RNA干扰Caspase-3基因抑制大鼠肝脏缺血再灌注损伤

目的 观察RNA干扰(RNAi)Caspase-3基因对大鼠肝脏缺血再灌注损伤(IRI)的保护作用.方法 构建针对大鼠Caspase-3基因的短发夹状RNA(shRNA)真核表达载体.肝脏IRI前48 h经门静脉注射磷酸盐缓冲液(PBS)或Caspase-3 shRNA,实验随机分为3组,假手术组、PBS组和shRNA组.阻断大鼠70%入肝血流40 min,再灌注6,12,24 h,3,5,7 d检测血清谷丙转氨酶(ALT)和谷草转氨酶(AST)水平,肝组织Caspase-3 mRNA的表达,细胞凋亡情况,丙二醛(MDA)以及超氧化物歧化酶(SOD)的含量.结果 与PBS组比较,shRNA组再灌注6,12,24 h血清ALT和AST水平显著降低(P<0.05),shRNA组大鼠肝组织的Caspase-3 mRNA水平、肝细胞凋亡指数(25.21%±3.18%vs 35.24%±2.33%,P<0.05)和肝组织中MDA含量[(96.3±12.8)nmol/mg vs(133.5±12.4)nmol/mg,(P<0.05)]显著降低,SOD活性显著升高[(22.5±3.4)U/mg vs(12.2±3.1)U/mg,(P<0.05)].结论 RNA干扰Caspase-3基因可以抑制细胞凋亡的发生,保护肝脏缺血再灌注损伤.     

抑制Caspase-8基因表达对减轻大鼠肝脏缺血再灌注损伤的作用

目的 观察抑制半胱氨酸天冬氨酸特异性蛋白酶8(Caspase-8)基因的表达对减轻大鼠肝脏缺血再灌注损伤的作用.方法 构建针对大鼠Caspase-8基因的短发夹状RNA(shRNA)真核表达载体.将Lewis大鼠随机分为3组,每组8只.(1)假手术组:麻醉后,取腹部正中切口,缝合关腹;(2)磷酸盐缓冲液(PBS液)组:阻断肝门血流前48 h经门静脉注射PBS液1 ml,然后行肝脏缺血再灌注;(3)shRNA组:阻断肝门血流前48 h经门静脉注射Caspase-8 shRNA 50 μg(总体积为1 ml),然后行肝脏缺血再灌注.肝脏缺血再灌注的方法为阻断大鼠70%入肝血流40min.于再灌注6 h、12 h、24 h、3 d、5 d、7 d时检测血清丙氨酸转氨酶(ALT)和天冬氨酸转氨酶(AST)水平;检测肝组织中Caspase-8 mRNA的表达、细胞凋亡情况、丙二醛(MDA)以及超氧化物岐化酶(SOD)的含量.结果 与PBS液组比较,shRNA组再灌注6、12、24 h,血清中ALT和AST水平显著降低(P<0.05);肝组织中Caspase-8 mRNA水平、肝细胞凋亡指数(shRNA组和PBS液组分别为22.33%±4.28%和35.24%±2.33%)以及肝组织中MDA含量[shRNA组和PBS液组分别为(94.5±11.2)nmol/mg和(133.5±12.4)nmol/mg]均显著降低(P<0.05),而肝组织中SOD活性显著升高[shRNA组和PBS液组分别为(21.6±3.7)U/mg和(12.2±3.1)U/mg,P<0.05].结论 通过RNA干扰Caspase-8基因的表达可以抑制肝细胞凋亡的发生,减轻肝脏缺血再灌注损伤.     

RNA干扰Kupffer细胞肿瘤坏死因子-α减轻大鼠肝脏缺血再灌注损伤

目的 观察RNA干扰肝脏Kupffer细胞肿瘤坏死因子-α(TNF-α)对大鼠肝脏缺血再灌注损伤的保护作用.方法 构建针对大鼠TNF-α基因的短发夹状RNA(shRNA)真核表达载体.肝脏缺血再灌注损伤前48 h经门静脉注射磷酸盐缓冲液(PBS)、空载体或TNF-α shRNA.实验随机分为4组,假手术组、PBS组、空载体组和shRNA组.阻断大鼠70%入肝血流40 min,再灌注6 h检测血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、肝脏Kupffer细胞TNF-α mRNA、血清TNF-α、肝组织中丙二醛(MDA)以及超氧化物岐化酶(SOD)含量.结果 与PBS组和空载体组比较,shRNA组再灌注6 h后血清ALT和AST水平显著降低(P＜0.05),Kupffer细胞TNF-α mRNA水平、血清TNF-α水平(56.6±6.7 pg/ml比87.8±8.7 pg/ml和96.5±7.3 pg/ml,P＜0.05)、肝组织中MDA含量(93.4±13.3 nmol/mg比133.5±12.4 nmo1/mg和136.7±13.6 nmol/mg,P＜0.05)显著降低,SOD活性显著升高(22.4±4.6 U/mg比12.2±3.1 U/mg和11.4±2.9 U/mg,P＜0.05).结论 RNA干扰Kupffer细胞TNF-α基因的表达可以减轻大鼠肝脏缺血再灌注损伤.     

高压氧对大鼠缺血再灌注损伤肾FasL和半胱氨酸蛋白酶3表达的影响

目的 研究高压氧(HBO)对大鼠缺血再灌注损伤(IRI)肾细胞凋亡相关基因(FasL)和细胞凋亡执行蛋白半胱氨酸蛋白酶3(caspase-3)表达的影响,并探讨其作用机制.方法 健康SD雄性大鼠随机分为假手术组(n=8)、IRI组(n=8)和IRI+HBO组(n=8).采用夹闭双侧肾动脉方法建立IRI模型.IRI+HBO组分别在再灌注后lh、24 h、48 h给予HBO处理,末次HBO后取双肾组织测定各组大鼠肾组织匀浆超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量 ;采用实时荧光定量PCR和免疫组织化学染色方法分别测定肾组织FasL mRNA、caspase-3蛋白表达.结果 与假手术组比较,IRI组SOD活性下降(P＜0.05),MDA含量升高(P＜0.05),经HBO治疗后SOD活性升高(P＜0.05),MDA含量降低(P＜0.05).FasL mRNA、caspase-3蛋白在假手术组呈低水平表达,而在IRI组表达显著上调(P＜0.01),IRI+HBO组表达较IRI组显著下调(P＜0.01).结论 大鼠肾缺血损伤后随着再灌注时间延长FasL mRNA 、caspase-3蛋白表达显著上调.早期HBO治疗后可以使FasL mRNA、caspase-3蛋白表达明显下调,抑制细胞凋亡,保护肾脏.     

肾上腺髓质素对大鼠肾缺血再灌注损伤中肾小管上皮细胞凋亡的影响及机制研究

目的 观察肾上腺髓质素(adrenomedullin,AM)对大鼠肾缺血再灌注损伤(ischemia reperfusion injury,IRI)中肾小管上皮细胞凋亡的影响,并探讨其机制.方法 Wistar大鼠32只,随机分为4组:假手术组和IRI组各6只,转染空质粒组和转染AM质粒组各10只.大鼠右肾切除后1周,用超声微泡造影剂介导的基因转染方法将大鼠AM真核表达质粒转染大鼠肾脏,1周后采用免疫组织化学方法检测转染效率.转染成功后夹闭左肾动脉45 min制作肾IRI模型,于再灌注24 h后留取肾组织标本.TUNEL染色检测肾组织细胞凋亡,RT-PCR检测肾组织Bcl-2、Bax和Fas的mRNA表达,蛋白质印迹法检测caspase-3、caspase-8和caspase-9蛋白质表达.结果 转染AM质粒组的AM表达显著高于转染空质粒组(0.51±0.09和0.23±0.05,P＜0.05).与假手术组相比,IRI组肾组织细胞凋亡率明显增加[(38.79±7.52)%和(2.89±0.52)%,P＜0.05];肾组织Bax、Bcl-2、Fas、caspase-3、caspase-8和caspase-9表达上调,分别为0.72±0.18和0.23±0.04、0.80±0.12和0.38±0.06、1.24±0.25和0.39±0.09、0.76±0.13和0.38±0.08、0.92±0.14和0.32±0.06、0.89±0.12和0.42±0.09(P＜0.05),Bax/Bcl-2升高(0.91±0.18和0.61±0.08,P＜0.05).转染AM质粒组肾组织凋亡细胞数、Bax、Fas、caspase-3、caspase-8和caspase-9表达下调,分别为(19.36±6.78)%、0.48±0.11、0.62±0.07、0.53±0.08、0.46±0.08、0.51±0.12,与IRI组比较差异均有统计学意义(P＜0.05);Bcl-2表达进一步上调为1.23±0.25,Bax/Bcl-2降低为0.44±0.12,与IRI组比较差异均有统计学意义(P＜0.05).转染空质粒组和IRI组比较,上述各指标差异均无统计学意义(P＞0.05).结论 AM能减轻肾IRI引起的肾小管上皮细胞凋亡,其部分机制可能是通过抑制caspase依赖的内、外源性凋亡途径实现的.

Abstract：

Objective To investigate the effect and mechanism of adrenomedullin (AM) on apoptosis of renal tubular epithelial cell in rats induced by renal ischemia reperfusion injury. Methods Thirty-two Wistar rats were randomly divided into 4 groups: control group, IRI group, empty plasmid group and AM group. One week after removing the right kidney, eukaryotic expression vector encoding rat AM gene was transfected into the left kidney using an ultrasound-microbubble mediated system. After 1 week the transfer efficiency was detected by immunohistochemical method . Renal IRI model induced by clamping left renal arteries for 45 min followed by reperfusion for 24 h. Tubular cell apoptosis was detected by TUNEL assay. Bcl-2, Bax and Fas expressions were examined by RT-PCR. The expressions of caspase-3, caspase-8 and caspase-9 were determined by Western bolt analysis. Results The expression of AM in the AM group was significantly higher than the empty plasmid group (0.51±0.09 vs 0.23±0.05; P<0.05). Compared with the control group, the apoptosis rate of renal tubular cell in the IRI group was significantly higher [(38.79±7.52)% vs (2.89±0.52)%; P<0.05]. The expressions of Bax, Bcl-2, Fas, caspase-3, caspase-8 and caspase-9 were also significantly increased (0.72±0.18 vs 0.23±0.04, 0.80±0.12 vs 0.38±0.06, 1.24±0.25 vs 0.39±0.09, 0.76±0.13 vs 0.38±0.08, 0.92±0.14 vs 0.32±0.06, 0.89±0.12 vs 0.42±0.09; P<0.05). Bax/Bcl-2 was also significantly increased (0.91±0.18 vs 0.61±0.08; P<0.05). Compared with the IRI group, AM pretreatment significantly decreased the apoptosis rate of renal tubular cells [(19.36±6.78)% vs (38.79±7.52)%; P<0.05]. AM inhibited the up-regulation of Bax, Fas, caspase-3, caspase-8 and caspase-9, while promoting the up-regulation of Bcl-2 (0.48±0.11 vs 0.72±0.18, 0.62±0.07 vs 1.24±0.25, 0.53±0.08 vs 0.76±0.13, 0.46±0.08 vs 0.92±0.14, 0.51±0.12 vs 0.89±0.12, 1.23±0.25 vs 0.80±0.12; P<0.05). Bax/Bcl-2 significantly decreased (0.44±0.12 vs 0.91±0.18; P<0.05). The above parameters had no significant diffe-rence between the empty plasmid group and the IRI group (P>0.05). Conclusion AM can reduce apoptosis of renal tubular epithelial cell induced by renal IRI, the mechanism of which might be achieved by inhibiting caspase-dependent intrinsic and extrinsic pathways.     

Fas及Caspase家族在泡球蚴感染宿主肝脏中的表达及其意义

目的 探讨细胞凋亡信号分子Fas及Caspase家族在肝泡球蚴病宿主中的表达及意义.方法 16例配对泡型包虫患者(AE)肝脏标本,16只雌性BALB/C小鼠.分析肝脏标本中肝细胞病理变化、凋亡及Fas、Caspase-3、Caspase-8和Caspase-9的表达.结果 AE患者病灶周围肝细胞水肿,汇管区周围纤维化,肝细胞造型坏死;病灶周围肝细胞发生显著凋亡[(74.0±15.0)％比(4.2±3.3)％],Fas[ (71.3±19.0)％比(5.2±1.6)％]、Caspase-3[ (39.4±18.3)％比(3.3±2.9)％]、Caspase-8[ (37.6±3.5)％比(4.0±1.8)％]和Caspase-9[ (24.7±13.8)％比(2.0±1.4)％]高表达(P＜0.05).泡球蚴感染小鼠病灶处可见囊泡,肝组织纤维化严重;肝细胞发生显著凋亡[(37.5±5.2)％比(13.9±2.7)％],Fas[ (48.3±6.2)％比(4.6±1.2)％]、Caspase-3[(29.7±6.3)％比(5.2±1.9)％]、Caspase-8[ (23.1±2.9)％比(3.5±1.4)％]和Caspase-9[( 16.7±3.2)％比(2.3±1.1)％]高表达(P＜0.05).结论 泡球蚴感染引起宿主肝细胞凋亡,提示Fas及Caspase家族介导的细胞凋亡信号通路可能参与了泡球蚴所致肝损伤进程.     

线粒体ATP敏感性钾通道开放预防脑缺血-再灌注引起的神经细胞凋亡

目的通过观察凋亡蛋白酶(caspase-3)活性的变化,探讨线粒体ATP敏感性钾通道开放预防脑缺血-再灌注引起的神经细胞凋亡的机制.方法选择雄性SD大鼠18只,随机分为三组:C组(n=6),单纯行大脑中动脉栓死(MCAO);D组(n=6),MCAO前30 min给予二氮嗪5 mg/kg腹腔注射;H组(n=6),先给予二氮嗪阻断剂(5-HD)10 mg/kg静脉注射,15 min后再给予二氮嗪5mg/kg腹腔注射,30min后行MCAO.所有大鼠缺血2 h再灌注22 h后行神经功能学评估,取大脑梗死灶半影区组织,提取胞浆液,酶标法测Caspase-3活性.结果D组与C组、H组相比神经功能学评分显著提高[(11.88±2.70)、(8.00±1.79)、(8.38士1.06)](P＜0.01),Caspase-3的活性明显降低[(9.57±3.80)、(29.80±17.69)、(16.25±6.76)FU·μg-1·min-1](P＜0.05).结论线粒体ATP敏感性钾通道开放通过抑制Caspase-3活性预防神经细胞凋亡的发生,对脑缺血-再灌注产生保护作用.     

PTEN-PI3K/AKT信号传导途径对胃癌细胞株MKN28凋亡的影响

目的 探讨PTEN-PI3 K/AKT信号传导途径对胃癌细胞株MKN28凋亡的调控作用及其机制.方法 构建PTEN真核表达载体,转染至胃癌细胞株MKN28中(转染组);同样方法转染空载体和等量的PBS分别作为阴性对照组和空白对照组,检测对胃癌细胞株MKN28凋亡的影响及对PI3K、AKT、Caspase-3、Caspase-9的影响.MTT检测细胞生长曲线,TUNEL染色法检测细胞凋亡,Western blot检测蛋白的表达.PI3K抑制剂LY294002处理未转染PTEN的胃癌细胞株MKN28(处理组);对照组加入等量的PBS,抑制PI3K活性后检测细胞凋亡蛋白及凋亡相关蛋白的表达.组间比较采用t检验,时间依赖性检验采用相关分析方法.结果 成功构建PTEN真核表达质粒并转染至胃癌细胞株MKN28中,获得稳定过表达PTEN的细胞模型.MTT检测发现转染组胃癌细胞株MKN28生长明显受到抑制,且呈时间依赖性(r =0.938,P＜0.05).转染组平均凋亡率达到27.86％ ±4.78％,明显高于阴性对照组的0.01％±0.01％(t=9.527,P＜0.05).转染PTEN后,转染组PI3K蛋白表达量为0.25±0.03,明显低于空白对照组的0.93 ±0.16及阴性对照组的0.96±0.15(t=7.235,8.883,P＜0.05).转染组活化的AKT (P-AKT)蛋白表达量为0.21±0.03,明显低于空白对照组的0.93 ±0.13及阴性对照组的0.91±0.12(t =9.347,9.802,P＜0.05).而转染组凋亡相关蛋白Caspase-3及Caspase-9表达量分别为0.86±0.11和0.87±0.12,明显高于阴性对照组的0.16±0.03和0.18 ±0.04及空白对照组的0.15±0.02和0.16 ±0.03(t=10.634,10.999,9.448,9.942,P＜0.05).抑制PI3K活性后,处理组细胞凋亡率为28.60％ ±4.50％,明显高于对照组的0.12％±0.06％(t=10.961,P＜0.05).Western blot检测发现处理组PI3K和P-AKT的蛋白表达量分别为0.18 ±0.02和0.11 ±0.01,明显低于对照组的0.93 ±0.14和0.90 ±0.12(t =9.186,11.363,P＜0.05);同时处理组PTEN的蛋白相对表达量为1.15 ±0.15,明显高于对照组的0.21±0.08(t =9.577,P＜0.05).处理组的凋亡相关蛋白Caspase-3及Caspase-9相对表达量分别为0.86 ±0.12和0.88 ±0.11,明显高于对照组的0.25±0.02和0.21±0.03(t =8.685,10.178,P＜0.05).结论 高表达PTEN可以抑制PI3K/AKT途径,促进胃癌细胞凋亡;抑制PI3K途径可以促进PTEN的表达,两者之间相互作用,共同调控着胃癌细胞的凋亡.     

前列地尔对兔肾缺血再灌注时细胞凋亡的保护作用

目的 观察前列地尔对兔肾缺血再灌注损伤时肾小管上皮细胞凋亡的保护作用.方法 建立兔肾缺血再灌注损伤动物模型,将实验兔随机分为3组:即对照组、缺血再灌注组和前列地尔组,每组10只.检测兔血清肌苷(Cr)、尿素氮(BUN)浓度及肾组织中丙二醛(MDA)、超氧化物歧化酶(SOD)和髓过氧化物酶(MPO)含量及肾组织中凋亡细胞.结果 与对照组比较,缺血再灌注组和前列地尔组在再灌注后Cr、BUN水平均大幅度上升(P＜0.05);但前列地尔组动物在再灌注60min后Cr水平(231.32±17.57)μmol/L明显低于缺血再灌注组(390.61±20.42)μmol/L(P＜0.05);肾小管上皮细胞bcl-2、bax、Caspase-3表达与对照组比较,缺血再灌注组明显增强(P＜0.05);前列地尔组与缺血再灌注组比较表达减弱,但仍强于对照组(P＜0.05).前列地尔组、缺血再灌注组与对照组比较凋亡细胞数增多,前列地尔组与缺血再灌注组比较凋亡细胞数减少.MDA、SOD与MPO的活性与对照组比较,缺血再灌注组与前列地尔组明显增强(P＜0.05);前列地尔组与缺血再灌注组比较,该两者活性明显减弱(P＜0.05).结论 前列地尔在肾脏缺血再灌注损伤时能有效的保护肾功能其作用机制可能是通过减少细胞脂质过氧化,从而降低bcl-2、bax、Caspase-3等凋亡基因的表达.

Abstract：

Objective To study the alprostadil effects of alprostadil on apoptosis by renal ischemia-reperfusion injury (IR[) in rabbits. Methods The rabbit IRI models were made, and randourly divided into three groups: control group, IR[group and prostavasin intervention group. The creatinine (Ct) and blood urea nitrogen (BUN) were determined. Malondialdehyde ( MDA), superoxide dismutase (SOD),myeloperoxidase ( MPO), bcl-2, bax, Caspase-3 and apoptosis were assayed at 60 min after reperfusion.Results The Cr and BUN levels in plasma in IRI group and Prostavasin intervention group were increased obviously after reperfusion. The Cr levels at 60 min after repeffusion in alprostadil intervention group (231.32 + 17. 57 ) μmol/L were significantly lower than in IRI group ( 390. 61 ± 20. 42 ) μ mol/L, ( P ＜0. 05 ). The levels of bcl-2, bax, Caspase-3 in the renal tissue in IRI group were significantly higher than in control group ( P ＜ 0. 05 ), and those in alprostadil intervention group were lower than in IRI group, but markedly higher than in control group (P ＜ 0. 05 ). The number of apoptotic cells in alprostadil intervention group and IRI group was increased as compared with control group, and that in alprostadil intervention group was reduced as compared with IRI group. The contents of MDA, SOD and MPO in renal tissue of IRI group and Prostavasin intervention group were significantly higher than in control group ( P ＜ 0. 05 ), and those in IRI group were significantly lower than in alprostadil intervention group (P ＜0. 05 ). Conclusion Alprostadil could be used to protect renal ischemia-reperfusion injury probably by decreasing oxygen free radicals generation, inhibiting neutrophils aggregating and activating in the renal tissues, thereby inhibiting the expression of bcl-2, bax, Caspase-3.     

中性粒细胞明胶酶相关脂质运载蛋白对大鼠肾脏缺血再灌注损伤肾小管上皮细胞的保护作用

目的 探讨中性粒细胞明胶酶相关脂质运载蛋白(NGAL)对大鼠缺血再灌注损伤肾脏肾小管上皮细胞凋亡的保护作用及机制.方法 建立大鼠肾脏缺血再灌注模型,雄性SD大鼠随机分为对照组、缺血再灌注模型组、NGAL组 ;HE染色观察3组大鼠肾组织病理变化 ;TUNEL法检测肾小管上皮细胞凋亡 ;实时定量PCR、Western印迹法检测凋亡蛋白fas、bcl-2的表达变化.结果 与缺血再灌注模型组比较,NGAL组肾小管上皮细胞凋亡数量显著减少[(8.6±3.4)/HP比(20.8±3.7)/HP,P＜0.05] ;NGAL组肾组织fas mRNA(2.34±0.51比6.84±2.34,P＜0.05)、fas蛋白(0.65±0.05比0.95±0.08,P＜0.05)表达显著下调,bcl-2蛋白(0.33±0.05比0.24±0.03,P＜0.05)表达显著上调,但bcl-2 mRNA表达无明显改变.结论 NGAL对大鼠缺血再灌注损伤肾小管上皮细胞有保护作用,其作用可能与减少细胞凋亡、改变凋亡蛋白的表达有关.     

氧自由基在异氟醚诱导小鼠前脑细胞凋亡中的作用

目的 观察小鼠暴露于异氟醚后前脑Caspase-3蛋白和氧自由基的变化,以了解氧自由基(reactive oxygen spceies,ROS)在异氟醚神经损伤中的作用.方法 56只雄性C57BL/6J小鼠按随机数字表法分为异氟醚组(Iso组,n=20)、二甲基硫脲±异氟醚组(DMTU±Iso组,n=8)、二甲基硫脲组...     

大鼠肝脏缺血再灌注过程中信号转导与转录激活子1的表达变化及其意义

目的 观察JAK-STAT信号通路蛋白STAT1和半胱氨酸天冬氨酸蛋白酶(Caspase)-3在大鼠肝脏缺血再灌注不同时限的表达及丹参对两者表达的影响.方法 将80只SD大鼠随机分为正常组、假手术组、缺血再灌注组和丹参组,建立肝缺血再灌注模型,采用免疫组织化学方法 检测STAT1及Caspase-3在肝缺血45 min再灌注0、3、12、24、72 h时的表达.结果 STAT1、Caspase-3在正常及假手术组中未见明显表达.缺血再灌注组STATI在再灌注0 h即有表达(PU=10.29±0.92),再灌注12～24 h表达最明显(PU=38.73±1.59～38.90±2.29),Caspase-3在再灌注0 h亦有表达(PU=8.18±0.95),峰值出现在再灌注24 h(PU=38.06±2.85).丹参组STAT1在各时限点均较同期缺血再灌注组低(P＜0.05),除再灌注72h外丹参组Caspase-3在各时限点均较同期缺血再灌注组低(P＜0.05).结论 STAT1在大鼠肝缺血再灌注损伤中被激活,丹参可能通过抑制STAT1的表达减轻缺血再灌注对肝的损伤.     

线粒体ATP敏感性钾通道阻断剂对在体大鼠肺缺血后处理的作用及其机制

目的 探讨缺血后处理(IPO)对大鼠在体肺缺血-再灌注损伤(I/R)的保护作用及线粒体ATP敏感性钾通道(mitoKATP)在缺血后处理效应中的作用.方法 将Wistar大鼠35只随机分为5组:假手术组(Sham组)、缺血再灌注损伤组(I/R组)、缺血后处理组(IPO组)、缺血再灌注损伤+5-羟基葵酸盐组(I/R+5-HD组)、缺血后处理+5-羟基葵酸盐组(IPO+5-HD组).观察各组肺组织中丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性、湿/干比值(W/D)以及病理形态学改变.结果 I/R组与Sham组比较MDA含量增加[(5.07±1.60)nmol/mg prot比(1.43±0.41)nmol/mgprot,P＜0.01],SOD活性减低[(12.38±2.24)U/mg prot比(45.51±5.42)U/mg prot,P＜0.01],W/D比值增高(5.45±0.82比3.05±0.47,P＜0.01),肺组织形态及超微结构明显受损;IPO+5-HD组与IPO组比较MDA含量增加[(3.74±0.71)nmol/mg prot比(2.60±0.43)nmol/mg prot,P＜0.01],SOD活性减低[(22.91±2.71)U/mg prot比(28.74±2.03)U/mg prot,P＜0.01],W/D比值增高(4.64±0.79比3.89±0.60,P＜0.01),肺组织形态及超微结构明显受损;IPO组与I/R组比较,肺组织MDA含量减少[(2.60±0.43)nmol/mg prot比(5.07±1.60)nmol/mg prot,P＜0.01],SOD活性增高[(28.74±2.03)U/mg prot比(12.38±2.24)U/mg prot,P＜0.01],W/D比值减低(3.89±0.60比5.45±0.82,P＜0.01),肺组织病理形态学改变轻于I/R组;I/R+5-HD组与I/R组比较,肺组织MDA含量[(5.14±1.30)mol/mg prot比(5.07±1.60)mol/mg prot,P＞0.05)、SOD活性[(11.65±1.82)U/mg prot比(12.38±2.24)U/mg prot,P＞0.05]、W/D比变化(5.54±0.61比5.45±0.82),差异无统计学意义(P＞0.05),肺组织病理形态学改变无明显差异.IPO+5-HD组的各项指标介于IPO组和I/R组之间.结论 缺血后处理能减轻大鼠在体肺缺血再灌注损伤,mitoKATP参与了肺缺血后处理效应.

Abstract：

Objective To investigate the protective effect of ischemic postconditioning (IPO) on lung ischemic reperfusion (L/R) in rats in vivo and the mechanism of mitochondrial ATP-sensitive potassium channel (mitoKATP) blocker in the ischemic postconditioning. Methods Thirty five Wistar rats were randomly divided into 5 groups: sham group, I/R group, ischemic postconditioning (IPO) group, I/R +5-hydroxydecanoate (I/R + 5-HD) group, IPO + 5-HD group. The concentration of malondialdehyde (MDA) and activity of superoide dismutase (SOD) were determined in the lung homogenate, wet to dry weight ratio (W/D) was measured and pathological changes were also observed. Results The levels of MDA[(5.07±1.60) vs (1.43 ±0.41) nmol/mg prot,P＜0. 01]and W/D (5.45 ±0.82 vs 3.05 ±0. 47,P ＜0. 01 ) were increased significantly in I/R group as compared with sham group, while the activity of SOD[( 12. 38 ±2. 24) vs (45.51 ±5.42) U/mg prot,P ＜0. 01]was decreased, and the injury of lung tissues was significantly aggravated in IPO + 5-HD group as compared with IPO group[MDA: (3.74 ±0. 71 ) nmol/mg prot vs (2. 60 ± 0. 43 ) nmol/mg prot , P ＜ 0. 01]; W/D: 4. 64 ± 0. 79 vs 3. 89 ± 0. 60,P＜0.01; SOD:[(22.91 ±2.71) U/mg prot vs (28.74±2.03) U/mg prot,P＜0. 01]. The levels of MDA[(2.60±0.43) vs (5.07 ±1.60) nmol/mg prot,P＜0. 01]and W/D (3.89 ±0.60 vs 5.45 ±0. 82,P ＜0. 01 ) were decreased significantly in IPO group as compared with I/R group, the activity of SOD[(28.74±2.03) vs (12.38 ±2.24) U/mg prot,P＜0. 01]increased and lung tissue histological damage attenuated. The difference in MDA[(5.14 ± 1.30) vs (5.07 ± 1.60) nmol/mg prot, P ＞ 0. 05],W/D (5.54±0.61 vs5.45 ±0.82,P＞0.05) and SOD[(11.65 ±1.82) vs (12.38 ±2.24) U/mgprot,P ＞ 0. 05]levels had no statistical significance between I/R + 5-HD group and I/R group, and the injury of lung tissues had no significant difference too. Each index in IPO + 5-HD group was between IPO and I/R groups. Conclusion Ischemic postconditioning can attenuate the lung I/R injury, and mitoKATP plays a vital role in the protective procession of ischemic postconditioning on lung ischemic reperfusion.     

大黄素对全身炎性反应综合征病人外周血中性粒细胞Fas/FasL、Caspase-3表达的影响

目的 探讨全身炎性反应综合征(SIRS)病人外周血中性粒细胞(PMN)凋亡信号Fas/FasL、Caspase-3表达的动态变化及大黄素对SIRS时PMN凋亡的影响及其作用机制.方法 采集6例健康志愿者及8例SIRS病人(均为急性胰腺炎病人)外周血液并从中分离PMN进行体外培养.分为3个实验组:正常组,SIRS组,大黄素干预组.观察各组PMN的凋亡情况;检测各组PMN Fas/FasL和Caspasc-3表达水平的变化.结果 SIRS组PMN凋亡百分率明显低于正常组(P＜0.05),大黄素干预后SIRS病人PMN凋亡百分率明显增加(P＜0.05);SIRS组PMN Fas和Caspase-3的表达水平明显低于正常组(P＜0.05),大黄素能诱导SIRS病人PMN Fas和Caspase-3的表达(P＜0.05).各组PMN在体外培养24 h后,经Western Blotting没有检测到FasL的表达.结论 SIRS病人外周血PMN凋亡存在异常,且与Fas和Caspase-3的表达降低有关;大黄素能通过诱导Fas和Caspase-3的表达,对SIRS时PMN凋亡延迟具有抑制作用.     

La核糖核蛋白6对人血管内皮细胞增殖与凋亡的作用

目的 研究La核糖核蛋白6(Achn)在人血管内皮细胞增殖和凋亡中的调节作用.方法 (1)DMEM无血清培养基培养人血管内皮细胞株Eahy926细胞,按随机数字表法(下同)分为Achn抑制组(转染Achn抑制表达载体psi-Achn)、psi4.1空载体组(转染psi4.1)、Achn诱导组(转染Achn诱导表达载体pcDNA-Achn)、pcDNA3.1空载体组(转染pcDNA3.1)、Achn与钙/钙调蛋白依赖性丝氨酸蛋白激酶(CASK)共转染组(转染pcDNA-Achn与CASK抑制表达载体psi-CASK)、空白对照组(PBS处理),分别于转染后1、24、48、72 h用噻唑蓝法测定各组细胞570 nm波长下的吸光度值.(2)取Eahy926细胞,裂解细胞总蛋白,二辛丁酸法定量后分为蛋白质印迹组(总蛋白量为20μg)、Achn蛋白沉淀组、CASK蛋白沉淀组、IgG对照组,后3组细胞蛋白总量各为100μg,免疫共沉淀法检测各组Achn、CASK蛋白水平.(3)取Eahy926细胞分为LPS组(5 mol/L LPS处理)、氯化钾组(5 mol/L氯化钾处理)、空白对照组(5 mol/L PBS处理)、Achn诱导转染组(转染pcDNA-Achn)、Achn与CASK共转染组(转染pcDNA-Achn与psi-CASK),转染组转染24 h后加入LPS刺激12 h,免疫组织化学法检测各组半胱氨酸天冬氨酸蛋白酶3(caspase-3)蛋白表达.(4)取Eahy926细胞分为Achn诱导组(转染pcDNA-Achn)、Achn抑制组(转染psi-Achn)、对照组(PBS处理),24 h后加入烧伤患者血清处理12 h,流式细胞仪检测各组细胞凋亡率.对实验数据行t检验和单因素方差分析.结果 (1)Achn抑制组细胞增殖水平从24 h开始低于psi4.1空载体组,48、72 h时差异均有统计学意义(t值分别为10.777、6.112,P值均小于0.05);转染后24、48、72 h Achn诱导组细胞增殖水平均显著高于pcDNA3.1空载体组(t值分别为5.367、6.053、9.831,P值均小于0.05);Achn与CASK共转染组细胞增殖水平48、72 h均显著低于Achn诱导组(t值分别为5.481、9.517,P值均小于0.05).(2)CASK蛋白沉淀组CASK抗体沉淀物中可同时检测到CASK蛋白和Achn蛋白,而Achn蛋白沉淀组Achn抗体沉淀物中可同时检测到Achn蛋白和CASK蛋白.(3)Achn诱导转染组血管内皮细胞caspase-3阳性表达率为(15.6±0.5)%,低于LPS组[(32.8±2.6)%,t=10.083,P＜0.05];Achn与CASK共转染组caspase-3阳性细胞表达率[(7.0±2.0)%]进一步降低,显著低于LPS组(t=9.827,P＜0.01).(4)Achn抑制组细胞凋亡率为(45.6±10.9)%,显著高于对照组的(13.2±4.3)%,t=7.043,P＜0.05;Achn诱导组的细胞凋亡率为(5.3±2.9)%,显著低于对照组(t=6.499,P＜0.05).结论Achn能促进入血管内皮细胞增殖,抑制LPS或烧伤血清诱导细胞凋亡并与CASK的作用相关联.

Abstract：

Objective To investigate regulatory effect of Acheron (Achn) on proliferation and apoptosis of human vascular endothelial cell. Methods ( 1 ) Eahy926 cells were cultured in serum-free DMEM medium (96-well plates) and were divided into Achn inhibition group (transfected with plasmid psi-Achn), psi4.1 group (transfected with psi4. 1 empty vector), Achn induction group (transfected with pcDNA-Achn), pcDNA3.1 group (transfected with pcDNA3.1 empty vector), cotransfection group [cotransfected with pcDNA-Achn + psi-calcium/calmodulin-dependent serine protein kinase (CASK)] , blank control group (treated with PBS) according to the random number table (the same method below). The cell proliferation was determined by MTT assay at post transfection hour (PTH) 1, 24, 48, 72, with expression of absorbance value. (2) Total protein of Eahy926 cells were extracted and quantitated by BCA assay, and then they were divided into Achn antibody precipitation group (100 μg protein) , CASK antibody precipitation group ( 100 μg protein), IgG antibody group ( 100 μg protein), Western blot group (20 μg protein).Achn and CASK protein levels were determined by immunoprecipitation and Western blot. (3) Synchronously cultured Eahy926 cells were divided into LPS induction group (treated with 5 mol/L LPS), Achn transfection group (transfected with pcDNA-Achn), cotransfection group (cotransfected with psi-CASK and pcDNA-Achn) , KCl group (treated with 5 mol/L KCl), and blank control group (treated with 5 mol/LPBS). Cells in transfection groups were stimulated by LPS for 12 hours after PTH 24. Caspase-3 protein level was detected by immunohistochemistry. (4) Synchronously cultured Eahy926 cells were divided into Achn inhibition group (transfected with psi-Achn vector), Achn induction group ( transfected with pcDNA-Achn vector), and blank control group ( treated with PBS). Apoptosis rate was determined by FITC/PI with flow cytometry. Data were processed with one-way analysis of variance and t test. Results ( 1 ) The cell proliferation in Achn inhibition group was lower than that in psi4.1 group from PTH 24, and the differences were statistically significant at PTH 48, 72 (with t value respectively 10. 777, 6.112, P values all below 0. 05 ).The cell proliferation in Achn induction group during PTH 24-72 were higher that in pcDNA3. 1 group (with t value respectively 5. 367, 6. 053, 9. 831, P values all below 0.05 ). The cell proliferation in cotransfection group at PTH 48, 72 were significantly lower than that in Achn induction group ( with t value respectively 5.481, 9. 517, P values all below 0. 05). (2) Achn protein was detected in CASK antibody precipitation group while CASK protein was also detected in Achn antibody precipitation group. (3) Caspase-3 level in Achn transfection group was lower [( 15.6 ± 0. 5 ) %] as compared with that in LPS induction group [(32. 8 ±2.6)%, t = 10. 083, P ＜ 0. 05], and that in cotransfection group showed further inhibition [(7.0 ±2.0)%,t =9.827, P ＜0.01]. (4) Apoptosis rate in Achn inhibition group[(45.6 ± 10.9)%] was higher than that in blank control group [(13.2±4.3) %, t =7.043, P ＜0.05]; while that in Achn inductiongroup [(5.3 ±2.9)%] was lower than that in blank control group ( t =6.499, P ＜0.05).Conclusions Achn can promote human vascular endothelial cell proliferation, and inhibit its apoptosis induced by LPS or burn serum, and the effect is related to CASK.     

甲氨蝶呤对人骨肉瘤MG-63细胞凋亡及28Livin和Caspase-3表达的影响

目的 观察甲氨蝶呤(MTx)对人骨肉瘤MG-63细胞凋亡及Livin和Caspase-3表达的影响.方法 体外培养骨肉瘤MG-63细胞株,用0、50、100、200和400μmol/L的MTX作用于骨肉瘤MG-63细胞24、48、72 h后,用噻唑蓝(MTT)比色法检测骨肉瘤MG-63细胞的增殖活性,用流式细胞仪测细胞的凋亡率,用Western blot检测不同浓度MTX作用于骨肉瘤MG-63细胞24 h后各组细胞的Livin和Caspase-3蛋白表达水平.结果 随MTX浓度增加骨肉瘤MG-63细胞的增殖活性降低,同时细胞的凋亡率增加(P＜0.05),随MTX浓度增加各组细胞中Livin蛋白的表达降低,Caspase-3蛋白表达增加(P＜0.05).结论 MTX诱导人骨肉瘤MG-63细胞凋亡,其机制可能与下调Livin表达继而上调Caspase-3表达有关.

Abstract：

Objective To observe the effect of Methotrexate (MTX) on apoptosis and expression of Livin and Caspase-3 in human osteosarcoma cell line MG-63. Methods After treatment of MG-63 cells with MTX at different concentrations (0, 50, 100,200,400 μmol/L) for 24, 48 and 72 h, methyl thiazol tetrazolium(MTT) assay was used to observe the growth inhibition of MG-63. The apoptosis was assessed by flow cytometry. The protein expression of Livin and Caspase-3 was detected by Western blotting. Results When MTX was added, growth inhibition and increased apoptosis of MG-63 cells were detected,which was showed in a dose- and time-dependent manner. MTX also down-regulated the level of the protein expression of Livin (P＜0.05), and elevated the protein expression of Caspase-3 (P＜0.05). Conclusion MTX can induce apoptosis of MG-63 cells, by down-regulating Livin expression and subsequently up-regulating Caspase-3 expression.