肝动脉桥式置管转流对供肝及胆道组织细胞凋亡的保护作用

目的验证肝动脉桥式置管转流这一方法是否能够减轻肝动脉缺血(hepatic artery ischemia,HAI)引起的肝胆细胞凋亡。方法应用简易犬肝自体原位移植模型,将24只杂交犬随机分为肝动脉缺血组(HAI组,8只)、肝动脉桥式置管转流组(TBB组,8只)及对照组(8只)。在制模后,3组动物均于冷灌注后不同时点切取肝脏、胆道组织,分别用戊二醛及40 g/L多聚甲醛溶液固定,进行电镜观察和TUNEL染色,观察肝胆组织形态学改变及细胞凋亡情况,并计算两组的凋亡指数。结果在冷灌注后2 h,HAI组电镜下出现较明显的肝胆细胞凋亡现象;肝动脉桥式置管转流组肝胆细胞凋亡现象少见;对照组难以找到凋亡肝胆细胞。TUNEL法染色切片显示,冷灌注后3组肝胆组织内均有少量凋亡细胞,其凋亡指数相差不大(P>0.05);随冷灌注后时间的延长,3组凋亡细胞的数量均有所增加,但HAI组凋亡指数的升高更为显著,肝动脉桥式置管转流组次之,对照组变化不大,3组间差别有统计学意义(P<0.01)。结论肝动脉桥式置管转流对肝移植时肝动脉缺血所导致的肝胆细胞凋亡具有显著保护作用,应在继续完善改进的基础上实施于临床。     

乌司他丁通过抑制肠黏膜细胞凋亡减轻大鼠肠道缺血-再灌注损伤

目的 探讨蛋白酶抑制剂乌司他丁(ulinastatin,UTI)对大鼠肠缺血-再灌注损伤后肠黏膜屏障功能的保护作用及其机制.方法 24只成年雄性SD大鼠夹闭肠系膜上动脉1 h,再灌注1 h,随机分为空白对照组、对照组和治疗组.治疗组于缺血后经阴茎背静脉缓慢泵入UTI(5万单位/kg);对照组给予等量等渗盐水;空白对照组仅做开关腹并给予等量生理盐水作为对照.各组大鼠均于制模后采集标本,动态浊度法检测血清内毒素含量,TUNEL法检测肠黏膜上皮细胞凋亡率,RT-PCR检测凋亡调控基因Bax、Bcl-2 mRNA的表达.结果 空白对照组血清内毒素水平和肠黏膜细胞凋亡指数均低于对照组(P<0.01);治疗组BaxmRNA表达低于对照组(P<0.01),而Bcl-2 mRNA表达高于对照组(P<0.01).结论 乌司他丁可能通过抑制肠黏膜上皮细胞的凋亡,维持肠黏膜屏障的完整性,从而防止缺血-再灌注损伤时细菌和内毒素的移居.     

藏红花素抑制谷氨酸盐诱导的视网膜神经节细胞凋亡

目的研究藏红花素通过影响Ca~(2+)内流对谷氨酸盐诱导的视网膜神经节细胞(RGCs)凋亡的影响及可能机制。方法分离大鼠RGCs,以0.1、1mmol/L的谷氨酸盐刺激RGCs 24、48h,建立RGCs凋亡模型,并用0.1、1.0、3.0μmol/L浓度梯度藏红花素分别处理。Annexin V-FITC/PI双标检测细胞凋亡率,Fluo-3/AM荧光标记Ca~(2+)检测胞内钙离子浓度,Western blot检测藏红花素对胞内钙离子介导的凋亡信号分子calpain和CaMKⅡ表达的影响。JC-1荧光染色和Western blot分别检测藏红花素对线粒体膜电位和线粒体凋亡相关信号分子Caspase-3、Caspase-9、Bcl-2/Bax表达的影响。结果 0.1mmol/L谷氨酸盐刺激24h,RGCs细胞凋亡率与对照组差异无统计学意义(P>0.05);而当刺激48h时,RGCs的凋亡率达到(43.050±2.616)%,差异有统计学意义(P<0.01)。高剂量谷氨酸盐(1mmol/L)刺激24、48h的RGCs凋亡率为(46.450±1.061)%和(45.500±3.253)%,较对照组均显著增加,差异有统计学意义(P<0.01)。用1mmol/L谷氨酸盐刺激RGCs 12h后加入0.1、1.0、3.0μmol/L藏红花素再处理12h,不同浓度藏红花素均可显著抑制细胞凋亡(P<0.01),且抑制效率具有剂量依赖性。另外,1.0μmol/L藏红花素组的谷氨酸盐诱导的胞外Ca~(2+)内流减少及钙依赖蛋白Calpain1和CaMKⅡ的表达减弱,线粒体膜电位增高,Caspase-3和Caspase-9的表达减少,Bcl-2/Bax表达上调。结论藏红花素抑制谷氨酸盐诱导的RGCs凋亡,其机制可能与阻止胞外Ca~(2+)内流,抑制钙依赖的凋亡信号通路和线粒体凋亡信号通路有关。     

甘氨脱氧胆酸盐诱导人正常肝细胞HL-7702凋亡及其机制的研究

目的 观察甘氨脱氧胆酸盐(glycodeoxycholate, GCDC)对人正常肝细胞株HL-7702凋亡的影响,并探讨其凋亡机制.方法 终浓度分别为100、150、200、250μmol/L的GCDC处理HL-7702细胞24h,光学显微镜观察GCDC对HL-7702细胞形态的影响,AnnexinV-FITC/PI双染色法检测HL-7702细胞的凋亡率 ,用荧光指示剂Fluo-3/AM测定HL-7702细胞内钙离子浓度,RT-PCR测定Bcl-2、Bax基因mRNA表达水平.结果 终浓度为150μmol/L的GCDC处理HL-7702细胞24h后,细胞出现典型的凋亡细胞形态学改变;100、150、200、250μmol/L的GCDC均可诱导HL-7702细胞凋亡,呈剂量依赖性,凋亡率分别为(13.16±2.9)% (t=6.41)、(20.3±3.0)% (t=10.22)、(25.02±2.1)% (t=18.11)、(45.02±3.5)% (t=28.89),较对照组(2.2±0.6)%显著升高(P＜0.05);GCDC能使HL-7702细胞内钙离子浓度增加,且具有浓度依赖性;GCDC使细胞内Bcl-2 mRNA的表达下降、Bax mRNA的表达增加.结论 GCDC可能是通过增加HL-7702细胞内钙离子浓度,并进一步下调Bcl-2、上调Bax表达水平,从而诱导细胞凋亡.     

重组人促红细胞生成素对兔缺血再灌注损伤视网膜结构的保护作用

目的 探讨皮下注射重组人促红细胞生成素(recombinant human erythropoietin, rhEPO)对兔眼缺血再灌注损伤视网膜结构的保护作用.方法 24只健康日本大耳白兔任选一眼造成视网膜缺血再灌注损伤模型(模型组,共24眼),对侧眼不作任何处理作为对照组(24眼).另24只健康日本大耳白兔任选一眼造模(EPO组,共24眼),于造模前第3天及造模结束时每只兔皮下注射rhEPO 100 IU/kg各1次.于造模后第1、3、7、14天分别摘除每组各6只兔眼,观察视网膜组织结构形态、测量内层视网膜(inner retina layer, IRL)厚度并计数视网膜节细胞(retinal ganglion cell, RGC).结果 自造模后第3天开始,模型组较对照组RGC数量减少、IRL厚度变薄(P<0.05,P<0.01).自造模后第3天开始EPO组IRL较模型组厚(P<0.05,P<0.01),自造模后第7天开始EPO组RGC数量较模型组多(P<0.05).结论 rhEPO可以显著改善缺血再灌注损伤兔眼视网膜结构,可能成为一种神经缺血再灌注损伤的保护剂.     

厄贝沙坦抑制高浓度葡萄糖诱导的脂质氧化效应和人脐静脉血管内皮细胞损伤

目的 探讨厄贝沙坦对高浓度葡萄糖诱导的脂质氧化效应及人脐静脉血管内皮细胞(HUVEC)损伤的影响.方法 取生长良好的HUVEC进行实验.将细胞分为4组:正常浓度葡萄糖对照组(葡萄糖终浓度为5.5mmol/L)、高糖组(葡萄糖终浓度为33.3mmol/L)、厄贝沙坦干预组和抗氧化剂N-乙酰半胱氨酸(N-acetyl-cysteine, NAC)干预组.干预组首先用厄贝沙坦(10-5mol/L)和NAC(10mmol/L)分别预作用1h,而后加入33.3mmol/L葡萄糖共同孵育24、48、72h.分别采用TBARS法和分光光度比色法检测培养上清液中丙二醛(MDA)含量及超氧化物歧化酶(SOD)活性.结果 与正常浓度葡萄糖组比较,高糖组MDA含量显著升高(P<0.01),SOD活性明显降低(P<0.01);高糖作用24h 时MDA含量已达到较高水平(P<0.01),且随葡萄糖作用时间的延长(48、72h)MDA含量呈上升趋势,但与高糖作用24h比较,差异无统计学意义(P>0.05).与高糖组比较,厄贝沙坦组和NAC组MDA含量均显著下降(P<0.05),SOD活性则均明显升高(P<0.05).与高糖组比较,厄贝沙坦组和NAC组各时点细胞凋亡率均显著降低(P<0.05),高糖对HUVEC细胞形态的损伤明显减轻.结论 厄贝沙坦部分通过抑制氧化应激作用降低高浓度葡萄糖诱导的脂质氧化效应,保护HUVEC.     

钙离子载体A23187对转化生长因子β1刺激的肝星状细胞增殖、周期及凋亡蛋白caspase-3表达的影响

目的研究钙离子载体A23187对转化生长因子β_1(TGF-β_1)刺激的肝星状细胞增殖、周期及凋亡蛋白caspase-3表达的影响。方法将液氮保存下的肝星状细胞株,于37℃、50mL/L CO_2孵箱中进行复苏传代培养,同步化后将细胞分为5组:空白组、TGF-β_1(5ng/mL)组、TGF-β_1+低、中、高剂量钙离子载体A23187组,即5ng/mL TGF-β_1刺激24h,再分别加入1、2、4μmol/L不同剂量钙离子载体A23187作用24h后,用MTT比色法检测细胞增殖,流式细胞仪检测细胞周期,蛋白免疫印记测定凋亡蛋白caspase-3表达。结果不同浓度钙离子载体A23187均能显著抑制细胞增殖(P<0.05),且随着剂量的增加,细胞的相对增殖率(RGR)降低,低、中、高剂量组分别为85.93%、61.71%、48.43%,组间两两比较差异均有统计学意义(P<0.05);不同处理组G1期细胞比例、S+G2期细胞比例差异均有统计学意义(P<0.05),钙离子载体A23187剂量越大,G1期细胞比例越高,S+G2期细胞比例越低(P<0.05)。随着钙离子A23187浓度的增加,细胞内caspase-3蛋白表达明显增加(P<0.05)。结论钙离子载体A23187可能通过阻滞大鼠肝星状细胞由G1期进入S期和G2期,抑制细胞增殖,同时上调凋亡蛋白caspase-3的表达。     

沉默SPARC基因抑制高氟介导的甲状腺细胞凋亡

目的研究富含半胱氨酸的酸性分泌蛋白(SPARC)对高氟介导的甲状腺细胞凋亡的作用及可能机制。方法培养人甲状腺细胞Nthy-ori 3-1,给予不同浓度的NaF(0.1、1、10 mmol/L)处理24 h,通过实时定量PCR和蛋白印迹法评价SPARC的表达,确定后续实验NaF作用浓度。另将细胞分组进行实验:对照组、NaF组(1 mmol/L孵育24 h)、si-SPARC组(转染SPARC siRNA 48 h用NaF处理)和si-NC组(转染Negative control siRNA 48 h用NaF处理)。CCK-8法和乳酸脱氢酶(LDH)试剂盒检测细胞毒性;细胞凋亡试剂盒检测细胞凋亡率,蛋白印迹法检测活化caspase3(c-caspase3)和IGF-1R的蛋白表达。此外,将siSPARC和siIGF-1R共同转染至甲状腺细胞,通过评价细胞凋亡进一步探讨SPARC的作用机制。结果随着NaF的浓度增大,SPARC mRNA和蛋白表达均逐渐升高(P<0.05)。si-SPARC组细胞活性较si-NC组增高[(84.02±9.51)%vs.(58.31±6.86)%,P<0.05],且LDH释放率减少[(134.25±18.98)%vs.(195.18±23.50)%,P<0.05]。si-SPARC组较si-NC组细胞凋亡率减少[(124.67±19.44)%vs.(175.24±16.46)%,P<0.05]。此外,si-SPARC组(1.95±0.24 vs. 0.93±0.08,P<0.05)IGF-1R蛋白表达上调,抑制IGF-1R逆转SPARC对细胞凋亡的作用。结论沉默SPARC基因降低高氟介导的细胞毒性、抑制细胞凋亡,其机制可能是通过负调控IGF-1R实现的。     

c-Jun氨基末端激酶信号通路在大鼠脑缺血再灌注过程中的作用

目的探讨c-Jun氨基末端激酶(JNK)信号通路在大鼠脑缺血再灌注过程中所发挥的作用。方法雄性SD大鼠108只,体重290-310 g,随机分成假手术组(SH组)、缺血再灌注组(IR组)和JNK抑制剂SP600125组(SP组),分别于缺血前30 min侧脑室注射10 mL/L二甲基亚砜(DMSO)1、0 mL/L DMSO及JNK抑制剂SP600125。每组再根据再灌注时间分为2、6、12、24、487、2 h 6个亚组,每亚组6只动物。采用4-VO法建立SD大鼠全脑缺血模型,在预定时间点行灌注、固定、取脑、石蜡包埋切片;免疫组化方法检测p-JNK的表达变化,光镜下计数海马CA1区存活细胞,TUNEL法检测CA1区凋亡细胞。结果脑缺血再灌注后海马CA1区p-JNK在IR组有明显表达,于再灌注2 h时即明显升高,6 h时略有降低,后逐渐上升,24 h到高峰,之后表达量减小。SP组p-JNK的表达则无明显增高,各时点与IR组比较均有显著性差异(P<0.01)。海马CA1区神经元存活数目SP组明显高于IR组(P<0.01),凋亡指数显著低于IR组(P<0.01)。结论在大鼠全脑缺血再灌注损伤过程中,JNK信号通路发挥了重要作用,抑制JNK通路的激活可对脑缺血再灌注损伤导致的细胞损伤起到保护作用。     

流式细胞仪测定血小板胞浆游离钙浓度

目的建立流式细胞仪测定血小板胞浆游离钙离子浓度[Ca2+]i的方法。方法用Fluo 3-AM作为钙指示剂,在有或无细胞外钙[Ca2+]o存在的条件下,测定静息和凝血酶激活状态下人血小板胞浆游离钙离子浓度[Ca2+]i的变化。结果Fluo 3-AM标记的血小板的平均荧光强度明显增加。凝血酶使负载Fluo 3-AM标记的血小板胞浆[Ca2+]i明显增加,且显示剂量依赖和对[Ca2+]o的依赖。结论凝血酶引起血小板胞浆[Ca2+]i增加的来源主要是[Ca2+]o,可能也有部分是内钙释放的参与。     

EFFECT OF STENT ABSORBED c-myc ANTISENSE OLIGODEOXYNUCLEOTIDE ON SMOOTH MUSCLE CELLS APOPTOSIS IN RABBIT CAROTID ARTERY

Ｉｎｔｈｅｐａｓｔｆｅｗ ｙｅａｒｓ ,ｗｅｈａｖｅｗｉｔｎｅｓｓｅｄａｄｒａ ｍａｔｉｃ ｐｒｏｌｉｆｅｒａｔｉｏｎｉｎｔｈｅｕｓｅｏｆｉｎｔｒａｃｏｒｏｎａｒｙｓｔｅｎｔｓ.Ｓｔｅｎｔｓｎｏｗａｃｃｏｕｎｔｆｏｒｏｖｅｒ 70 %ｏｆｐｅｒｃｕｔａ ｎｅｏｕｓｔｒａｎｓｌｕｍｉｎａｌｃｏｒｏｎａｒｙａｎｇｉｏｐｌａｓｔｙ (ＰＴＣＡ ) .Ｈｏｗｅｖｅｒ,ｉｎ ｓｔｅｎｔｒｅｓｔｅｎｏｓｉｓｒｅｍａｉｎｓａｍａｊｏｒｐｒｏｂ ｌｅｍ ,ｏｃｃｕｒｉｎｇｉｎ 2 0 %～ 30 %ｏｆｔｈｅｓｅ ｐｒｏｃ ｅｄｕｒｅｓ[1-3] .Ｉ…     

短发卡RNA抑制survivin表达并诱导CNE-2细胞凋亡

目的观察以短发卡RNA(shRNA)抑制鼻咽癌细胞survivin表达对细胞凋亡与增殖的影响。方法构建特异性survivin shRNA,转染CNE-2细胞。分组:A组为空白对照组;B组为阴性干扰组,转染pSIREN-survivin/nonsenseshRNA;C组为阳性干扰24h组,转染pSIREN-survivin/shRNA;D组为阳性干扰48h组,转染pSIREN-survivin/shRNA。以RT-PCR、Western-blot分别测定细胞survivin mRNA及蛋白,PI、TUNEL及MTT检测细胞周期、凋亡率、细胞增殖。结果转染效率约(70.90±4.76)%;C组survivin mRNA和蛋白抑制率分别为(41.58±0.63)%、(68.29±0.52)%;D组抑制率分别为(63.64±0.96)%、(70.83±0.48)%。C组凋亡率为(36.24±0.78)%,D组为(50.37±0.85)%,显著高于B组和A组;S期细胞减少,G2/M期比例增高;MTT结果提示细胞增殖受到明显抑制。结论特异性shRNA可有效干扰CNE-2内survivin的表达,诱导细胞凋亡并抑制其过度增殖。     

Effect of ulinastatin on hepatic ischemia-reperfusion injury in rats

Objective To investigate the effect of ulinastatin (UTI) on hepatic ischemia-reperfusion injury in rats. Methods Totally 24 adult Sprague-Dawley rats were randomly divided into 3 groups: sham-operated control group (SO group), ischemia-reperfusion group (I/R group) and ulinastatin group (UTI group). Liver in I/R group underwent 1 h of reperfasion after 30 min of ischemia. In UTI group, UTI (2×104 U/kg) was administered to rats 30 min before modeling. The levels of alanine aminotransferase, aspartate aminotransferase and tumor necrosis factor-alpha (TNF-α) in serum were measured and the levels of nitric oxide and malondialdehyde in liver were determined. The histological changes of liver were observed. Results The levels of alanine aminotransferase, aspartate aminotransferase and TNF-α in serum were significantly increased in I/R group compared with those in UTI group (P<0.05). The levels of nitric oxide and malondialdehyde in liver were significantly higher in I/R group than in UTI group (P<0.05).Histological examination of liver indicated that the damages were more severe in I/R group than in UTI group.Conclusion UTI has the ability to inhibit the production of TNF-α and oxyradical, and ameliorate microcirculatory dysfunction in rats with hepatic ischemia-reperfasion injury.     

Effects of erigeron breviscapus （Vant.） Hand-Mazz pretreatment on pathology and oxyradical level following spinal cord ischemia-reperfusion injury in rabbits

Objective To investigate the effects of erigeron breviscapus (Vant.) Hand-Mazz (erigeron breviscapus) pretreatment on pathology and oxyradical level in the spinal cord after ischemia-reperfusion (I/R) injury in rabbits. Methods A total of 40 New Zealand white rabbits were randomly divided into three groups: sham-operation group with 10 rabbits treated with only abdominal aorta exposure without occlusion, control group with 15 rabbits that underwent ischemia for 50 minutes and treated with matched saline, and experimental group with 15 rabbits that underwent ischemia for 50 minutes and treated with erigeron breviscapus (9mg/kg) injection before ischemia. Malondialdehyde (MDA) level and superoxide dismutase (SOD) activity in the spinal cord were examined at 6 and 24 hours after I/R, respectively. The morphological changes and the number of the spinal cord anterior horn motor neurons were observed and counted under the light microscope and electron microscope, respectively. Results The level of MDA was markedly decreased and SOD activity was increased in the experimental group compared with those in the control group (P<0.01). Compared with that in the control group, the number of motor neurons in the experimental group significantly increased at 24h after I/R (P<0.01) and the morphous of the motor neurons improved. Conclusion Erigeron breviscapus can reduce oxyradical production and the apoptosis of nerve cells, and protect nerve tissue structure and function after spinal cord I/R.     

CHONDROCYTE APOPTOSIS IN ARTICULAR CARTILAGE WITH KASHIN-BECK DISEASE

Kashin BeckDisease(KBD)isachronic,en demicosteoarthritisaffectingover0.81millionspa tientsand101millionsofpeopleatriskinChina[1]. Thebasicpathologicalfeaturesofthediseaseis chondrocytesdegenerationandnecrosisinthedeep zoneofarticularcartilageandgrowthpl…     

川芎嗪对成体大鼠脑缺血再灌注损伤后海马齿状回细胞增殖的影响

目的研究川芎嗪(tetramethylpyrazine,TMP)对大鼠脑缺血再灌注损伤后海马齿状回(dentate gyrus,DG)细胞增殖的影响。方法成年雄性SD大鼠行2 h大脑中动脉阻塞手术,术后2 h开始腹腔注射TMP[40 mg/(kg.d)]。手术后腹腔注射5-溴脱氧尿核苷(5-bromodeoxyuridine,BrdU),末次注射24 h后处死动物,免疫组化染色观察TMP对脑缺血再灌注损伤后DG细胞增殖的作用。结果正常组和假手术组在DG有少量BrdU阳性细胞,对照组缺血后1 d阳性细胞开始增加,14 d达到高峰(P<0.05),TMP治疗组缺血后1 d损伤侧BrdU阳性细胞数开始增加,7 d达高峰(P<0.05)。结论TMP能促进缺血再灌注损伤大鼠海马齿状回内源性神经干细胞增殖。     

Oleanolic acid-induced apoptosis and its relation with intracellular calcium in human lung adenocarcinoma A549 cells

Objective To investigate the effect of oleanolic acid (OA) on apoptosis, correlation between apoptosis and intracellular calcium, and its mechanism in human lung adenocarcinoma cell line A549. Methods Human lung adenocarcinoma A549 cells were incubated in vitro and assigned with OA concentrations of 0, 10, 20 and 40μg/mL. The apoptosis status of A549 cell line was detected with Annexin V-FITC/PI by flow cytometry (FCM); fluorescence intensity (FI) of A549 cells was assessed and the level of intracellular calcium was calculated at 24 hour of OA intervention. The relation between apoptosis and calcium FI was illustrated by curve fitting. Results FCM showed that 10, 20 and 40μg/mL of OA could induce A549 cell apoptosis, which followed a concentration-effect pattern; 24-hour intervention with 20μg/mL and 40μg/mL OA showed increased A549 cell apoptosis, and was significantly different from that with 0μg/mL OA (P<0.01). The FI of intracellular calcium concentration in 10, 20 and 40μg/mL OA groups was significantly higher than that in 0μg/mL group after 24 hours' intervention, and the FI showed a trend of increase with increased OA concentration (P<0.01). Curve fitting showed a significant correlation between apoptosis rate and intracellular calcium concentration in A549 cells (r=0.981, P<0.01). Regression equation was Y=0.508X-1.627. Conclusion OA plays a role in inducing apoptosis of human lung adenocarcinoma cells in a concentration-dependent manner. The OA-induced apoptosis is responsible for intracellular calcium overload of the tumor.