小鼠神经管发生中程序性细胞死亡的时空规律

目的：研究小鼠神经管发生中程序性细胞死亡(programmed cell death．PCD)的时空规律。方法：分别取交配后9．0d、交配后9．5d、交配后10．5d鼠胚做石蜡包埋连续切片的TUNEL染色。进行神经上皮凋亡细胞的计数分析。结果：观察到交配后9．0d神经上皮的细胞凋亡率(0．1815&;#177;0．14)最高，交配后9．5d后细胞凋亡率(0．1377&;#177;0．09)降低，交配后10．5d(0．1284&;#177;0．07)降至更低。从空间分布看，神经管头端的凋亡细胞明显多于尾端。结论：交配后9．0d神经管的高细胞凋亡率，可能与神经管的关闭有关；神经管颅侧的细胞死亡与脑泡的塑形有关。     

小鼠神经管发生中抑凋亡基因bcl-2的表达及其对细胞生长的作用

目的:研究小鼠神经管形成过程中原癌基因bcl-2表达及与程序性细胞死亡(programedcelldeath,PCD)的关系。方法:分别取交配后7.5,8.5,9.0,9.5,10.5d鼠胚做全胚胎交实验。结果:交配后7.5d鼠胚,Bcl-2mRNA杂交信号弥散分布于整个胚体,但胚体前1/2的杂交信号较弱,后1/2则相对较强。交配后8.5d鼠胚Bcl-2mRNA有较强的杂交信号沿逐渐升高的神经褶分布(权重为19)。到交配后9.0d,随着神经管的关闭,Bcl-2mRNA在神经褶的表达下降(权重为8)。到交配后9.5dBcl-2mRNA的表达开始回升(权重为14),到交配后10.5dBcl-2mRNA的表达在胚胎颅神经管升高(权重为19)。结论:Bcl-2在早期神经管发生中的作用是多方面的,参与对细胞凋亡的调节和促进细胞的生长和分化。     

小鼠神经管发生中抑凋亡基因bcl-2的表达及其对细胞生长的作用

目的：研究小鼠神经管形成过程中原癌基因bcl-2表达及与程序性细胞死亡(programed cell death，PCD)的关系。方法：分别取交配后7．5，8．5，9．0，9．5，10．5d鼠胚做全胚胎交实验。结果：交配后7．5d鼠胚，Bcl-2mRNA杂交信号弥散分布于整个胚体，但胚体前1／2的杂交信号较弱，后1／2则相对较强。交配后8．5d鼠胚Bcl-2mRNA有较强的杂交信号沿逐渐升高的神经褶分布(权重为19)。到交配后9．0d，随着神经管的关闭，Bcl-2mRNA在神经褶的表达下降(权重为8)。到交配后9．5dBcl-2mRNA的表达开始回升(权重为14)，到交配后10．5dBcl-2mRNA的表达在胚胎颅神经管升高(权重为19)。结论：Bcl-2在早期神经管发生中的作用是多方面的，参与对细胞凋亡的调节和促进细胞的生长和分化。     

中孕早期胎儿神经管畸形的超声筛查

目的：探讨中孕早期胎儿神经管畸形的超声诊断价值。方法：对深圳市福田妇幼保健院2006-01/2008-12期间5421例中孕早期（14-18孕周）胎儿的产前超声筛查的相关资料进行统计分析。结果：5421例中孕早期孕妇经超声筛查检出胎儿神经管畸形10例,检出率为91%,并经引产所证实,阳性率为0.184%。结论：超声筛查对诊断中孕早期胎儿神经管畸形有重要价值,对优生优育具有重要的意义。     

正常神经管发育和神经管缺陷中转染重组bcl-2的作用

目的:探讨bcl-2在正常神经管发育和异常神经管缺陷中的作用。方法:构建了重组bcl-2的真核表达载体,建立了维A酸致小鼠神经管缺陷(neuraltubedefects,NTD)模型。用全胚胎培养结合显微注射技术将重组的正义bcl-2表达质粒导入NTD鼠胚后,观察神经管缺陷的改善程度。结果:经转染bcl-2基因后,前脑的关闭率由原来的34%增至80%,中脑的关闭率由原来的17%增至70%,后脑的关闭率也由原来的25%增至增加了70%,脊神经管的关闭率达到100%。结论:RA介导的神经管缺陷可部分被重组的bcl-2基因缓解。     

帕金森病中多巴胺能神经元的程序性细胞死亡机制

帕金森病(PD)是常见的神经退行性疾病,其病理基础是黑质多巴胺能神经元的变性死亡。近年来研究发现PD中多巴胺能神经元变性死亡主要是程序性细胞死亡,涉及细胞凋亡、自噬样细胞死亡、程序性细胞坏死等多种类型。本文将对PD中多巴胺能神经元的程序性细胞死亡机制进行综述,以期更好的认识PD的发病机制和干预靶点。     

过氧化氢诱导C2C12细胞凋亡的机制初探

[目的]探讨氧化应激诱导小鼠胚胎肌原细胞株C2C12细胞凋亡的分子机制。[方法]采用0．5mmol/L过氧化氢(hydrogen peroxide．H2O2)作用于C2C12细胞；通过Hoechst荧光染色检测C2C12细胞凋亡，Caspase活性定量分析及Western-blot检测Caspase-3，-8，-9活化情况，Western-blot及间接免疫荧光检测细胞色素C在细胞内的分布情况。[结果]本实验发现H2O2能明显诱导C2C12细胞凋亡，同时Caspase-3，-8，-9被激活．而细胞色素C从线粒体释放入胞浆。[结论]氧化应激可通过同时激活线粒体通路与死亡受体通路导致C2C12细胞凋亡．这为临床防治凋亡相关的心血管疾病提供了新的信息。     

超声在中孕早期胎儿神经管畸形免费筛查中的应用

目的:探讨超声筛查中孕早期胎儿神经管畸形的价值.方法:回顾性分析我院2006年1月至2008年12月期间,对中孕早期即14～18孕周胎儿进行神经管畸形的产前超声筛查的相关资料.结果:5 421例中孕早期孕妇经超声筛查检出胎儿神经管畸形10例,并经引产所证实,其中无脑儿4例,露脑畸形2例,脑积水1例,脊柱裂1例,脑膜脑膨出合并足内翻1例,无脑儿合并脐膨出1例,漏诊1例,检出率为91%,漏诊率为9%.结论:超声免费筛查对诊断中孕早期胎儿神经管畸形有重要价值,对优生优育具有重要的意义.     

扶正抑瘤颗粒对小鼠肿瘤细胞凋亡和细胞周期的影响

目的：研究扶正抑瘤颗粒对小鼠移植性肿瘤S180的抑制作用及对细胞凋亡和细胞周期的影响。方法：用小鼠体内肿瘤试验观察扶正抑瘤颗粒对S180肉瘤的肿瘤抑制率，通过流式细胞术和末端脱氧核苷酸转移酶介导的缺口末端标记(TUNEL)法测定细胞凋亡率、凋亡指数，分析细胞周期。结果：扶正抑瘤颗粒显著抑制S180生长，最高肿瘤抑制率为56．04％(P&;lt;0．01)，显著提高S180肉瘤的细胞凋亡率，最高凋亡率为18．37％(P&;lt;0．001)，细胞周期分析表明扶正抑瘤颗粒将肿瘤细胞阻滞于Go／G1期而降低S期比率。TUNEL检测证实扶正抑瘤颗粒明显提高S180肉瘤的细胞凋亡指数(P&;lt;0．001)。结论：扶正抑瘤颗粒具有明显的体内抗肿瘤活性，其抗肿瘤作用与调控肿瘤细胞周期和诱导细胞凋亡有关。     

"斯利安"片预防胎儿神经管畸形效果观察

目的探讨孕前服用“斯利安”片对降低神经管畸形的效果。方法对3年间出生缺陷病例进行回顾性临床分析。结果3年间出生缺陷平均发生率为21．84‰,其中神经管畸形和先天性心脏病的发生率为5．72‰,早产儿、低出生体重儿的发生率为6．24‰,窒息、疾病感染的发生率为8．84‰。结论出生缺陷儿的发生与季节、营养、感染、环境及遗传因素有关,其防治应以预防为主,特别是在孕前普服“斯利安”片,可降低神经管畸形的发生率。     

Caspase-9 takes part in programmed cell death in developing mouse kidney

Programmed cell death is a mechanism by which organisms dispose of unwanted cells, and it is thought to be an important process in organogenesis. We have already reported the role of caspase-3 in the developing metanephros. While caspase-3 is thought to be positioned downstream of the caspase-activating cascade, the upstream caspase for programmed cell death in the developing kidney is still unknown. In an attempt to identify it, we blocked caspase activity in metanephric explants with caspase inhibitors. Administration of a caspase-9 inhibitor (Ac-IETD-CHO) effectively prevented both ureteric bud branching and nephrogenesis, the same as a caspase-3 inhibitor (Ac-DEVD-CHO). On the other hand, administration of a caspase-8 inhibitor (Ac-LETD-CHO) did not inhibit ureteric bud branching or nephrogenesis. Apaf-1, which executes programmed cell death in the caspase-9-related pathway, was detected in the cells exhibiting caspase-9 activity, and our results suggest that Apaaf-1/caspase-9 activates caspase-3 in kidney organogenesis.     

细胞凋亡在帕金森病发病机制中的作用

目的研究帕金森病(PD)病因中细胞凋亡的发生及作用机制。方法应用6－OHDA毁损大鼠纹状体,以制备PD大鼠模型,应用酪氨酸羟化酶(TH)和DNA原位末端标记免疫组化双标染色检测黑质内多巴胺(DA)神经元细胞凋亡的发生及其变化规律。结果成功复制出符合临床特点的PD大鼠模型,其黑质内DA神经元的丢失是以细胞凋亡为主要形式,且于术后2周及1月为最明显,术后2个月仍然存在细胞凋亡情况。结论黑质内DA神经元细胞凋亡的发生将使其数目减少,从而导致帕金森病的发生,其机制可能与纹状体区病变有关。     

Stretch-induced programmed myocyte cell death.

To determine the effects of loading on active and passive tensions, programmed cell death, superoxide anion formation, the expression of Fas on myocytes, and side-to-side slippage of myocytes, papillary muscles were exposed to 7-8 and 50 mN/mm2 and these parameters were measured over a 3-h period. Overstretching produced a 21- and a 2.4-fold increase in apoptotic myocyte and nonmyocyte cell death, respectively. Concurrently, the generation of reactive oxygen species increased 2.4-fold and the number of myocytes labeled by Fas protein 21-fold. Moreover, a 15% decrease in the number of myocytes included in the thickness of the papillary muscle was found in combination with a 7% decrease in sarcomere length and the inability of muscles to maintain stable levels of passive and active tensions. The addition of the NO-releasing drug, C87-3754, prevented superoxide anion formation, programmed cell death, and the alterations in active and passive tensions with time of overloaded papillary muscles. In conclusion, overstretching appears to be coupled with oxidant stress, expression of Fas, programmed cell death, architectural rearrangement of myocytes, and impairment in force development of the myocardium.     

The machinery of programmed cell death.

Apoptosis or programmed cell death is an essential physiological process that plays a critical role in development and tissue homeostasis. However, apoptosis is also involved in a wide range of pathological conditions. Apoptotic cells may be characterized by specific morphological and biochemical changes, including cell shrinkage, chromatin condensation, and internucleosomal cleavage of genomic DNA. At the molecular level, apoptosis is tightly regulated and is mainly orchestrated by the activation of the aspartate-specific cysteine protease (caspase) cascade. There are two main pathways leading to the activation of caspases. The first of these depends upon the participation of mitochondria (receptor-independent) and the second involves the interaction of a death receptor with its ligand. Pro- and anti-apoptotic members of the Bcl-2 family regulate the mitochondrial pathway. Cellular stress induces pro-apoptotic Bcl-2 family members to translocate from the cytosol to the mitochondria, where they induce the release of cytochrome c, while the anti-apoptotic Bcl-2 proteins work to prevent cytochrome c release from mitochondria, and thereby preserve cell survival. Once in the cytoplasm, cytochrome c catalyzes the oligomerization of apoptotic protease activating factor-1, thereby promoting the activation of procaspase-9, which then activates procaspase-3. Alternatively, ligation of death receptors, like the tumor necrosis factor receptor-1 and the Fas receptor, causes the activation of procaspase-8. The mature caspase may now either directly activate procaspase-3 or cleave the pro-apoptotic Bcl-2 homology 3-only protein Bid, which then subsequently induces cytochrome c release. Nevertheless, the end result of either pathway is caspase activation and the cleavage of specific cellular substrates, resulting in the morphological and biochemical changes associated with the apoptotic phenotype.     

Targeting the programmed cell death 1: programmed cell death ligand 1 pathway reverses T cell exhaustion in patients with sepsis

Introduction

A major pathophysiologic mechanism in sepsis is impaired host immunity which results in failure to eradicate invading pathogens and increased susceptibility to secondary infections. Although many immunosuppressive mechanisms exist, increased expression of the inhibitory receptor programmed cell death 1 (PD-1) and its ligand (PD-L1) are thought to play key roles. The newly recognized phenomenon of T cell exhaustion is mediated in part by PD-1 effects on T cells. This study tested the ability of anti-PD-1 and anti-PD-L1 antibodies to prevent apoptosis and improve lymphocyte function in septic patients.

Methods

Blood was obtained from 43 septic and 15 non-septic critically-ill patients. Effects of anti-PD-1, anti-PD-L1, or isotype-control antibody on lymphocyte apoptosis and interferon gamma (IFN-γ) and interleukin-2 (IL-2) production were quantitated by flow cytometry.

Results

Lymphocytes from septic patients produced decreased IFN-γ and IL-2 and had increased CD8 T cell expression of PD-1 and decreased PD-L1 expression compared to non-septic patients (P<0.05). Monocytes from septic patients had increased PD-L1 and decreased HLA-DR expression compared to non-septic patients (P<0.01). CD8 T cell expression of PD-1 increased over time in ICU as PD-L1, IFN-γ, and IL2 decreased. In addition, donors with the highest CD8 PD-1 expression together with the lowest CD8 PD-L1 expression also had lower levels of HLA-DR expression in monocytes, and an increased rate of secondary infections, suggestive of a more immune exhausted phenotype. Treatment of cells from septic patients with anti-PD-1 or anti-PD-L1 antibody decreased apoptosis and increased IFN-γ and IL-2 production in septic patients; (P<0.01). The percentage of CD4 T cells that were PD-1 positive correlated with the degree of cellular apoptosis (P<0.01).

Conclusions

In vitro blockade of the PD-1:PD-L1 pathway decreases apoptosis and improves immune cell function in septic patients. The current results together with multiple positive studies of anti-PD-1 and anti-PD-L1 in animal models of bacterial and fungal infections and the relative safety profile of anti-PD-1/anti-PD-L1 in human oncology trials to date strongly support the initiation of clinical trials testing these antibodies in sepsis, a disorder with a high mortality.     

Western印迹技术研究神经管发育和神经管畸形鼠胚Bcl-2蛋白的表达

目的:探讨转染外源性bcl-2基因后,能否通过Bcl-2蛋白表达抑制凋亡,从而减轻和改善鼠胚神经管缺陷的程度。方法:分别取维甲酸致神经管缺陷鼠胚、转染正义bcl-2DNA的神经管缺陷鼠胚、正常鼠胚、转染反义bcl-2DNA的正常鼠胚神经管组织,用Western免疫印迹实验技术检测Bcl-2蛋白和Bax蛋白的表达情况。结果:经转染外源性bcl-2后,神经管缺陷鼠胚的Bcl-2蛋白表达升高,神经管缺陷程度得到改善。结论:维甲酸对内源性bcl-2的抑制包括了mRNA水平和蛋白水平。本实验从蛋白表达水平证实了外源性bcl-2转染成功并有表达。