钙敏感受体在缺氧/复氧诱导乳鼠心肌细胞凋亡中的作用

目的 探讨钙敏感受体(CaSR)对缺氧/复氧(A/R)所诱导乳鼠心肌细胞凋亡的影响及其机制.方法 原代培养乳鼠心室肌细胞缺氧2 h/复氧24 h,建立A/R心肌细胞损伤模型.心肌细胞随机分为三组:对照组、A/R组和CaSR激动剂氯化钆(GdCl3)组.四氮唑嗅盐法(MTT)检测细胞的存活率,原位缺口末端标记法(TUNEL)分析细胞凋亡率,蛋白印迹法检测CaSR、半胱氨酸天冬酶(caspase)-3、9和细胞色素c(Cytc)蛋白的表达.结果 TUNEL染色显示A/R后心肌细胞凋亡为(17±3)%,与对照相比,心肌细胞凋亡明显增加,CaSR表达也增加.CaSR激动剂GdCl3使心肌细胞凋亡进一步增加至(28±4)%,MTT检测表明心肌细胞存活率仅为(51.2±6.8)%;CaSR、caspase-3、caspase-9和Cytc表达进一步上调,明显高于A/R组.结论 CaSR通过线粒体Cytc-caspase-3途径参与了A/R所诱导的心肌细胞凋亡.     

钙敏感受体参与乳鼠心肌细胞缺氧/复氧损伤的机制研究

目的 观察钙敏感受体(CaSR)对缺氧/复氧(A/R)乳鼠心肌细胞内钙的影响,探讨CaSR参与缺氧/复氧乳鼠心肌细胞损伤的机制及信号传导途径.方法 原代培养乳鼠的心肌细胞,建立A/R乳鼠心肌细胞模型(采用95%N2+5%CO2饱和2 h的低糖、无血清DMEM液孵育60 min,再更换成正常氧合细胞培养液孵育30 min的方法).心肌细胞随机分为6组:(1)正常对照组;(2)A/R组;(3)A/R+GdCl3组:在复氧开始时给予GdCl3(CaSR激动剂)300 μmol/L;(4)A/R+NiCl2+CdCl2+GdCl3组:在复氧时细胞培养液内加入10 mmol/L NiCl2(Na+-Ca2+交换阻断剂)和200 μmol/L CaCl2(L-Ca2+阻断剂)孵育10min,再加入300/μmol/L GdCl3;(5)A/R+U73122+GdCl3组:在复氧时细胞培养液内加入10μmol/L磷脂酶C(PLC)阻断剂U73122孵育10 min,再加入300μmol/L GdCl3;(6)A/R+thapsigargin+GdCl3组:在复氧时细胞培养液内加入10μmol/L肌浆网三磷酸肌醇(IP3)敏感Ca2+泵阻断剂thapsigargin孵育10 min,再加入300μmol/L GdCl3.应用Fluo-3/AM荧光指示剂负载心肌细胞,激光共聚焦显微镜连续观察细胞内钙荧光强度的动态变化.结果 A/R使心肌细胞内钙离子浓度([Ca2+];)增加,GdCl3使A/R乳鼠心肌细胞[Ca2+]?I进一步增加;NiCl2和CdCl2对GdCl3引起的心肌细胞[Cd2+]I增加无影响;而U73122和thapsigargin则抑制了GdCl3引起的心肌细胞[Ca2+]I增加.结论 CaSR通过磷脂酶C-三磷酸肌醇途径参与了A/R所致心肌细胞钙超载.     

还原型谷胱甘肽对大鼠深低温暂停循环的脑保护作用

目的 探讨还原型谷胱甘肽(GSH)对深低温暂停循环(DHCA)大鼠是否具有脑保护作用.方法 SD大鼠24只,随机分为3组,每组8只,A组:常温体外循环(CPB)组;B组:DHCA组;C组:GSH处理组.各组大鼠转流前及转流结束后30 min留取静脉血检测S-100 β及神经元特异性烯醇化酶(NSE).术后留取脑组织,用于测定脑含水量及病理变化.结果 DHCA组病理变化较常温CPB组明显,而与GSH处理组差别不大.转流后DHCA组大鼠脑含水量较常温CPB组显著增加(P ＝ 0.012),GSH处理组大鼠脑含水量与常温CPB组差异无统计学意义.各组转流后S-100 β和NSE均较术前增加,DHCA组增加最为明显,但3组间差异无统计学意义.结论 DHCA对大鼠的脑损伤严重;GSH预处理对施行DHCA的大鼠可能有一定的脑保护作用.     

还原型谷胱甘肽治疗新生儿缺氧缺血性心肌损害的疗效观察

新生儿缺氧缺血性心肌损害是新生儿窒息的常见并发症,其发生率可高达65．5％,起病急而临床表现不典型,进展快且很容易出现心力衰竭,是新生儿科常见的危急重症。已有较多文献报道用果糖、维生素C等改善心肌代谢,清除氧自由基的药物来治疗该病。但尚未见到用还原型谷胱甘肽治疗新生儿缺氧缺血性心肌损害的报道。我院收治的66例缺氧缺血性心肌损害患儿应用还原型谷胱甘肽治疗,取得满意疗效,现报道如下。     

复方丹参注射液减轻缺氧/复氧性HK-2细胞损伤的作用及机制

目的：探讨丹参注射液减轻缺氧/复氧性人肾近曲小管上皮细胞（HK-2）细胞损伤的作用机制。方法：以人近曲肾小管上皮细胞为研究对象,采用液体石蜡覆盖法建立缺氧/复氧模型。实验分为3组：对照组、损伤组和丹参组,每个组均设立7个时间点：缺氧4,12,24 h和缺氧24 h后复氧4,12,24,48 h,其中丹参组设立4个浓度亚组（生药浓度）：0.05%,0.10%,0.15%,0.20%。在倒置相差显微镜下观察细胞的形态学改变,MTT法进行活细胞计数,生化法检测培养上清液中乳酸脱氢酶（LDH）含量,放射免疫法检测肿瘤坏死因子α（TNF-α）的含量。结果：缺氧处理后,损伤组在各时间点细胞数量均明显降低,在缺氧24 h达最低值;与损伤组比较,在丹参各组中,HK-2细胞数量较多,其中以0.10%丹参组细胞数量在各时间点均为最高。与对照组比较,损伤组LDH,TNF-α水平升高,在缺氧24 h/复氧4 h达最高值;与损伤组相比,丹参组在缺氧/复氧过程各时间点中LDH,TNF-α水平降低。结论：复方丹参注射液对HK-2细胞缺氧／复氧性损伤具有保护作用,作用机制可能与丹参注射液抑制炎症细胞因子的产生有关。［中国当代儿科杂志,2007,9（6）：559-562］     

钙敏感受体对乳鼠心肌细胞凋亡的诱导作用

探讨在缺氧/复氧所诱导乳鼠心肌细胞凋亡中,钙敏感受体（calcium - sensing receptor,CaSR）对Fas/Fas配体（Fas L）途径的影响。方法对8只2日龄Wistar大鼠心肌细胞进行原代培养,后随机分为三组：(1)对照组：细胞不经缺氧/复氧处理,在其他组复氧开始时培养基内加入与用药组等体积的生理盐水;(2)缺氧/复氧组：心室肌细胞缺氧2h/复氧24h,细胞培养基内于复氧开始时加入与用药组等体积的生理盐水;(3) CaSR激动剂氯化钆(GdCl3)组：细胞培养基内于复氧开始时加入300μmol/L CaSR激动剂GdCl3。流式细胞仪分析细胞凋亡率,透射电镜观察细胞超微结构,蛋白印迹法检测CaSR、Fas和FasL蛋白的表达。结果流式细胞仪检测显示,缺氧/复氧组心肌细胞凋亡为12.18％±1.54％,与对照组相比,心肌细胞凋亡明显增加(P＜0.01)。CaSR激动剂GdCl3使心肌细胞凋亡进一步增加至20.25％±2.87％ (P＜0.01)。透射电镜下可见线粒体絮状变、线粒体嵴溶解、空泡样变;CaSR激动剂使CaSR、Fas和FasL表达进一步上调,明显高于缺氧/复氧组(P＜0.05)。结论CaSR激活参与了缺氧/复氧所诱导的乳鼠心肌细胞凋亡,CaSR可通过激活Fas/FasL死亡受体途径导致乳鼠心肌细胞凋亡。     

心肌缺氧／复氧时pH反常和钙超载的关系

缺氧／复氧可导致心肌细胞的钙超载和ｐＨ反常，ｐＨ反常是Ｎａ＾＋／Ｈ＾＋交换和Ｎａ＾＋／Ｃａ＾２＋交换的共同作用产生钙超载而引起的，且可能是细胞损伤的始动环节。缺氧期应用Ｎａ＾＋／Ｈ＾＋或Ｎａ＾＋２＋／Ｃａ＾２＋交换抑制剂可有效防治心肌细胞缺氧／复氧时ＰＨ反常造成的损伤。     

体外牛磺酸对缺氧诱导心肌细胞凋亡的影响

为探讨牛磺酸( Tau)对缺氧引起的心肌细胞凋亡是否有抑制作用,将原代培养的乳鼠心肌细胞分为正常对照组( n=5)、 Tau对照组( n=5)、缺氧+ Tau组 (n=10)和缺氧组 (n=10)4组,用 Annexin V-FITC/PI双染色流式细胞术 (flow cytomeutry,FCM)、 Hoechst/PI荧光染色镜检判断细胞凋亡,同时测定培养液中 LDH含量.结果显示,早期凋亡细胞百分率缺氧组、缺氧+ Tau组分别为 24.03%± 0.60%、 22.85%± 0.95%,差异无统计学意义 (P>0.05),但均明显高于正常对照组 (3.57%± 0.26%,均 P<0.01);Annexin V+细胞分别为 39.33%± 0.72%、 34.49%± 1.71% (P>0.05),也均高于正常对照组 (6.89%± 0.17%,均 P<0.01);心肌细胞凋亡指数缺氧组、缺氧+ Tau组分别为 28.25± 2.2、 27.25± 2.2(P>0.05);细胞培养液中 LDH含量缺氧+ Tau组、缺氧组分别为( 42.82± 5.79) U/L、( 63.27± 10.89) U/L(P<0.01).提示 Tau能使心肌细胞 LDH漏出减少,对心肌损伤有保护作用,但 Tau对心肌细胞凋亡无抑制作用.     

脑氧利用率对新生儿缺氧缺血性脑病的预测作用

目的 探讨脑氧利用率(O2UCc)对新生儿缺氧缺血性脑病(HIE)的预测作用.方法 回顾性分析2007年3月至2009年2月我院收治的45例HIE新生儿的临床资料,按治疗结果分为痊愈组40例,后遗症组5例.对照组20例为健康足月儿.45例HIE患儿在入院治疗前24 h内和治疗第28天,对照组在出生后24 h内,均采血测定血氧饱和度,计算O2UCc.结果 治疗前HIE患儿O2UCc高于对照组、痊愈组、后遗症组(P＜0.01),后遗症组O2UCc低于痊愈组和对照组(P＜0.01),痊愈组O2UCc与对照组比较差异无统计学意义(P＞0.05).结论 O2UCc监测可作为HIE发病及判断预后的一项指标.     

Effects of omeprazole and gentamicin on the biochemical and histopathological alterations of the hypoxia/ reoxygenation induced intestinal injury in newborn rats

We utilized a newborn rat model of hypoxia/reoxygenation (H/R) that resembles human necrotizing enterocolitis (NEC) to investigate the effects of omeprazole and/or gentamicin on the formation of free oxygen radicals (FOR) and bowel histopathology. For H/R, 1-day-old rats were placed into a chamber of 100% CO₂ for 5 min, then they were reoxygenized for the next 5 min. The rats (n=70) were divided into seven groups: group 1 (control), group 2 (H/R), group 3 (omeprazole), group 4 (H/R+omeprazole), group 5 (gentamicin), group 6 (H/R+gentamicin), group 7 (H/R+omeprazole+gentamicin). Gentamicin and/or omeprazole were given orally for 3 days, then all animals were killed; bowel specimens were harvested. Histopathologic injury scores (HIS) and malonyldialdehyde (MDA) and XO/(XO+XDH) rates (XO; xanthine oxidase, XDH; xanthine dehydrogenase) were measured, which reflect the FOR levels. In group 2, the HIS was significantly higher than groups 4 and 6. The mean MDA values in groups 1–7 were as follows: 54.16, 104.2, 56.85, 63.43, 62.31, 76.85, 79.13, respectively. The mean XO/(XO+XDH) levels were 0.306, 0.461, 0.286, 0.335, 0.323, 0.410, 0.375 from groups 1 –7, respectively. Group 2 rats had significantly more MDA and XO/(XO+XDH) rates versus other groups (P<001). Histopathologic injury and biochemical results were significantly more severe in group 2 than in groups 4 and 6 (P<001). There was no difference between groups 1 and 4 according to XO/(XO+XDH) rates. In newborn rats, H/R produces FOR, which cause serious intestinal damage. Omeprazole and/or gentamicin reduce biochemical and histopathologic bowel damage. This effect was more obvious in omeprazole treated rats. We think omeprazole may open new insights into the treatment of H/R related bowel injuries like NEC.     

Salubrinal抑制脂多糖诱导H9C2心肌细胞凋亡的保护机制

目的 研究脓毒症中内质网应激诱导心肌细胞凋亡的机制.方法 建立脂多糖(lipopolysacchride,LPS)H9C2心肌细胞脓毒症模型.通过实时定量聚合酶链反应(RTq-PCR)和流式细胞技术分别观察LPS干预H9C2心肌细胞24 h后的促凋亡蛋白CHOP mRNA、线粒体膜电位(mitochondrial membrane potential,MMP)和细胞凋亡的变化.使用内质网应激抑制剂Salubrinal预处理H9C2心肌细胞,观察以上指标变化.结果 LPS组CHOP mRNA表达(2.40±0.30)与对照组比较(1.00±0)显著上调(P＜0.05),LPS组MMP(1.07 ±0.33)与对照组(2.62±0.51)比较显著下降(P＜0.05),LPS组细胞凋亡率[(16.58±2.71)％]与对照组[(3.85±1.07)％]比较显著上升(P＜0.05).Salubrinal+ LPS组CHOP mRNA表达(1.60±0.23)与LPS组(2.40±0.30)比较显著下调(P＜0.05),Salubrinal+ LPS组MMP(1.85 ±0.31)与LPS组比较,显著上调(P＜0.05);Salubrinal+ LPS组细胞凋亡率[(6.05±1.48)％]与LPS组比较,显著下调(P＜0.05).结论 在H9C2心肌细胞脓毒症模型中,Salubrinal通过抑制LPS诱导CHOP mRNA的表达,减轻线粒体的损伤,从而降低心肌细胞凋亡率.脓毒症心肌细胞中内质网应激可能通过线粒体途径诱导细胞凋亡.     

MMP-2在缺氧缺血/复氧再灌注损伤新生大鼠星形胶质细胞表达及FDP干预研究

目的 研究新生大鼠星形胶质细胞(AC)基质金属蛋白晦2(MMP-2)在缺氧缺血/复氧再灌注(复氧)损伤中的变化和1,6-二磷酸果糖(FDP)对AC缺氧/复氧损伤MMP-2表达的影响,为缺氧缺血性脑损伤的机制和治疗提供线索.方法 采用免疫激光共聚焦的方法检测体外培养的AC在缺氧/复氧不同时段和给予FDP干预时MMP-2的表达.结果 随着缺氧/复氧时间延长,MMP-2蛋白存AC的表达明显增高;FDP保护组及治疗组较相应时段缺氧复氧组MMP-2蛋白表达均明显降低.且随着缺氧/复氧时间延长,AC形态变化明显,主要表现为细胞间连接减少,足突减少;FDP保护12 h组及FDP治疗6 h组,AC形态变化明显减轻.结论 在缺氧/复氧损伤时,AC的MMP-2表达增高,AC间的相互连接破坏,影响AC正常形态,功能的维持.FDP可通过降低MMP-2的表达,以维持Ac问相互连接和AC的正常形态,减轻缺氧/复氧对AC的损伤.     

Protective effects of vitamin E and omeprazole on the hypoxia/reoxygenation induced intestinal injury in newborn rats

Evaluation of prophylactic effects of omeprazole and/or vitamin E on the formation of free oxygen radicals (FOR) and bowel histopathology in the newborn rat model of hypoxia/reoxygenation (H/R) that resembles human necrotizing enterocolitis (NEC). Eighty newborn rats were randomly divided into eight groups. H/R was done using airtight chamber. Rats were exposed to 100% CO(2) for 15 min followed by a reoxygenation for the next 15 min with 100% O(2). Group 1 (n = 10) was the control group. Group 2 (n = 10) rats received vitamin E. In Group 3 (n = 10) omeprazole was administrated. Group 4 (n = 10) rats received omeprazole and vitamin E. Group 5 (n = 10) rats were subjected to H/R two times for 2 days and one time for 3 days. Group 6 (n = 10) received vitamin E in addition to H/R for 5 days and in Group 7 (n = 10) omeprazole in addition to H/R for 5 days. In Group 8 (n = 10), vitamin E and omeprazole and H/R were applied for 5 days. Rats were killed at the end of the each process and bowel specimens were harvested for histopathological and biochemical investigations. We administrated vitamin E intramuscularly 300 unit/kg per day and omeprazole orally 20 mg/kg per day. Malondialdehyde (MDA), xanthine oxidase (XO), xanthine dehydogenase (XDH) and XO/(XO + XDH) were measured. Vitamin E and/or omeprazole treated rats had significantly less XO% levels than H/R only group (0.36, 0.38 and 0.57, respectively). Similarly, the MDA levels were significantly lower in vitamin E and/or omeprazole received rats than H/R only rats (88.8, 97.9 and 122.6, respectively). All rats treated with omeprazole and/or vitamin E had better biochemical and histopathological levels compared to H/R rats (p < 0.05). Histopathological results show that Group 5 (H/R only) had significantly more intestinal damage when compared with Group 6 (vitamin E + H/R), Group 7 (omeprazole + R/H) and Group 8 (vitamin E + omeprazole + H/R) (p < 0.001). Grade 2 and 3 intestinal damages were only in Group 5 and there were no statistical difference between in Groups 6, 7 and 8 (p > 0.001). Omeprazole and/or vitamin E may protect the biochemical and histopathological intestinal damage of H/R injury in rats. These drugs may be beneficial in the prophylaxis of NEC in humans as well.     

Impact of reoxygenation with oxygen and air on the extent of the brain damage after hypoxia-ischaemia in neonatal rats

Brain damage after hypoxia-ischaemia develops partly during the state of reoxygenation. The generation of free oxygen radicals is considered to be one possible mechanism. In order to evaluate the role of hyperoxygenation, a comparison was made between reoxygenation with pure oxygen and with air after hypoxia-ischaemia in a rat model of unilateral cerebral hemisphere damage. Brain damage was induced in 7-day-old rats. The animals were treated during reoxygenation with either 100% oxygen for 0.5 h or air. The extent of the brain damage was determined at 3 weeks of age by weighing the left and right hemispheres separately. No significant difference in weight deficit of the hemispheres was seen in the oxygen-treated group (15.5%, median) compared to the air-treated group (25.0%). Reoxygenation with pure oxygen after hypoxia-ischaemia in neonatal rats does not cause increased brain damage compared with reoxygenation with room air.     

肾上腺素对内毒素致大鼠肠道损害的保护作用

目的 探讨肾上腺素对内毒素(lipopolysaccharide,LPS)所致大鼠肠道损害的保护作用及其作用机制.方法 50只SD大鼠随机分为5组(每组10只):对照组:大鼠静脉输注0.9％生理盐水24 ml/(kg·h);LPS组:大鼠静脉注射LPS 6m/kg后,静脉输注生理盐水2.4 ml/(kg·h);低、中和高剂量肾上腺素组:大鼠静脉注射LPS 6 mg/kg后,分别静脉输注肾上腺素0.12 μg/(kg·min)、0.3μg/(kg·min)和0.6μg(kg· min).在LPS注射前、注射后2h和注射后6h3个时点取血,检测血清肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β和IL-10水平,并在24h观察肠道的组织病理学变化.结果 LPS组大鼠在LPS注射后2h血清TNF-α浓度为(1164±145) ng/L,IL-1β浓度为(521±68)ng/L,IL-10浓度为(303±20) ng/L,较对照组均明显升高(P＜0.05).病理结果显示,LPS组小肠黏膜充血、水肿、出血及炎症细胞浸润,上皮细胞坏死、脱落.高剂量肾上腺素组2h血清TNF-α浓度为(576±105) ng/L,2h和6h的IL-10浓度分别为(424±29) ng/L和(24.5±14) ng/L,与LPS组相比血清TNF-α水平明显降低(P＜0.05)且IL-10水平明显升高(P＜0.05),但IL-1β水平无明显变化(P＞0.05).高剂量肾上腺素组大鼠肠道病理损伤明显减轻.中、低剂量肾上腺素组各时间点血清细胞因子水平与LPS组比较,差异无统计学意义(P＞0.05),未见肠道病理损害减轻.结论 肾上腺素可以通过抗炎作用减轻LPS素诱导的肠道损害.