒素处理引起的支气管肺泡上皮渗透性增高(P<0.05),但Gln+LPS组支气管肺泡渗透性仍较对照组及Gln组增高(P<0.05)。免疫印迹和RT-PCR显示在LPS组的occludin和E-cadherin蛋白和mRNA表达水平均较对照组及Gln组的低(P<0.01);在Gln+LPS组中occludin和E-cadherin的蛋白和mRNA表达水平较LPS组的高(P<0.05),而较对照组及Gln组的表达水平低(P<0.05)。结论:实验提示内毒素通过降低TJ分子occludin和E-cadherin的mRNA、蛋白表达水平导致肺泡上皮屏障功能受损,补充谷氨酰胺通过上调其mRNA、蛋白表达水平而对肺泡上皮屏障功能有保护作用。     

核因子-kB活化在重症急性胰腺炎肠道屏障损伤中的作用

目的:探讨NF-κB的活化在重症急性胰腺炎时肠道屏障功能损伤中的作用。方法:Wistar大鼠随机分为假手术(SO)组、重症急性胰腺炎(SAP)组、四氢化吡咯二硫代氨基甲酸脂治疗(SAP PDTC)组。术后分批行下腔静脉采血后处死,取胰腺和回肠末段组织。偶氮显色法测定血浆内毒素;改良分光光度法测定血浆D-乳酸;免疫组织化学S-P法检测回肠黏膜NF-κB的活化。结果:SAP组血浆内毒素和D-乳酸则随发病时间延长而持续升高,较SO组差异显著(P<0.05)。PDTC治疗组血浆内毒素和D-乳酸浓度较SAP组明显下降,差异显著(P<0.05)。SO组回肠黏膜少见NF-κB活化的细胞。而SAP组术后1 h即出现大量核内NF-κB P65染色阳性细胞,3 h时最多,较SO组差异显著(P<0.05)。PDTC治疗组NF-κB活化细胞明显少于SAP组,差异显著(P<0.05)。结论:NF-κB作为一多种炎症因子基因的转录因子,在SAP时肠道屏障功能损伤中起重要调控作用。     

NF-κB在重症急性胰腺炎小鼠肠黏膜屏障损伤中的作用

目的探讨NF-κB在重症急性胰腺炎小鼠肠黏膜屏障功能损伤中的调控机制。方法 36只BALB/C小鼠随机分为对照组、模型组、NF-κB干预组,每组12只。18h后处死小鼠,比较各组的腹腔内大体改变、肠黏膜病理改变,肠道通透性的变化及血清细胞因子水平,肠上皮紧密连接蛋白occludin的表达。结果模型组小鼠腹腔内呈明显炎症反应,肠管水肿,肠黏膜水肿,肠道通透性显著增高,NF-κB特异性阻断剂能降低肠道损伤,改善肠黏膜水肿,上调肠上皮紧密连接蛋白occludin的表达,显著降低肠道通透性,降低细胞因子水平。结论 NF-κB阻断剂能够通过选择性的抑制NF-κB活性,改善受损的肠屏障功能。这一作用通过上调肠上皮紧密连接蛋白occludin的水平而实现。     

Specificity of protein C and protein S assays

Summary Specific tests for the measurement of protein C antigen and activity and protein S antigen are used in the clinical laboratory for the routine diagnosis of hereditary protein C and protein S deficiency. The performance of these tests is reviewed and discussed. Special attention is paid to the application of these tests for the analysis of patients on oral anticoagulant therapy. Presented at the ‘2nd International Symposium on Standardization and Quality Control of Coagulation Tests: Implications for the Clinical Laboratory’, Rome, September 28–29, 1989.     

Plasma protein turnover and tissue exchange; influence of dietary protein and protein depletion

The rate of plasma protein turnover is more rapid in dogs receiving adequate dietary protein than when a diet devoid of protein is fed. Both albumin and combined globulins are involved in this change. The difference in turnover is reflected in a total protein half-life of 4.8 days with protein feeding versus 7.8 days without protein in the diet and in the metabolism of 1.0 and 0.65 gm. per kilogram of body weight per day on the respective diets. Additions of dietary protein from 10 to 30 per cent caused no further increase in the rate of plasma protein turnover. With protein depletion due to plasmapheresis and a very low protein diet there is evidence of reduced protein metabolism as indicated by nitrogen retention as well as a reduction in total plasma protein breakdown and interchange of isotope between plasma and tissue proteins. Following introduction of labeled plasma protein into the circulation the net amount of isotope transferred to tissues has been computed from the difference between total plasma protein breakdown and combined C¹⁴ excretion in urine and expired air. In animals receiving adequate dietary protein, tissue transfer amounts to 70 per cent of the total lost from the plasma proteins each day while the percentage rises to 85 in depleted dogs deprived of protein. In dogs with both plasma and tissue proteins labeled it can be estimated that, under conditions of protein feeding, an amount of C¹⁴ approximately equal to that lost from the plasma must recycle to account for the observed decrease in Apparent plasma protein turnover rate, (t½ of 15 versus 5 days). Without protein in the diet the isotope contribution of the tissues to the maintenance of plasma protein levels must be as great as or greater than that transferred in the opposite direction.     

Acquisition of inhibitor-sensitive protein kinases through protein design.

Protein phosphorylation is the major post-translational modification used by eukaryotic cells to control cellular signaling. Protein kinases have emerged as attractive drug targets because heightened protein kinase activity has been associated with several proliferative diseases, most notably cancer and restenosis. Until now, it has been very difficult to confirm the utility of protein kinases as inhibitor targets because very few small molecules that selectively inhibit one particular kinase are known. Discovery of highly specific kinase inhibitors has been slow because the protein family contains approximately 2000 members, all of which share a conserved active site fold. Recent work in several laboratories has sought to circumvent the problem of kinase structural degeneracy by engineering drug sensitivity into Src family tyrosine kinases and mitogen-activated protein kinases through site-directed mutagenesis. By introducing a unique non-naturally occurring amino acid into a conserved region of the enzyme's binding site, a target protein kinase can be rapidly sensitized to a small molecule. Introduction of the engineered kinase into a cell line or animal model should greatly expedite the investigation of protein kinase inhibition as a viable drug treatment. The purpose of this review is to summarize these recent advances in protein kinase drug sensitization.     

Modulation of protein kinase signaling by protein phosphatases and inhibitors

Protein tyrosine phosphatases (PTPs) form a large family of enzymes that serve as key regulatory components in signal transduction pathways. Defective or inappropriate regulation of PTP activity leads to aberrant tyrosine phosphorylation, which contributes to the development of many human diseases. In addition to controlling the phosphorylation states of protein kinase substrates, PTPs can also directly modulate protein kinase activity. Evidence suggests that PTPs can exert both positive and negative effects on a signaling pathway. Thus, further understanding of the fundamental role of protein tyrosine phosphorylation in complex and critical signal transduction pathways requires detailed studies of both the kinases and the phosphatases. In this review, we first summarize our current understanding of PTP structure and function. We then discuss the molecular basis of PTP substrate specificity, focusing primarily on mitogen-activated protein (MAP) kinase phosphatase 3. We demonstrate that the MAP kinase phosphatases display exquisite substrate specificity requiring extensive protein-protein interactions for precise down-regulation of MAP kinase activity. We also highlight our recent progress in developing small molecule PTP1B inhibitors. Using a novel combinatorial approach that is designed to target both the active site and a unique peripheral site in PTP1B, we have obtained a PTP1B inhibitor with 2.4 nM affinity and orders of magnitude selectivity against a panel of PTPs. Currently, some of the compounds are being evaluated in both cell and animal models to further define the role of PTP1B in insulin signaling.     

磷酸腺苷活化蛋白激酶在内毒素血症大鼠蛋白调节中的作用

内毒素血症是现代临床危重病医学面临的棘手问题,也是导致危重症的常见原因和对预后产生严重影响的重要因素,内毒素血症患者往往因为严重感染和应激,处于一个高分解、高消耗的状态.这种能量消耗增加的高分解代谢状态和对代谢底物利用的改变共同构成了此类患者的代谢特征.蛋白质是人体三大能量物质之一,也是各种代谢活动的基础和构成人体的最重要的物质之一.     

Treatment of experimental encephalomyelitis with a novel chimeric fusion protein of myelin basic protein and proteolipid protein.

It has been shown that peripheral T cell tolerance can be induced by systemic antigen administration. We have been interested in using this phenomenon to develop antigen-specific immunotherapies for T cell-mediated autoimmune diseases. In patients with the demyelinating disease multiple sclerosis (MS), multiple potentially autoantigenic epitopes have been identified on the two major proteins of the myelin sheath, myelin basic protein (MBP) and proteolipid protein (PLP). To generate a tolerogenic protein for the therapy of patients with MS, we have produced a protein fusion between the 21.5-kD isoform of MBP (MBP21.5) and a genetically engineered form of PLP (deltaPLP4). In this report, we describe the effects of treatment with this agent (MP4) on clinical disease in a murine model of demyelinating disease, experimental autoimmune encephalomyelitis (EAE). Treatment of SJL/J mice with MP4 after induction of EAE either by active immunization or by adoptive transfer of activated T cells completely prevented subsequent clinical paralysis. Importantly, the administration of MP4 completely suppressed the development of EAE initiated by the cotransfer of both MBP- and PLP-activated T cells. Prevention of clinical disease after the intravenous injection of MP4 was paralleled by the formation of long-lived functional peptide-MHC complexes in vivo, as well as by a significant reduction in both MBP- and PLP-specific T cell proliferative responses. Mice treated with MP4 were resistant to disease when rechallenged with an encephalitogenic PLP peptide emulsified in CFA, indicating that MP4 administration had a prolonged effect in vivo. Administration of MP4 was also found to markedly ameliorate the course of established clinical disease. Finally, MP4 therapy was equally efficacious in mice defective in Fas expression. These results support the conclusion that MP4 protein is highly effective in suppressing disease caused by multiple neuroantigen epitopes in experimentally induced demyelinating disease.     

腺苷酸活化蛋白激酶与骨骼肌蛋白质降解

背景:机体运动时骨骼肌收缩,ATP被大量消耗,产生大量腺苷一磷酸,导致腺苷酸活化蛋白激酶的激活。目的:综述不同运动过程中腺苷酸活化蛋白激酶活性的变化,以及腺苷酸活化蛋白激酶对骨骼肌蛋白质降解的研究成果。方法:检索中国期刊网、维普期刊数据库、www.ncbi.nlm.nih.gov/pubmed和http://highwire.stanford.edu/网站与腺苷酸活化蛋白激酶、运动、蛋白质降解研究相关的文章。并对腺苷酸活化蛋白激酶的结构与作用,不同运动过程中腺苷酸活化蛋白激酶活性的变化,以及腺苷酸活化蛋白激酶升高对骨骼肌蛋白质降解的内容进行分析综述。结果与结论:共纳入相关文献35篇。本文综述了腺苷酸活化蛋白激酶的结构、作用的研究进展;在抗阻运动和中到大强度的周期运动中,腺苷酸活化蛋白激酶活性都可能升高,而在小强度周期运动过程中腺苷酸活化蛋白激酶活性可能不升高;腺苷酸活化蛋白激酶的活化可能对骨骼肌蛋白质的降解有促进作用。