低温液体灌注产生亚低温的进展

低温对脑损伤的保护作用已被众多动物实验所证实,在临床方面,尤其是发现轻度和中度低温的保护作用之后,应用逐渐增多.近年来,亚低温被用于治疗新生儿缺氧性脑病、心肺复苏后脑保护、脑外伤及脑卒中等领域.目前临床及实验中所应用的低温方式主要为全身亚低温,如降温毯、冰袋等,这些方式降温效率低,不易迅速实现亚低温,而且全身低温可伴有多种并发症,如心律紊乱、凝血功能障碍等.低温液体或自体血液灌注是目前最有效的降温方式,而且局部动脉内灌注低温液体更可以快速实现局部亚低温.介入技术的进步使得快速局部动脉内低温液体灌注成为可能.我们详细回顾相关动物实验及临床应用文献,介绍低温液体灌注产生亚低温的研究进展.     

颅脑外伤后亚低温治疗对Calpain表达的影响

目的 探讨亚低温治疗对颅脑外伤后Calpain基因和蛋白表达的影响.方法 将27只SD大鼠按随机数字表法分为正常对照组、常温脑损伤组和亚低温脑损伤组.脑损伤组分别接受右顶部侧方中度液压损伤打击,其中亚低温组在伤后即刻接受持续4 h的亚低温治疗[肛温(32±0.5)℃].在伤后6,12,24和72 h四个时间点分别通过荧光PCR、Western blot定量检测各组打击伤灶周围皮层钙蛋白酶Calpain基因转录和蛋白分子表达的水平,同时运用免疫荧光观察Calpain蛋白分布情况.结果 与常温组比较,亚低温使颅脑外伤后12 h及24 h Calpain基因的转录较正常对照和同时间点常温脑损伤组显著增加(P<0.05).然而,伤后6,12,24和72 h亚低温治疗均使Calpain在蛋白水平较同时间点常温脑损伤组表达升高的程度显著抑制(P<0.05).结论颅脑外伤后亚低温治疗的脑保护机制可能与其在Calpain基因转录后明显抑制Calpain蛋白水平的表达有关.     

脑源性神经营养因子对亚低温处理后移植干细胞的存活及分化的影响

目的 研究脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)对亚低温处理后移植干细胞的存活及分化的影响.方法 脂质体介导BDNF表达质粒转染293T细胞,ELISA法确定BDNF高峰表达时相,收集高表达时相阶段细胞上清液.建立SD大鼠液压脑创伤模型,分为A组(干细胞移植组)和B组(干细胞移植+BDNF组),两组同时给予亚低温治疗(33℃)5 d.对第4天和第8大(复温后第3天)脑组织标本行增殖细胞核抗原(PCNA)、巢蛋白(nestin)、神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)的免疫组织化学及原位细胞凋亡检测,实验动物予以神经功能缺陷评分.结果 ELISA结果显示细胞转染48～60 h后BDNF持续高表达.亚低温处理第4天两组PCNA、巢蛋白均高表达,NSE及GFAP无或极低表达,细胞凋亡少见,但B组较A组更少(P＜0.05).复温后第3天两组PCNA、巢蛋白均表达下降,NSE及GFAP表达升高,但B组较A组NSE阳性率更高,细胞凋亡更少(P＜0.05).B组获得最佳动物神经功能评分.结论 BDNF可增加低温环境下的干细胞存活率,并诱导干细胞向神经元分化.

Abstract：

Objective To study the effect of brain-derived neurotrophic factor (BDNF) on the environmental nutrition and neural differentiation of the transplanted stem cells under hypothermia.Methods The BDNF gene mediated by liposome was transfected into 293T cell line, and ELISA assay was applied to find the peak time of BDNF expression. When BDNF was highly expressed, the supernatant was collected for establishment of SD rat models of brain injury. The rats were divided into Group A (stem cell transplantation group) and Group B (stem cell transplantation and BDNF group). Rats in both groups were under hypothermia treatment for five days. Four and eight days later ( three days from rewarming), rat brain tissues were obtained to detect the expressions of proliferating cell nuclear antigen (PCNA), nestin, neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) by immunohistochemical method and to detect the apoptosis by in situ hybridization. Finally, the nerve function scores were obtained for evaluation of the nerve function. Results The ELISA showed that the high level of BDNF expression was at 48 to 60 hours after gene transfection. PCNA and nestin were highly expressed, while NES and GFAP showed nil or low level of expression in both groups at the fourth day after hypothermia, with little apoptotic cells especially in the Group B (P ＜0.05). The expressions of PCNA and nestin were decreased, but the expressions of NSE and GFAP were increased at the third day after rewarming. The positive rate of NSE expression in the Group B was much higher and the apoptotic cells were much less compared with the Group A ( P ＜ 0. 05 ). A better nerve score was obtained in the Group B. Conclusion BDNF can enhance the survival rate of the transplanted stem cells and induce their differentiation into neurons under hypothermia.     

亚低温对创伤性脑损伤后线粒体呼吸功能和超微结构的影响

目的 研究亚低温治疗对创伤性脑损伤(TBI)大鼠线粒体超微结构和呼吸功能的影响.方法 利用液压打击(FPI)制作中度TBI大鼠模型,并随机分为假手术对照组、常温TBI组(肛温36～37℃)、亚低温TBI组(肛温31～33℃,低温持续2 h).各组于脑外伤后2,24 h、3 d和7 d断头取脑,取伤侧海马组织固定,透射电镜观察线粒体形态改变.差速离心法提取伤侧大脑线粒体,采用Clark氧电极法测定线粒体的氧呼吸速率,计算呼吸控制比(RCR)值和磷氧比值(P/O).结果 (1)电镜下常温TBI组线粒体结构形态损伤程度较重,亚低温TBI组线粒体形态基本完好;(2)线粒体RCR值和P/O值在TBI后2 h就显著下降,以24 h为最低(P＜0.01),在7 d时RCR仍然显著低于假手术对照组,P/O值恢复正常.亚低温TBI组线粒体RCR值变化趋势同常温TBI组,但在3 d内RCR值均显著高于常温TBI组,P/O值在3 d后恢复正常.结论 TBI后,线粒体的呼吸功能明显下降,亚低温可以明显改善线粒体的呼吸功能,保护线粒体结构完整性.     

The Influence of Hypothermia Involving Minimal Hypoxia on the Radiosensitivity of Leucocytes in the Rat

Summary

Rats were irradiated with 357 rads of gamma-rays, some at normal body-temperatures and some at body-temperatures of less than 5°c. The method of induction of hypothermia was designed to produce minimal anoxia. White-cell counts were performed up to seventeen days after irradiation. Depression of the lymphocyte count was seen in all animals irradiated, recovery being nearly complete at the end of the study. The depression observed was similar in the cooled and uncooled groups, apart from the values obtained two days after irradiation. It is concluded that hypothermic radioprotection of the lymphopoietic tissues is dependent on anoxia.     

基因芯片在同型半胱氨酸诱导动脉粥样硬化基因表达谱变化中的应用

目的　应用基因芯片技术研究同型半胱氨酸诱导动脉粥样硬化的基因表达谱变化。方法　选取 3例无冠心病传统危险因素的住院患者 ,其中 1例为血同型半胱氨酸正常的冠状动脉造影正常者 ,1例为血同型半胱氨酸正常的冠心病患者 ,1例为高同型半胱氨酸血症的冠心病患者。分离外周血淋巴细胞并抽提总RNA ,逆转录制备杂交探针 ,应用含有 115 2条基因的人类表达谱芯片进行差异表达谱分析。结果　高同型半胱氨酸血症的冠心病患者与血同型半胱氨酸正常的冠状动脉造影正常者比较差异表达的基因 177条 (组 1) ,高同型半胱氨酸血症的冠心病患者与血同型半胱氨酸正常的冠心病患者比较差异表达的基因有 15 1条 (组 3)。组 1与组 3基因芯片平行比较 ,两者共同的差异表达基因 4 8条 ,其中上调基因 2 3条 ,下调基因 2 5条。结论　在差异表达的基因中 ,有些是凋亡、信号转导、免疫、蛋白质合成及原癌基因等的相关基因 ,这些基因可能与同型半胱氨酸致动脉粥样硬化发生有关。应用基因芯片技术研究同型半胱氨酸诱导动脉粥样硬化差异表达谱 ,可能为动脉粥样硬化的发病机制研究提供新的思路     

Expression of Hsp27 and Hsp70 and vacuolization in the pituitary glands in cases of fatal hypothermia

Hypothermia causes systemic cellular stress. The pituitary gland is an endocrine gland and plays an important role in thermoregulation. When the core body temperature drops, the pituitary gland is activated by stimulation of hypothalamic hormones. In this study, we investigated morphological alterations of the pituitary gland in cases of fatal hypothermia. Several morphological alterations of the anterior lobe of the pituitary gland, such as hemorrhage, vacuolization, and hyperemia, have been previously described in fatal hypothermia. However, the diagnostic value of these findings is controversial. We compared 11 cases of fatal hypothermia with 10 cases lacking antemortem hypothermic influences. In the presence of thermal cellular stress, the expression of heat shock proteins increases to protect cellular structures. Therefore, we immunohistochemically analyzed Hsp27 and Hsp70. Hsp27 expression was detected in 27.3% of the cases of fatal hypothermia and in 10.0% of the control cases, whereas Hsp70 expression was not detected in any case. Additionally, Sudan staining was performed to quantify fatty degeneration. A positive reaction was found in 45.5% of the study group and in 10.0% of the control group. This indicates that fatty degeneration might be a valuable marker when other macroscopic signs of hypothermia are absent.     

纤溶酶原激活抑制因子1基因在人脑星形细胞瘤中的表达及意义

采用Ｎｏｒｔｈｅｒｎ ｂｌｏｔ分子杂交和ＡＢＣ免疫组化方法检测３６例人脑星形细胞瘤纤溶酶原激活抑制因１基因ｍＲＮＡ和蛋白表达，所有星形细胞瘤组织均可表达３．０ｋｂ和２．２ｋｂ的ＰＡＩ－１ ｍＲＮＡ转录物，高级别星形细胞瘤ＰＡＩ－１ ｍＲＮＡ表达水平显著高于低级别星形细胞瘤，ＰＡＩ－１display status     

基质金属蛋白酶-2,9及其抑制因子在增生性瘢痕中的表达特征及意义

 目的 探讨3种不同的基质金属蛋白酶-2, 9(MMP-2, 9)及其抑制因子(TIMP-2)在不同形成时期的增生性瘢痕中的基因表达和蛋白含量的变化.方法 用病理学技术检测增生性瘢痕和正常皮肤的结构特征后,分别用RT-PCR和Western blot方法检测这3种基因在16例不同发生时期的增生性瘢痕和8例正常皮肤组织中的基因表达和蛋白含量的变化规律.结果 在正常皮肤组织内,MMP-2, 9和TIMP-2的基因表达较弱,蛋白含量较低;而在增殖期的增生性瘢痕中这2种MMPs和TIMP2基因表达水平和蛋白含量都显著升高(P＜0.05),在成熟期的瘢痕中,MMP-2, 9基因表达量降低至正常皮肤水平,但蛋白含量仍保持较高水平,明显高于正常皮肤(P＜0.05),TIMP-2的mRNA水平和蛋白含量则降低至正常皮肤相近的水平.结论 MMP-2, 9和TIMP-2表达增强可能是增生性瘢痕形成的机制之一,而MMP-2, 9蛋白含量的增多,TIMP-2蛋白含量减少可能与增生性瘢痕达到相对稳定的成熟期有关.     

内毒素诱导小鼠急性肺损伤早期基因表达谱的研究

目的 利用改良基因表达的综合分析(serial analysis of gene expression,SAGE)技术检测内毒素诱导的小鼠急性肺损伤早期的基因表达状况,探讨急性肺损伤的分子机制.方法 气管内注射内毒素(25 mg/kg)复制急性肺损伤模型.正常对照动物气管内注射等体积等渗盐水.内毒素注射6小时后以氯胺酮深麻醉处死动物,整块取出肺脏用于病理、SAGE及逆转录-聚合酶链式反应(RT-PCR)研究.建立正常和急性肺损伤SAGE标记物文库.结果 正常文库包括24 670个SAGE标记物,代表12 168个基因.急性肺损伤文库包括23 346个SAGE标记物,代表12 434个基因.急性肺损伤组有8种基因升高＞10倍,152种基因升高5～10倍,1 187种基因升高2～5倍;4种基因表达降低＞10倍,75种基因降低5～10倍,1 643种基因降低2～5倍.RT-PCR法进一步证实TNFα、IL-1β、IL-6等细胞因子表达明显增高.结论 证实了内毒素诱导的急性肺损伤早期发生机制中各种基因表达的变化,尤其是细胞因子表达的变化,并发现了以往没有报道的基因.细胞因子表达的变化及其引发的全身炎症反应是急性肺损伤早期的重要发生机制.白蛋白等代表机体防御能力的指标的下降提示机体防御能力的变化也在急性肺损伤的发生发展过程中起重要作用.     

常温体外循环在先心病直视手术中的应用

目的探讨全身常温、心脏局部低温体外循环(CPB)在先天性心脏病直视手术治疗中的应用价值。方法将20例先天性心脏病手术患者随机分为常温组(10例)和低温组(10例),对比研究两组CPB时间、主动脉阻断时间、手术时间、术后体温、胸腔引流量、血细胞水平等检测指标间的差别。结果常温组CPB时间较低温组短[(37.8±11.5)m in与(50.6±11.9)m in],P<0.05,手术时间也较低温组明显缩短[(2.3±0.5)h与(2.8±0.4)h],P<0.01,两组患者术后总引流量,术后前3 h胸腔引流量和血细胞水平比较差别无统计学意义。结论全身常温、心脏局部低温体外循环明显缩短了手术的时间,具有临床应用价值。     

Accidental fatal hypothermia in elderly people with Alzheimer's disease

We report two forensic autopsy cases of fatal accidental hypothermia in an 89-year-old woman and a 76-year-old man who were found dead and unclothed. In both cases, Alzheimer's disease (AD) was diagnosed by neuropathological examination. Wandering due to AD was determined as the cause of these accidents. Although paradoxical undressing in hypothermic victims is known to occur as a result of cold exposure, in our patients, undressing was attributed to dementia due to AD before they became hypothermic. These cases indicate that neuropathological examination is crucial to determining the cause of such accidents and that undressing is not always the result of hypothermia in elderly victims.     

轻度低温联合低压复苏对非控制出血性休克大鼠的保护作用及与线粒体功能改善的关系

目的 观察轻度低温联合低压复苏对非控制出血性休克大鼠的保护作用及对线粒体功能的影响.方法 通过断脾复制非控制性出血性大鼠休克模型(时相Ⅰ),以乳酸林格液(LR)和羟乙基淀粉(HES)(体积比2:1)在37℃和32℃分别进行维持50mmHg 1小时的低压复苏和维持80mmHg 1小时的常压复苏(时相Ⅱ),彻底止血后用全血...     

Plasma and urine catecholamines and cerebrospinal fluid amine metabolites as hypothermia markers in guinea-pigs

Changes in catecholamines (CA) in the plasma and urine and metabolites of CA and serotonin (5-HT) in the cerebrospinal fluid of guinea-pigs in hypothermia (Trec 30 degrees C) and after subsequent rewarming were determined with HPLC in order to obtain data on early stress reactions and their timing. Both noradrenaline (NA) and adrenaline (A) were low in the plasma but high in the urine after the hypothermic period. These had normalized in the plasma after rewarming but were still high in the urine. Dopamine values tended to be low (not significant). Methoxyhydroxyphenylglycol and homovanilic acid were elevated in the cerebrospinal fluid both after hypothermia and following rewarming, and 5-hydroxyindoleacetic acid after rewarming. The ratio of adrenaline to noradrenaline, the catecholamine hypothermia index, in the urine had risen 24-fold after hypothermia and 40-fold after rewarming. The results support the view that elevated catecholamine concentrations in the urine and elevated values of their metabolites in the cerebrospinal fluid could be regarded as hypothermia markers. However, other stress conditions, which have lasted at least a few hours, should be excluded in the final interpretation.     

Effect of temperature in focal ischemia of rat brain studied by 31P and 1H spectroscopic imaging

³¹P, ¹H and lactate spectroscopic imaging was used to evaluate the effects of hypothermia on focal cerebral ischemia produced by middle cerebral artery occlusion. The effects on high energy phosphate metabolism, pH, lactate and NAA were investigated in 24 spontaneously hypertensive rats subjected to either permanent or transient ischemia. Under either normothermic (37.5°C) or hypothermic (32°C) conditions, with permanent 6-h occlusion, there was little difference between groups in either the NMR measurements or the volume of infarction. In animals that underwent 3 h of ischemia followed by 12 h of reperfusion, the ischemic changes in lactate, pH, NAA, and high-energy phosphate returned toward control values, and there was a protective effect of hypothermia (infarct volume of 211 ± 26 and 40 ± 14 mm³ in normothermic and hypothermic groups, respectively). Thus, hypothermia did not ameliorate the changes in lactate, pH, NAA, or high energy phosphate levels occurring during ischemia, however, during reperfusion there was an improvement in both the recovery of these metabolites and pathological outcome in hypothermic compared with normothermic animals.