同步辐射显微红外光谱研究6-OHDA诱导帕金森病细胞模型

神经毒素6-hydroxydopamine (6-OHDA)处理的人神经瘤母细胞系SH-SY5Y是一种经典的帕金森病细胞模型。利用同步辐射红外显微光谱研究6-OHDA处理的SH-SY5Y细胞系的生物化学成分。与正常细胞相比较,所含磷脂的平均饱和水平显著提高,蛋白质的二级结构发生显著变化,其中的β-sheet的比例明显增高,核酸的含量明显下降,提示神经毒素6-OHDA对细胞造成了严重的氧化损伤。同步辐射红外显微光谱能够精确有效的检测到细胞生化成分的改变,从而对细胞的病理损伤进行评估。     

超声空化增强川芎嗪对脑缺血再灌注损伤保护作用机制的研究

川芎的生物活性成分(川芎嗪)已广泛应用于治疗心脑血管疾病。基于谷氨酸诱导PC12细胞损伤建立脑缺血再灌注损伤的细胞模型,探讨超声增强川芎嗪对谷氨酸损伤PC12细胞的保护作用机制。研究结果表明,超声能进一步加强川芎嗪对细胞的保护,其主要作用机制为:(1)抑制氧化应激和细胞凋亡相关的Bcl-2蛋白和Bax蛋白的变化从而达到抗凋亡的效果;(2)降低炎症因子(TNF-α和IL-8)的表达,减轻炎症反应损伤;(3)适当的声压可以增强川芎嗪对谷氨酸损伤PC12细胞的保护作用,但过高的声压会引起细胞损伤,导致细胞凋亡。本文的工作表明超声能够增强川芎嗪对脑缺血再灌注损伤的保护作用,为临床脑缺血再灌注损伤的治疗提供了新治疗策略。      

同步辐射显微红外光谱研究6-OHDA诱导帕金森病大鼠海马

利用同步辐射红外显微光谱研究6-hydroxydopamine(6-OHDA)毁损内侧前脑束所致帕金森病大鼠脑海马区神经元的生物化学成分改变。研究样本为6-OHDA诱导的帕金森大鼠模型海马区神经元。与正常大鼠海马区神经元比较,PD大鼠样品在属于脂类的CH2反对称和对称伸缩振动的2 924和2 850cm-1的振动吸收积分面积以及在峰值位于1 736cm-1的伸缩振动吸收积分强度比正常大鼠都有增加,提示PD样品脂质含量升高,而在属于核酸的PO2反对称伸缩振动和对称伸缩振动的强度比正常大鼠样品下降,提示PD样品中核酸的含量比正常样品减少。蛋白质的振动没有发现异常。该研究表明帕金森病大鼠模型海马神经元存在生物化学成分改变,这种改变可能与神经元的病理改变密切相关。     

重离子对人胃癌细胞DNA错配修复基因MSH2 表达的影响

采用高传能线密度(LET) 重离子辐照人胃癌SGC7901 细胞,应用流式细胞技术、蛋白质印迹法(Western blot) 及反转录聚合酶链式反应(RT-PCR) 观察重离子诱导人胃癌SGC7901 细胞周期、凋亡和MSH2 表达状况。结果表明: 与对照组相比,SGC7901 细胞在辐射后72 h G2/M 期所占细胞比率(33.26±0.08) 和凋亡率(24.16±0.64) 均达到峰值,且呈时间依赖性增加;经重离子照射后,DNA错配修复基因MSH2 mRNA 和蛋白表达水平在6 h 最高。结果提示:重离子在体外诱导SGC7901 细胞周期阻滞和凋亡,且具有显著的时间依赖性效应;重离子在一定剂量和时间下,诱导了SGC7901 细胞MSH2 基因表达。DNA错配修复基因MSH2 可能参与了重离子辐照诱导胃癌细胞DNA损伤的修复应答。Human gastric cancer cell SGC7901 were irradiated with high linear energy transfer (LET) carbon ion. Apoptotic cells after irradiation were analyzed by flow cytometry and expression of MSH2 genes in the irradiated cells was detected by western blot and RT-PCR assay. Compared with the control group, we found that the number of G2/M (33.26±0.08) or apoptosis (24.16±0.64) of SGC7901 cells reached a maximum after irradiation at 72 h in a dose dependent manner. And heavy ion irradiation efficiently up-regulated the expression of MSH2 gene at 4.0 Gy after being irradiated 6 h. These results imply that heavy ion beam could induce cell apoptosis and cell cycle arrest in time-dependent manners. Furthermore, expression of MSH2 genes activated by carbon ion irradiation suggests that DNA mismatch repair gene MSH2 might be involved in DNA repair pathways.     

荧光过氧化氢酶纳米传感器的制备及性能（英文）

过氧化氢(H_2O_2)是活性氧类的主要标志物,它与多种疾病如神经退行性疾病密切相关。本文设计了一种检测细胞内过氧化氢的荧光过氧化氢酶纳米传感器。这种纳米传感器由含多聚赖氨酸、辣根过氧化物酶的生物相容性外壳和含有氧探针的多孔聚合物基质的氧传感核组成。辣根过氧化物酶(HRP)催化H_2O_2生成氧,进而通过荧光氧气探针进行探测。该酶纳米传感器的流体动力学尺寸约为270 nm,zeta电位为-18 mV,具有良好的生物相容性。它的荧光比率和时间分辨荧光均对H_2O_2高度敏感。此外,该纳米传感器可以被活细胞有效地摄取,从而可以用TRF方式灵敏地检测细胞内的H_2O_2浓度。结果表明,本实验制备的酶纳米传感器有望进一步用于监测H_2O_2相关的细胞生化反应,如氧化应激。     

碱性成纤维细胞生长因子对PC12细胞氧化损伤的保护作用和机制

探讨碱性成纤维细胞生长因子(bFGF)对H<,2>O<,2>诱导PC12细胞凋亡的影响及其作用机制.利用H<,2>O<,2>诱导的PC12细胞建立细胞氧化损伤模型,用不同浓度的bFGF进行药物干预,CCK-8法和流式细胞技术检测细胞存活率和凋亡率,试剂盒测定超氧化物歧化酶(SOD)活性,Western blot检测ER...     

重离子诱导细胞衰老研究

综述了细胞衰老诱导因素和衰老细胞的特征,提出细胞衰老在癌症发生发展过程中具有双重作用效应。同时,以X射线和12C6+离子束对黑色素瘤细胞系92-1或人正常成纤维细胞系MRC5进行辐照,观察分析DNA损伤应答效应和细胞衰老诱导。实验结果表明,相对于X射线,12C6+离子束能导致DNA团簇损伤,持续激活DNA损伤应答,更容易诱导细胞衰老;12C6+离子束能同时诱导癌细胞和正常细胞衰老。这些实验结果暗示开展12C6+离子束诱导细胞衰老研究具有紧迫性。     

X射线致HeLa细胞损伤过程中NADPH氧化酶的作斥

以HeLa细胞为实验材料,探讨了NADPH氧化酶在X射线诱导细胞损伤过程中的作用。结果显示,12Cry X射线辐照后细胞内活性氧（ROS）明显增加,在用NADPH氧化酶抑制剂处理后再辐照,则细胞内ROS降低到未辐照水平;同时辐照后NADPH氧化酶细胞质亚基p47^phox在细胞质积聚并和细胞膜亚基gp91^phox结合;Western blotting检测结果显示,NADPH氧化酶的关键亚基gp91^phox的表达量明显增加。以上结果说明,NADPH氧化酶可以被X射线激活,由其介导产生的ROS在X射线诱导HeLa细胞损伤过程中扮演重要角色。     

基于DNA损伤的蛋白调控网络研究

细胞生长的每个阶段都离不开蛋白质相互作用.研究细胞周期的功能、调控机理及参与调控的蛋白质之间的关系对生物工程等领域有重大的应用价值.本文通过研究电离辐射下生物体细胞的DNA损伤后,细胞内以p53为核心的扩展蛋白调控网络的功能、原理及其自修复机理,在现有蛋白网络基础上引入更多蛋白网络调控因子来建立蛋白调控网络,仿真模拟更为全面的细胞周期进程;并且从复杂网络图论和细胞周期调控两个方面分析扩展PMP调控网络的抗扰能力及自修复机理,结果表明:1)蛋白网络在对抗环境中出现的小扰动时具有较强的稳定性.但在面对蓄意攻击时网络的稳定性较差.2)受损的DNA能否被修复取决于p53蛋白的动力学行为,即低损伤与中损伤情况下,p53可诱导细胞周期进程阻滞来完成细胞的自修复;而当高损伤或过损伤时,p53蛋白浓度表现为周期振荡行为并诱导细胞凋亡.     

细胞色素c甲硫氨酸氧化机制的NMR研究

细胞在呼吸作用中产生活性氧，活性氧含量处于低水平时有利于信号传导，累积过量时会引发蛋白质氧化修饰．细胞色素c是位于线粒体内的多功能金属蛋白，其发生氧化修饰特别是甲硫氨酸Met80的亚砜化修饰可能影响蛋白构象变化，但其机制仍不清楚．本研究通过13C选择性标记细胞色素c上甲硫氨酸的末端甲基，利用液体核磁共振技术，追踪了细胞色素c在氧化环境中的氧化修饰变化．发现在氧化环境中，该蛋白质先由还原态转化为氧化态，当活性氧达到一定量时，再发生甲硫氨酸Met80的亚砜化修饰，但在活性氧作用初期未导致明显的蛋白结构变化．这表明细胞色素c具有一定抵御活性氧的作用．     

茶多酚对鼠心肌缺血再灌产生氧自由基的清除

用电子顺磁共振(Electron Spin Resonance,缩写ESR)在低温110K条件下,以离体大鼠心脏缺血再灌为模型,直接测量了缺血再灌损伤时产生的活性氧自由基信号.实验发现,缺血再灌损伤的心肌中氧自由基的含量比正常灌流心肌增加了188%,茶多酚可以使其降低71%。这说明茶多酚对氧自由基引发的缺血再灌损伤有保护作用。     

Evidence for neuroprotective properties of human umbilical cord blood cells after neuronal hypoxia <Emphasis Type="Italic">in vitro</Emphasis>

Background  

One of the most promising options for treatment of stroke using adult stem cells are human umbilical cord blood (HUCB) cells that were already approved for therapeutic efficacy in vivo. However, complexity of animal models has thus far limited the understanding of beneficial cellular mechanisms. To address the influence of HUCB cells on neuronal tissue after stroke we established and employed a human in vitro model of neuronal hypoxia using fully differentiated vulnerable SH-SY5Y cells. These cells were incubated under an oxygen-reduced atmosphere (O₂< 1%) for 48 hours. Subsequently, HUCB mononuclear cells (MNC) were added to post-hypoxic neuronal cultures. These cultures were characterized regarding to the development of apoptosis and necrosis over three days. Based on this we investigated the therapeutic influence of HUCB MNC on the progression of apoptotic cell death. The impact of HUCB cells and hypoxia on secretion of neuroprotective and inflammatory cytokines, chemokines and expression of adhesion molecules was proved.     

Agmatine protects retinal ganglion cells from hypoxia-induced apoptosis in transformed rat retinal ganglion cell line

Background  

Agmatine is an endogenous polyamine formed by the decarboxylation of L-arginine. We investigated the protective effects of agmatine against hypoxia-induced apoptosis of immortalized rat retinal ganglion cells (RGC-5). RGC-5 cells were cultured in a closed hypoxic chamber (5% O₂) with or without agmatine. Cell viability was determined by lactate dehydrogenase (LDH) assay and apoptosis was examined by annexin V and caspase-3 assays. Expression and phosphorylation of mitogen-activated protein kinases (MAPKs; JNK, ERK p44/42, and p38) and nuclear factor-kappa B (NF-κB) were investigated by Western immunoblot analysis. The effects of agmatine were compared to those of brain-derived neurotrophic factor (BDNF), a well-known protective neurotrophin for retinal ganglion cells.     

Differential biological responses of green tea polyphenol in normal cells vs. cancer cells

Green tea has shown remarkable anti-inflammatory and cancer chemopreventive effects in many animal tumor bioassays, cell culture systems and epidemiological studies. Many of these biological effects of green tea are mediated by epigallocatechin 3-gallate, the major polyphenol present therein. In this study, the differential biological responses of green tea polyphenols (GTP) were examined in normal rat osteoblasts (NRO) vs. human osteosarcoma (MG-63 and Saos-2) cells. The GTP treatment with micromolar concentrations (0.1, 1, 10 and 100 μM for 24 h) resulted in dose-dependent inhibitions of cell growth and alkaline phosphatase activity, morphological alterations, G0/G1-phase arrest of the cell cycle and induction of apoptosis in both osteosarcoma cells, but not in the NRO. These results suggest that the GTP treatment may be contributed to the differential regulation of cell cycle in normal cells and cancer cells, which can be exploited to craft strategies for the physiological preservation of cells or tissues by GTP.     

2D spatially controlled polymer micro patterning for cellular behavior studies

A simple and effective method to functionalize glass surfaces that enable polymer micropatterning and subsequent spatially controlled adhesion of cells is reported in this paper. The method involves the application of laser induced forward transfer (LIFT) to achieve polymer patterning in a single step onto cell repellent substrates (i.e. polyethyleneglycol (PEG)). This approach was used to produce micron-size polyethyleneimine (PEI)-patterns alternating with cell-repellent areas. The focus of this work is the ability of SH-SY5Y human neuroblastoma cells to orient, migrate, and produce organized cellular arrangements on laser generated PEI patterns.     

Synthesis and properties of small interfering RNA duplexes carrying 5-ethyluridine residues

Oligoribonucleotides carrying 5-ethyluridine units were prepared using solid-phase phosphoramidite chemistry. The introduction of the tert-butyldimethylsilyl group at the 2′-OH position proceeded in good yield and very high 2′-regioselectivity. RNA duplexes carrying 5-ethyluridine either at the sense or the guide strands display RNAi activity comparable to or slightly better than that of unmodified RNA duplexes. Gene suppression experiments using luciferase targets in SH-SY5Y cells show that the ethyl group is generally well accepted at all positions although a small decrease in RNA interference activity is observed when one 5-ethylU residue is incorporated in the 3′ overhangs.     

Effect of glutaphen on generation of active oxygen species, photosynthetic electron transport, and the functional activity of photosystem 2 in Chlorella cells

Treatment of chlorella green algae (Chlorella sp.) for 2 h in the dark with the photodynamic herbicide glutaphen (GTP), consisting of 0.3 mM 1,10-phenanthroline and 0.6 mM glutamic acid, followed by illumination leads to efficient generation of active oxygen species (AOS). After 15 min of illumination, AOS accumulation reaches 200% compared with the level in cells of the control culture, then it decreases down to 130% and 115% after 1 day and 3 days of illumination. During the first few days after treatment, we observe inhibition of synthesis of a specific precursor of chlorophyll (Chl): 5-aminolevulinic acid molecules, and then we observe stimulation of the synthesis. The effect of GTP on the photosynthetic activity of chlorella does not depend on the AOS level but rather remains uniform, in contrast to its effect on the Chl biosynthesis system. GTP does not change the efficiency of light harvesting and charge separation at the reaction centers of photosystem (PS) 2, but significantly lowers the functional efficiency of the electron transport chain: the photochemical quenching constants for Chl a fluorescence and the effective quantum yield of photochemical reactions in photosystem 2 decrease. The major mechanism of action for GTP is probably displacement of the secondary quinone acceptor Q_B from its binding site on the D1 protein by the 1,10-phenanthroline, consequent inhibition of electron efflux from Q _A ⁻ and increase in the fraction of Q_B-nonreducing centers of PS 2. The active oxygen species generated in the cells have a photodegradative effect on the Chl biosynthesis system, in particular on its initial steps, and do not involve already formed pigment-protein complexes of PS 2. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 73, No. 6, pp. 801–806, November–December, 2006.     

Photosensitizer Targeting: Mitochondrion-Targeted Photosensitizer Enhances Mitochondrial Reactive Oxygen Species and Mitochondrial Calcium-Mediated Apoptosis

Using laser scanning imaging microscopy, our previous studies have demonstrated visible laser irradiation induced significant generation of mitochondrial reactive oxygen species (mROS) and apoptosis. In addition, photodynamic therapy (PDT) with a mitochondrion-targeted photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA), significantly enhanced visible laser irradiation induced mitochondrial dysfunction and apoptosis. To validate the critical role of the mitochondrion as well as how mitochondrial mechanisms were involved in the PDT of BPD-MA, subcellular PDT with BPD-MA were performed using region irradiation and recorded by multi-photon imaging microscopy coupled with mitochondrial specific fluorescent probes in single live C6 glioma cells. Specifically, mROS and mitochondrial free calcium (mCa²⁺) were simultaneously measured from cells received subcellular PDT with BPD-MA in different compartments including the mitochondrion, the cytosol and the nucleus. Results revealed that significant formation of mROS accompanied by elevation of mCa²⁺ was found only in the mitochondrial PDT with BPD-MA. Subsequently, mitochondrial PDT with BPD-MA resulted in severe mitochondrial swelling, plasma membrane blebing and apoptotic death. We conclude that via augmented mROS formation and mCa²⁺ elevation PDT with a mitochondrion-targeted photosensitizer extensively enhances mitochondria-mediated apoptosis and, thus, may provide high eradication efficacy for clinical cancer treatment.