蛋白激酶C激活在兔内毒素休克诱发急性肾损伤中的作用

目的:探讨蛋白激酶C(PKC)在兔内毒素休克诱发急性肾损伤中的作用及其可能作用机制。方法:健康清洁级雄性新西兰大白兔40只,采取随机数字表法分为对照组(CON组)、内毒素休克致急性肾损伤组(AKI组)、PKC-α阻断剂-白屈菜赤碱加AKI组(CHA)、PKC-α激活剂-佛波酯加AKI组(PMA)。PMA组经兔耳缘静脉注射佛波酯5μg/kg,CHA组静脉注射白屈菜赤碱5 mg/kg,CON组与AKI组注射各自溶媒1%DMSO 0.5 m L。30 min后,AKI组、PMA组及CHA组静脉注射脂多糖(LPS)5 mg/kg(溶于2 m L生理盐水),CON组给予等容量生理盐水。静脉注射LPS或生理盐水6 h时,采集动脉血,检测血清尿素氮(BUN)和肌酐(Cr)浓度,处死动物后取肾组织,进行病理学观察及肾损伤评分,测定肾组织PKC-α蛋白、HO-1蛋白、Nrf2核蛋白及总蛋白的表达水平。结果:与CON组比较,AKI组、CHA组及PMA组血清BUN、Cr浓度升高,肾组织病理学评分升高,PKC-α蛋白(1.37±0.26)、HO-1蛋白(0.89±0.11)、Nrf2核蛋白(0.97±0.26)及总蛋白的表达均上调(P0.05);与AKI组比较,PMA组血清BUN、Cr浓度降低,肾组织病理学评分降低,PKC-α蛋白、HO-1蛋白、Nrf2核蛋白及总蛋白的表达均上调(P0.05),CHA组血清BUN、Cr浓度升高,肾组织病理学评分(9.8±3.9)升高,PKC-α蛋白、HO-1蛋白、Nrf2核蛋白及总蛋白的表达均下调(P0.05)。结论:PKC-α激活是内毒素休克诱发急性肾损伤时机体的适应性调节反应机制之一,其机制可能与激活Nrf2/ARE通路、上调HO-1的表达有关。     

活化蛋白-1在大鼠内毒素性肺损伤时血红素加氧酶-1上调中的作用

目的 评价活化蛋白-1(AP-1)在大鼠内毒素性肺损伤时血红素加氧酶-1(HO-1)上调中的作用.方法 健康清洁级雄性SD大鼠48只,体重200 ～ 220 g,2.5 ～ 3.0月龄,采用随机数字表法,将其随机分为4组(n=12):正常对照组(C组)腹腔注射0.1％二甲基亚砜(姜黄素溶媒)0.5 ml,30 min时股静脉注射生理盐水(LPS溶媒)0.5 ml;内毒素性肺损伤组(ALI组)腹腔注射0.1％二甲基亚砜0.5ml,30 min时股静脉注射10 mg/kg LPS0.5 ml;姜黄素+内毒素性肺损伤组(Cur+ ALI组)腹腔注射20mg/kg姜黄素0.5 ml,30 min时股静脉注射10 mg/kg LPS 0.5 ml;姜黄素组(Cur组)腹腔注射姜黄素20mg/kg,30 min时股静脉注射生理盐水0.5 ml.静脉注射LPS 6 h时处死大鼠取肺组织,行病理学评分,测定MDA含量和SOD活性;采用Western blot法测定HO-1和AP-1表达;采用荧光定量PCR法测定HO-1 mRNA表达.结果 与C组比较,ALl组和Cur +ALI组肺组织病理学评分和MDA含量升高,SOD活性降低,HO-1 mRNA、HO-1和AP-1表达上调(P＜0.05),Cur组上述各指标差异无统计学意义(P＞0.05);与ALl组比较,Cur+ ALl组肺组织病理学评分和MDA含量升高,SOD活性降低,HO-1mRNA、HO-1和AP-1表达下调(P＜0.05).结论 内毒素性肺损伤时HO-1上调的机制与转录因子AP-1活化有一定关系.     

缺血后处理对小鼠肠缺血再灌注致肾损伤时Nrf2蛋白表达的影响

目的 评价缺血后处理对小鼠肠缺血再灌注致肾损伤时核因子E2相关因子2(Nrf2)蛋白表达的影响.方法 健康雄性C57BL/6J小鼠36只,9～12周,采用随机数字表法,将其随机分为3组(n=12):假手术组(S组)、缺血再灌注组(I/R组)、缺血后处理+缺血再灌注组(IPO组).采用夹闭肠系膜上动脉根部45 min恢复灌注的方法制备小鼠肠缺血再灌注损伤模型,IPO组于缺血45 min时再灌注30s,缺血30s,重复3次后恢复灌注.于再灌注2h时采集颈动脉血样,然后处死小鼠,取肾组织,测定血清BUN、Cr和中性粒细胞明胶酶相关脂质运载蛋白(NGAL)水平,检测肾组织Nrf2和HO-1蛋白表达、MDA含量、SOD活性、TNF-α、IL-6和IL-10的含量.显微镜下观察肾组织病理学结果,并行病理学损伤评分.结果 与S组比较,I/R组血清BUN、Cr和NAGL浓度升高,肾脏组织Nrf2及HO-1蛋白表达上调,MDA含量升高,SOD活性降低,肾脏组织病理学损伤评分升高(P＜0.05);与I/R组比较,IPO组血清BUN、Cr和NAGL浓度降低,肾脏组织Nrf2及HO-1蛋白表达上调,MDA含量降低,SOD活性升高,肾脏组织病理学损伤评分降低(P＜0.05).各组肾脏组织TNF-α、IL-6和IL-10含量比较差异无统计学意义(P＞0.05).结论 缺血后处理可减轻小鼠肠缺血再灌注致肾损伤,其机制可能与促进Nrf2蛋白表达,从而上调HO-1蛋白表达有关.     

左卡尼汀减轻大鼠肾缺血再灌注损伤及其机制的研究

目的 研究左卡尼汀对大鼠肾缺血再灌注损伤(IRI)的影响及其机制.方法 将Wistar大鼠分为3组:L组大鼠制成IRI模型,于夹闭肾动静脉前5 min及松开动脉夹后30 min,分2次经尾静脉注射左卡尼汀,各500mg/kg;I组大鼠制成IRI模型,仅注射生理盐水;C组仅分离双侧肾动、静脉,注射生理盐水.分别于再灌注后3、6和24 h处死各组大鼠.处死前经下腔静脉取血,检测血清肌酐(Cr)、尿素氮( BUN)、超氧化物歧化酶(SOD)及丙二醛(MDA)含量.获取肾组织样本,进行病理学观察;应用逆转录聚合酶链反应检测肾组织核因子E2相关因子2(Nrf2)、血红素加氧酶-1(HO-1)、γ-谷氨酰半胱氨酸合成酶(γ-GCS)的mRNA水平;蛋白质印迹法检测细胞核中Nrf2含量;免疫组织化学法检测肾组织中Nrf2的表达及定位.结果 再灌注后3h时,L组与I组血清Cr和BUN均高于C组(P＜0.01);再灌注后6h时,L组血清Cr和BUN高于C组(P＜0.01),而低于I组(P＜0.01);再灌注后24 h时,L组血清Cr和BUN仍低于I组(P＜0.05).再灌注后6和24 h时,L组与I组SOD水平低于C组(P＜0.05),MDA水平高于C组(P＜0.05).再灌注后各时间点,L组SOD水平均高于I组(P＜0.05),MDA水平均低于I组(P＜0.05).再灌注后24 h时,L组肾组织病理改变较I组轻.再灌注后6h时,I组Nrf2、HO-1、γ-GCS的mRNA相对含量均高于C组(P＜0.05),而L组各基因mRNA的相对含量高于I组(P＜0.05).L组细胞核内Nrf2的相对含量高于I组(P＜0.05).结论 左卡尼汀可减轻大鼠肾脏IRI,其机制可能与激活Nrf2-ARE通路,进而增强下游抗氧化基因的表达有关.     

血红素氧合酶-1在感染性休克大鼠肾功能损伤中的作用

目的 探讨血红素氧合酶-1(HO-1)在感染性休克大鼠肾功能损伤中的作用.方法 健康清洁级SD大鼠80只,月龄3～4月,体重260～330 g,雌雄不拘,随机分为4组(n=20):对照组(C组)、感染性休克组(S组)、感染性休克+ZnPP-Ⅸ组(SZ组)和ZnPP-Ⅸ组(Z组).C组静脉注射生理盐水0.5 ml,2 h后腹腔注射50 mmol/L碳酸氢钠溶液1 ml;S组静脉注射LPS 10 mg/kg,2 h后腹腔注射50 mmol/L碳酸氢钠溶液1 ml;SZ组静脉注射LPS 10 mg/kg,2 h后腹腔注射ZnPP-Ⅸ10 μmol/kg;Z组静脉注射生理盐水0.5 ml,2 h后腹腔注射ZnPP-Ⅸ10 μmol/kg.于腹腔注射碳酸氢钠溶液或ZnPP-Ⅸ后4 h时,检测碳氧血红蛋白浓度(COHb)、血清尿素氮(BUN)、肌酐(Cr)的浓度和尿α1-微球蛋白(α1-M)浓度、N-乙酰-a-D-氨基葡萄糖酐酶(NAG)活性、视黄醇结合蛋白(RBP)浓度、γ-谷氨酰转肽酶(γ-GT)活性、肾组织丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性、HO-1 mRNA、HO-2 mRNA、HO-1和HO-2的表达水平.结果 与C组比较,S组和SZ组血清Cr、BUN的浓度、尿α1-M、RBP、NAG、γ-GT水平、COHb、肾组织MDA、HO-1 mRNA、HO-1水平升高,肾组织SOD活性降低(P<0.05),Z组上述指标差异无统计学意义(P>0.05);与S组比较,SZ组血清Cr、BUN的浓度、尿α1-M、RBP、NAG、γ-GT水平和肾组织MDA含量升高,肾组织SOD活性、COHb和肾组织HO-1 mRNA、HO-1水平降低(P<0.05);4组大鼠肾组织HO-2 mRNA、HO-2表达差异无统计学意义(P>0.05).结论 肾组织HO-1对感染性休克大鼠的肾功能可产生一定的保护作用.     

Nrf2 / HO-1通路在电针减轻肢体缺血再灌注诱发兔肺损伤中的作用

目的:观察核因子E2相关因子2(Nrf2)/血红素氧合酶-1(HO-1)信号通路在电针减轻肢体缺血再灌注诱发兔肺损伤中的作用。方法:40只健康雄性新西兰大白兔,采用随机数字表法分为假手术组、模型组、电针组和全反式维甲酸组(n=10)。采用夹闭股动脉3 h、再灌注4 h的方法制备兔肢体缺血再灌注模型;电针组、全反式维甲酸组于模型制备前1～4 d(30 min/次,1次/d)及模型制备过程中电针足三里穴和肺俞穴,采用疏密波2/15 Hz,强度以兔出现轻微肌颤为宜;全反式维甲酸组于模型制备前30 min腹腔注射Nrf2抑制剂全反式维甲酸7 mg/kg。再灌注4 h时采集颈动脉血样,随后处死兔取肺组织,观察病理学结果并行肺损伤评分,计算肺湿/干重(W/D)比值;测定肺组织MDA含量及SOD活性,采用Western blotting法检测肺组织Nrf2总蛋白、Nrf2核蛋白及HO-l蛋白表达水平。结果:模型组、电针组和全反式维甲酸组再灌注4 h时肺损伤评分分别为5.43±0.82、4.03±0.56、6.21±0.75,均明显高于假手术组(0.92±0.21,P0.05);肺组织W/D比值分别为6.42±1.00、4.45±0.68、7.08±1.12,均明显高于假手术组(2.04±0.29,P0.05);MDA含量分别为(4.82±0.51)nmol/mg、(3.56±0.45)nmol/mg、(5.18±0.47)nmol/mg,均明显高于假手术组(2.10±0.23)nmol/mg,P0.05;SOD活性分别为(50.6±6.3)U/mg、(63.1±7.7)U/mg、(47.2±5.6)U/mg,均明显低于假手术组(72.6±6.3)U/mg,P0.05;Nrf2总蛋白、Nrf2核蛋白及HO-l蛋白表达水平均明显高于假手术组(P0.05)。与模型组比较,电针组再灌注4 h时肺损伤评分、肺组织W/D比值、MDA含量均明显降低(P0.05),SOD活性、Nrf2总蛋白、核蛋白及HO-l蛋白表达水平均明显升高(P0.05)。与电针组比较,全反式维甲酸组再灌注4 h时肺损伤评分、肺组织W/D比值、MDA含量均明显升高(P0.05),SOD活性、Nrf2总蛋白、核蛋白及HO-l蛋白表达水平均明显降低(P0.05)。结论:Nrf2/HO-1信号通路激活参与了电针减轻肢体缺血再灌注诱发兔肺损伤的过程。     

左卡尼汀通过激活Nrf2/ARE通路改善大鼠横纹肌溶解急性肾损伤

目的探讨左卡尼汀对大鼠横纹肌溶解急性肾损伤的保护作用及其作用机制。方法取36只健康雄性成年大鼠,将其随机分为空白组、模型组及左卡尼汀治疗组,每组12只。模型组和治疗组通过肌肉注射50%甘油法(10 ml/kg)制备横纹肌溶解急性肾损伤模型,此外治疗组腹腔注左卡尼汀(200 mg/kg)。处死大鼠后心内采血并留取肾脏组织。应用自动生化仪检测血清肌酸激酶(creatine kinase,CK)、尿素氮(BUN)和肌酐(SCr)水平。光镜下观察大鼠肾脏组织的病理变化;用实时定量聚合酶链反应和免疫组化法分别检测大鼠肾脏组织的核因子NF-E2相关因子2(NF-E2-related factor 2,Nrf2)及血红素加氧酶(heme oxygenase 1,HO-1);酶联免疫吸附法检测大鼠肾脏组织的超氧化物歧化酶(superoxide dismutase,SOD)及丙二醛(malonaldehyde,MDA)。结果与空白组相比,模型组及治疗组的血清CK、BUN、SCr,肾脏组织的Nrf2、HO-1、MDA水平和急性肾小管坏死(acute tubular necrosis,ATN)评分明显升高,SOD水平明显下降,差异具有统计学意义(P0.05);与模型组相比,治疗组血清CK、BUN、SCr,肾脏组织MDA水平和ATN评分明显降低,肾脏组织Nrf2、HO-1和SOD明显升高,差异具有统计学意义(P0.05)。结论左卡尼汀对横纹肌溶解急性肾损伤具有保护作用,其机制可能与激活Nrf2/ARE通路增强HO-1的表达及抑制MDA的合成有关系。     

PI3K/Akt/Nrf2信号通路在电针刺减轻兔内毒素休克诱发急性肺损伤中的作用

目的:评价磷脂酰肌醇-3激酶(PI3K)/蛋白激酶B(Akt)/核因子E2相关因子2(Nrf2)信号通路在电针刺减轻兔内毒素休克诱发急性肺损伤中的作用。方法:健康清洁级雄性新西兰大白兔80只,随机分成8组(n=10):对照组(C组)、内毒素休克诱发急性肺损伤模型组(L组)、渥曼青霉素+模型组(WL组)、渥曼青霉素组(W组)、二甲基亚砜组(D组)、模型+电针刺组(EL)、模型+电针刺激非穴位组(SEL)、模型+电针刺+渥曼青霉素组(ELW)。EL组、ELW组于模型制备前1~4 d及制备过程中电针刺激兔双侧足三里和肺俞穴,SEL组用同样方法电针刺激足三里和肺俞穴旁开0.5 cm处。L组、WL组、EL组、SEL组和ELW组经耳缘静脉注射脂多糖(LPS)5 mg/kg,C组、W组和D组给予等容量生理盐水。WL组、W组和ELW组在模型制备前30 min静注渥曼青霉素0.6 mg/kg,D组静注0.08 mL/kg DMSO,C组、L组静注生理盐水0.08 mL/kg。注射LPS或生理盐水后6 h,采集动脉血,检测TNF-α和IL-10浓度,处死动物后取肺组织,进行病理学损伤评分,测定SOD活性及MDA含量及检测p-Akt蛋白、HO-1蛋白、Nrf2核蛋白及总蛋白的表达。结果:L组、WL组、EL组、SEL组和ELW组肺损伤评分分别为(6.8±0.9)、(8.3±1.1)、(4.5±0.6)、(6.5±0.7)、(5.9±0.9),血清TNF-α浓度分别为(3.14±0.23)ng/mL、(4.62±0.32)ng/mL、(2.03±0.18)ng/mL、(3.18±0.25)ng/mL、(3.96±0.28)ng/mL,以及肺组织MDA含量分别为(4.34±0.39)nmol/mg、(6.29±0.54)nmol/mg、(3.09±0.26)nmol/mg、(4.31±0.35)nmol/mg、(5.74±0.47)nmol/mg,均较C组升高,IL-10浓度分别为(5.37±0.47)pg/mL、(3.14±0.25)pg/mL、(6.98±0.54)pg/mL、(5.34±0.43)pg/mL、(5.19±0.39)pg/mL,以及SOD含量分别为(67.6±3.8)U/mg、(42.3±2.6)U/mg、(87.5±5.6)U/mg、(62.3±4.1)U/mg、(57.4±3.5)U/mg,较C组降低,肺组织p-Akt蛋白分别为(1.58±0.29)、(1.23±0.21)、(1.93±0.32)、(1.52±0.28)、(1.46±0.24),HO-1蛋白分别为(0.67±0.09)、(0.48±0.07)、(1.05±0.12)、(0.69±0.08)、(0.74±0.09),Nrf2核蛋白分别为(0.84±0.11)、(0.61±0.09)、(1.37±0.18)、(0.89±0.12)、(0.94±0.13),以及总蛋白分别为(2.14±0.43)、(1.82±0.32)、(3.25±0.58)、(2.18±0.45)、(2.46±0.49),表达水平较C组上调(P0.05),W组、D组上述各指标差异无统计学意义(P0.05);与L组比较,EL组肺损伤评分、血清TNF-α浓度及肺组织MDA含量降低,而IL-10浓度及SOD活性升高,肺组织p-Akt蛋白、HO-1蛋白、Nrf2核蛋白及总蛋白表达水平上调,WL组肺损伤评分、血清TNF-α浓度及肺组织MDA含量升高,而IL-10浓度及SOD活性降低,肺组织p-Akt蛋白、HO-1蛋白、Nrf2核蛋白及总蛋白表达水平下调(P0.05),而SEL组上述各指标差异无统计学意(P0.05);与EL组比较,ELW组肺损伤评分、血清TNF-α浓度及肺组织MDA含量升高,而IL-10浓度及SOD活性降低,肺组织p-Akt蛋白、HO-1蛋白、Nrf2核蛋白及总蛋白表达水平下调(P0.05);与ELW组比较,WL组肺损伤评分、血清TNF-α浓度及肺组织MDA含量升高,而IL-10浓度及SOD活性降低,肺组织p-Akt蛋白、HO-1蛋白、Nrf2核蛋白及总蛋白表达水平下调(P0.05)。结论:PI3K/Akt/Nrf2信号通路介导电针刺激足三里和肺俞穴减轻兔内毒素休克诱发急性肺损伤的作用,机制可能与上调HO-1表达有关。     

左卡尼汀对肾缺血再灌注损伤的抗氧化作用及其机制

目的研究左卡尼汀对大鼠肾缺血再灌注损伤的抗氧化作用并探讨其机制。方法将大鼠随机分为3组：对照组（C组）,缺血再灌注组（IR组）,左卡尼汀组（LC组）。C组不予缺血再灌注处理,IR组及LC组建立肾脏IR模型。再灌注6h后检测各组血清肌酐（Cr）和尿素氮（BUN）水平;测定肾组织超氧化物歧化酶（SOD）活性及丙二醛（MDA）含量;RT-PCR检测肾组织核因子E2相关因子2（Nrf2）、血红素氧化酶-1（HO-1）mRNA含量;Western-blot检测各组肾组织Nrf2及HO-1蛋白表达水平。结果 LC组血清Cr、BUN水平低于IR组[（74.17±12.80）μmol/L、（24.28±2.58）mmol/L vs.（112.83±17.45）μmol/L、（35.13±6.01）mmol/L],差异具有统计学意义（P〈0.01）。LC组肾组织SOD活性高于IR组[（39.55±6.61）kU/g vs.（28.05±4.37）kU/g],差异具有统计学意义（P〈0.01）;MDA显著降低于IR组[（4.15±0.69）μmol/g vs.（6.12±1.08）μmol/g],差异具有统计学意义（P〈0.01）。IR组Nrf2、HO-1mRNA及蛋白表达水平高于C组（P〈0.01）,低于LC组（P〈0.01）。结论左卡尼汀对肾脏缺血再灌注损伤具有明显保护作用,其机制可能为激活Keapl-Nrf2-ARE通路进而诱导HO-1的表达。     

白藜芦醇对梗阻性黄疸大鼠肾损害的保护机制

目的研究白藜芦醇在大鼠梗阻性黄疸肾损害中的保护作用机制。方法雄性Wistar大鼠45只,随机分为3组:假手术组、梗阻性黄疸组及梗阻性黄疸+白藜芦醇干预组。取各组血清检测总胆红素(TBIL)、直接胆红素(DBIL)、血尿素氮(BUN)、血肌酐(Cr)水平,取肾组织匀浆检测超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量、谷胱甘肽(GSH)水平,Western blot法检测肾组织中沉默信息调控因子1(SIRT1)、核因子-κB(NF-κB)蛋白表达,RT-PCR法检测肾组织中SIRT1 m RNA表达,TUNEL法检测肾细胞凋亡。结果 1与假手术组比较,梗阻性黄疸组血清中TBIL、DBIL及BUN、Cr明显升高(P0.05),肾组织中SOD活性、GSH水平及SIRT1m RNA和蛋白明显降低(P0.05),MDA含量、NF-κB蛋白和细胞凋亡率明显升高(P0.05)。2与梗阻性黄疸组比较,梗阻性黄疸+白藜芦醇干预组血清中TBIL、DBIL及BUN、Cr水平明显降低(P0.05),肾组织中SOD活性、GSH水平及SIRT1 m RNA和蛋白表达明显升高(P0.05),NF-κB蛋白和细胞凋亡率明显降低(P0.05)。结论白藜芦醇能减轻梗阻性黄疸肾损害,可能通过激活SIRT1抑制NF-κB表达、提高SOD活性而发挥抗炎、抗氧化、抗凋亡作用。     

基于慢病毒的SHARP-2特异的RNA干扰延长肾移植受者大鼠存活时间

Objective To investigate if rat enhancer of split- and hairy-related protein-2 (SHARP-2) short hairpin RNA interference (shRNAi) prolongs the survival time of rat kidney transplant recipients. Methods Gene recombinant procedures, transfection and co-transfection were carried out to introduce short hairpin RNA interference sequences target for SHARP-2 into 3rd generation self-inactivated lentiviral-ViraPower packaging mix. Limiting dilution method was used for viral titration. Real-time PCR was employed for quantification of gene expression. Rat kidney transplantation was utilized to investigate the effect of SHARP-2 gene silence on the recipient survival. Results A lentiviral-based shRNAi construct LV-SHARP-2iC showed 84% SHARP-2 gene silence efficiency in normal rat kidney cells. At multiplicity of infection 20, 57% T cells could be transfected by lentivirus with spinoculation method. In activated T cells, SHARP-2 g ene silence resulted in 61.3% and 68.7% reduction of intedeukin 2 (IL-2) and interferon γ (IFN-γ) gene expression. When donor kidney was perfused with 5×107 TU LV-SHARP-2iC, the median survival time prolonged for 4-5 days as compared to blank and scramble control groups. Conclusions A recombinant lentivirus LV-SHARP-2iC that effectively silence SHARP-2 gene expression is constructed successfully, leading to the inhibition of IL-2 and IFN-γ. LV-SHARP-2iC treatment can prolong the survival time of rat kidney transplant recipients.     

基于慢病毒的SHARP-2特异的RNA干扰延长肾移植受者大鼠存活时间

Objective To investigate if rat enhancer of split- and hairy-related protein-2 (SHARP-2) short hairpin RNA interference (shRNAi) prolongs the survival time of rat kidney transplant recipients. Methods Gene recombinant procedures, transfection and co-transfection were carried out to introduce short hairpin RNA interference sequences target for SHARP-2 into 3rd generation self-inactivated lentiviral-ViraPower packaging mix. Limiting dilution method was used for viral titration. Real-time PCR was employed for quantification of gene expression. Rat kidney transplantation was utilized to investigate the effect of SHARP-2 gene silence on the recipient survival. Results A lentiviral-based shRNAi construct LV-SHARP-2iC showed 84% SHARP-2 gene silence efficiency in normal rat kidney cells. At multiplicity of infection 20, 57% T cells could be transfected by lentivirus with spinoculation method. In activated T cells, SHARP-2 g ene silence resulted in 61.3% and 68.7% reduction of intedeukin 2 (IL-2) and interferon γ (IFN-γ) gene expression. When donor kidney was perfused with 5×107 TU LV-SHARP-2iC, the median survival time prolonged for 4-5 days as compared to blank and scramble control groups. Conclusions A recombinant lentivirus LV-SHARP-2iC that effectively silence SHARP-2 gene expression is constructed successfully, leading to the inhibition of IL-2 and IFN-γ. LV-SHARP-2iC treatment can prolong the survival time of rat kidney transplant recipients.     

基于慢病毒的SHARP-2特异的RNA干扰延长肾移植受者大鼠存活时间

Objective To investigate if rat enhancer of split- and hairy-related protein-2 (SHARP-2) short hairpin RNA interference (shRNAi) prolongs the survival time of rat kidney transplant recipients. Methods Gene recombinant procedures, transfection and co-transfection were carried out to introduce short hairpin RNA interference sequences target for SHARP-2 into 3rd generation self-inactivated lentiviral-ViraPower packaging mix. Limiting dilution method was used for viral titration. Real-time PCR was employed for quantification of gene expression. Rat kidney transplantation was utilized to investigate the effect of SHARP-2 gene silence on the recipient survival. Results A lentiviral-based shRNAi construct LV-SHARP-2iC showed 84% SHARP-2 gene silence efficiency in normal rat kidney cells. At multiplicity of infection 20, 57% T cells could be transfected by lentivirus with spinoculation method. In activated T cells, SHARP-2 g ene silence resulted in 61.3% and 68.7% reduction of intedeukin 2 (IL-2) and interferon γ (IFN-γ) gene expression. When donor kidney was perfused with 5×107 TU LV-SHARP-2iC, the median survival time prolonged for 4-5 days as compared to blank and scramble control groups. Conclusions A recombinant lentivirus LV-SHARP-2iC that effectively silence SHARP-2 gene expression is constructed successfully, leading to the inhibition of IL-2 and IFN-γ. LV-SHARP-2iC treatment can prolong the survival time of rat kidney transplant recipients.     

基于慢病毒的SHARP-2特异的RNA干扰延长肾移植受者大鼠存活时间

Objective To investigate if rat enhancer of split- and hairy-related protein-2 (SHARP-2) short hairpin RNA interference (shRNAi) prolongs the survival time of rat kidney transplant recipients. Methods Gene recombinant procedures, transfection and co-transfection were carried out to introduce short hairpin RNA interference sequences target for SHARP-2 into 3rd generation self-inactivated lentiviral-ViraPower packaging mix. Limiting dilution method was used for viral titration. Real-time PCR was employed for quantification of gene expression. Rat kidney transplantation was utilized to investigate the effect of SHARP-2 gene silence on the recipient survival. Results A lentiviral-based shRNAi construct LV-SHARP-2iC showed 84% SHARP-2 gene silence efficiency in normal rat kidney cells. At multiplicity of infection 20, 57% T cells could be transfected by lentivirus with spinoculation method. In activated T cells, SHARP-2 g ene silence resulted in 61.3% and 68.7% reduction of intedeukin 2 (IL-2) and interferon γ (IFN-γ) gene expression. When donor kidney was perfused with 5×107 TU LV-SHARP-2iC, the median survival time prolonged for 4-5 days as compared to blank and scramble control groups. Conclusions A recombinant lentivirus LV-SHARP-2iC that effectively silence SHARP-2 gene expression is constructed successfully, leading to the inhibition of IL-2 and IFN-γ. LV-SHARP-2iC treatment can prolong the survival time of rat kidney transplant recipients.     

基于慢病毒的SHARP-2特异的RNA干扰延长肾移植受者大鼠存活时间

Objective To investigate if rat enhancer of split- and hairy-related protein-2 (SHARP-2) short hairpin RNA interference (shRNAi) prolongs the survival time of rat kidney transplant recipients. Methods Gene recombinant procedures, transfection and co-transfection were carried out to introduce short hairpin RNA interference sequences target for SHARP-2 into 3rd generation self-inactivated lentiviral-ViraPower packaging mix. Limiting dilution method was used for viral titration. Real-time PCR was employed for quantification of gene expression. Rat kidney transplantation was utilized to investigate the effect of SHARP-2 gene silence on the recipient survival. Results A lentiviral-based shRNAi construct LV-SHARP-2iC showed 84% SHARP-2 gene silence efficiency in normal rat kidney cells. At multiplicity of infection 20, 57% T cells could be transfected by lentivirus with spinoculation method. In activated T cells, SHARP-2 g ene silence resulted in 61.3% and 68.7% reduction of intedeukin 2 (IL-2) and interferon γ (IFN-γ) gene expression. When donor kidney was perfused with 5×107 TU LV-SHARP-2iC, the median survival time prolonged for 4-5 days as compared to blank and scramble control groups. Conclusions A recombinant lentivirus LV-SHARP-2iC that effectively silence SHARP-2 gene expression is constructed successfully, leading to the inhibition of IL-2 and IFN-γ. LV-SHARP-2iC treatment can prolong the survival time of rat kidney transplant recipients.     

直肠癌远端移形黏膜COX-2及BCL-2蛋白的表达与临床意义

目的探讨直肠癌远端移形黏膜COX-2及BCL-2蛋白的表达情况,判断直肠癌远端移形黏膜是否为癌前病变。方法应用高铁二胺-阿辛蓝染色检测54例直肠癌远端2cm处黏膜．将远端黏膜分为移形黏膜（TM）组及非移形黏膜（NTM）组,通过免疫组织化学染色检测TM中COX-2和BCL-2蛋白的表达．比较TM与NTM、肿瘤组织以及正常黏膜组织（20例直肠良性息肉旁肠黏膜组织）内BCL-2以及COX-2的表达的差异。结果54例直肠癌远端2cm黏膜处组织中有19例存在TM．35例为NTM。COX-2蛋白在肿瘤组织、TM、NTM、正常组织中的阳性率分别为81．5％（44／54）、21．1％（4／19）、17．1％（6／35）、10．0％（2／20）;BCL-2蛋白在上述4种组织中的阳性率分别为77．8％（42／54）、21．1％（4／19）、22．9％（8／35）、5．0％（1／20）。TM内的COX-2及BCL．2蛋白的表达与肿瘤组织相比,差异有统计学意义[（0．737±0．895）比（3．519±1．998）;（0．632±0．955）比（2．833±1．756）,均P〈0．01];与NTM、正常肠黏膜组织相比,差异无统计学意义（均P〉0．05）。结论直肠癌远端TM内的COX-2和BCL-2蛋白的表达无特异性．TM是癌前病变的证据不足。     

基于慢病毒的SHARP-2特异的RNA干扰延长肾移植受者大鼠存活时间

Objective To investigate if rat enhancer of split- and hairy-related protein-2 (SHARP-2) short hairpin RNA interference (shRNAi) prolongs the survival time of rat kidney transplant recipients. Methods Gene recombinant procedures, transfection and co-transfection were carried out to introduce short hairpin RNA interference sequences target for SHARP-2 into 3rd generation self-inactivated lentiviral-ViraPower packaging mix. Limiting dilution method was used for viral titration. Real-time PCR was employed for quantification of gene expression. Rat kidney transplantation was utilized to investigate the effect of SHARP-2 gene silence on the recipient survival. Results A lentiviral-based shRNAi construct LV-SHARP-2iC showed 84% SHARP-2 gene silence efficiency in normal rat kidney cells. At multiplicity of infection 20, 57% T cells could be transfected by lentivirus with spinoculation method. In activated T cells, SHARP-2 g ene silence resulted in 61.3% and 68.7% reduction of intedeukin 2 (IL-2) and interferon γ (IFN-γ) gene expression. When donor kidney was perfused with 5×107 TU LV-SHARP-2iC, the median survival time prolonged for 4-5 days as compared to blank and scramble control groups. Conclusions A recombinant lentivirus LV-SHARP-2iC that effectively silence SHARP-2 gene expression is constructed successfully, leading to the inhibition of IL-2 and IFN-γ. LV-SHARP-2iC treatment can prolong the survival time of rat kidney transplant recipients.     

自由基清除剂对创伤后异位骨化形成中BMP-2及COX-2表达的影响

目的 研究自由基清除剂依达拉奉对创伤后异位骨化(HO)形成中骨形态发生蛋白(BMP)-2及环氧化酶(COX)-2表达的调节作用.方法 54只新西兰白兔随机分成实验组、模型组、对照组,每组18只.实验组和模型组兔适应性喂养1周后建立HO模型,对照组兔不建立HO模型,操作后第1天即开始给药.实验组给予依达拉奉30mg/次,...     

熊果酸对肝癌细胞株Bel-7404转移及侵袭能力的影响

目的 观察熊果酸(UA)对肝癌细胞株Bel-7404细胞转移侵袭能力及相关基因基质金属蛋白酶(MMP)-2、金属蛋白酶组织抑制因子(TIMP)-2 mRNA和蛋白表达水平的影响.方法 选用3个组:A组(UA 50 μmol/L)、B组(顺铂10mg/L)和C组(DMEM空白对照)对Bel-7404细胞进行处理,用细胞迁移实验及Transwell小室法检测细胞的迁移及侵袭能力,逆转录-聚合酶链反应(RT-PCR)及Western blot法检测细胞中MMP-2、TIMP-2 mRNA及蛋白的表达水平.结果 对Bel-7404细胞作用48 h后,A、B两组细胞的迁移速率(35.2±8.7)、(30.5±8.6)μm/h及侵袭穿膜细胞数(21.2±5.3)、(20.2±5.7)个、MMP-2 mRNA 0.24±0.06、0.23±0.05及蛋白0.21±0.01、0.24±0.04表达水平均较C组(75.6±8.7)μm/h、(54.8±7.8)个、0.46±0.11、0.42±0.06明显降低,差异均有统计学意义(P<0.01),而TIMP-2 mRNA0.87±0.05、0.83±0.06及蛋白0.98±0.06、0.95±0.09表达水平均比C组0.31±0.02、0.59±0.02高,差异均有统计学意义(P<0.01);A、B两组间细胞的迁移速率、侵袭穿膜细胞数、MMP-2和TIMP-2 mRNA及蛋白表达水平的比较差异均无统计学意义(P>0.05).结论 UA可抑制肝癌Bel-7404细胞株的迁移侵袭,其机制可能与MMP-2表达下调而TIMP-2表达上调有关.UA 50 μmol/L对Bel-7404细胞迁移侵袭能力的抑制作用与顺铂10 mg/L具有相同的效果及机制.     

地氟醚维持麻醉对SjvO2和CSFP的影响

研究地氟醚维持麻醉时ＰａＣＯ２对ＳｊｖＯ２和ＣＳＦＰ的影响。方法：４３例脑肿瘤病人用地氟醚维持麻醉,术中持续监测颅内压和动脉压。当人为改变ＰＥＴＣＯ２时,取颈内静脉血和同步采集动脉血作血气分析和测定两者乳酸含量。