褪黑素和6-羟褪黑素保护神经细胞抗缺血再灌注损伤比较研究

目的研究褪黑素(Mel)和6-羟褪黑素(6-OHMel)神经保护作用及作用机理。方法体外培养N2a细胞,模拟缺血再灌注(OGSD),加入Mel和6-OHMel,检测以下指标:①细胞生存能力:MTT法、乳酸脱氢酶释放;②细胞凋亡分析:DNA片断化,细胞色素C,Caspase3活性;③活性氧(ROS)和线粒体跨膜电位。结果①Mel和6-OHMel都能减轻OGSD诱导的N2a细胞损伤,Mel的作用强于6-OHMel。②Mel和6-OHMel均能抑制细胞色素C释放,但6-OHMel强于Mel。③Mel和6-OHMel都能稳定线粒体跨膜电位,但Mel作用时间比6-OHMel长。④Mel和6-OHMel能清除ROS,6-OHMel表现为直接作用,Mel表现为间接作用。⑤Mel和6-OHMel均能抑制caspase3的活性,但是作用时间不同。6-OHMel表现在OGSD后12h,Mel在OGSD后24h。结论Mel和6-OHMel的神经保护作用与其抗氧化、稳定线粒体功能相关,Mel的作用机制更复杂。     

蛋白激酶C在软脂酸诱导HepG2细胞胰岛素抵抗中的作用

目的从蛋白激酶C(PKC)信号通路角度,探讨游离脂肪酸(FFA)引起肝脏胰岛素抵抗(IR)的可能机制。方法培养HepG2细胞,同时设立对照组、软脂酸(PA)组、高胰岛素组。软脂酸组、高胰岛素组分别用250μmol/L PA、5×10-7mol/L胰岛素处理24h。然后对照组、软脂酸组再根据胰岛素刺激前加( )与不加(-)PKC抑制剂白屈菜红碱盐酸盐(chelerythrine chloride,CC)5μmol/L预处理1h,随机分为两亚组:对照组(-)、对照组( )、PA组(-)、PA组( )。葡萄糖氧化酶法测定胰岛素刺激后12h葡萄糖消耗量,蒽酮法测定胰岛素刺激后3h点细胞内糖原含量,Western blotting技术检测15min点细胞内P-Ser473PKB、P-Ser21/9GSK-3α/β水平。结果PA组(-)与高胰岛素组葡萄糖消耗量无统计学差异(P=0.523)。葡萄糖消耗量、细胞内糖原含量、P-Ser473PKB、P-Ser21GSK-3α、P-Ser9GSK-3β水平均显示,PA组(-)与对照组(-)比较显著降低(P值依次为0.000,0.000,0.004,0.004,0.028),对照组( )与对照组(-)比较略有升高但无显著性差异;PA组( )与PA组(-)比较显著升高(P值依次为0.000,0.014,0.043,0.041,0.035)。结论PA(250μmol/L)体外成功诱导了HepG2细胞产生IR,PKC信号通路在FFA引起肝脏IR中起着重要作用。     

花生四烯酸对游离脂肪酸诱导肝细胞胰岛素抵抗的缓解作用研究

目的：探讨游离脂肪酸（FFA）诱导肝细胞胰岛素抵抗及花生四烯酸（从）在软脂酸（PA）诱导的肝细胞胰岛素抵抗中的作用。方法：分别用不同浓度的PA和从与HepG2细胞共同培养不同的时间，并设立正常对照组（Coatrol组）。采用葡萄糖氧化酶法测定培养液中的葡萄糖浓度，蒽酮法测定细胞内糖原含量以确定HepG2细胞产生胰岛素抵抗程度．结果：1．0．25mmol／L PA组中的葡萄糖浓度高于Control组和PA＋AA（20umol／L）组（P〈0．05），糖原含量低于Control组和PA＋从（20umol／L）组（P〈0．05）。2，PA从（20umol／L）组与0．25mmol／L PA组比较，PA＋AA（50umol／L）组与Control组比较，葡萄糖浓度和糖原含量变化有显著意义（P〈0．05）。结论：高浓度的PA可以诱导肝细胞产生胰岛素抵抗，并呈现剂量依赖性；一定浓度的从能够缓解PA引起的胰岛素抵抗。     

Inhibitory Effect of Melatonin on the Growth of H22 Hepatocarcinoma Cells by Inducing Apoptosis

Whether melatonin not only inhibits the growth of H22 hepatocarcinoma cells but also induces apoptosis in vitro was assessed. The anti-proliferative effects of melatonin on tumor cells was observed by MTT assay and tumor cells growth curve assay. And the apoptosis of the cells was studied by acridine orange fluorescence assay and flow eytometry. The cell cycle of the tumor cells was also observed by flow eytometry. It was found that melatonin could significantly inhibit the growth of H22 hepatoeareinoma cells. Incubated with melatonin, ehromatin condensation of the tumor cells was observed by fluorescence microscopy. Compared with control, the percentage of apoptotic cells was increased, and the proportion of G0/S increased but that of G0/M decreased. It was suggested that melatonin could directly inhibit the growth of H22 hepatoearcinoma cells by inducing apoptosis and extending the length of cell cycle of the tumor cells.     

Fluvoxamine对褪黑素生物转化的抑制及其药物动力学研究

采用人肝微粒体温育实验，ＣＹＰ４５０１Ａ２特异性抑制法以及ＨＯＰＬＣ和电化学分析仪检测的结果表明，（１）抗抑郁药Ｆｌｕｖｏｘａｍｉｎｅ（三氟志胺）对ＣＹＰ４５０１Ａ２具和ＣＹＰ４５０１Ａ２特异抑制剂Ｆｕｒｐａｈｙｌｌｉｎ（呋拉茶碱）同样的对ＭＴ生物转化的抑制作用并求测其动力学参数。Ｆｌｕｖｏｘａｍｉｎｅ对ＭＴ郑化反应有很强的抑制作用，其Ｋｉ＝０．０２４μｍｏｌ／Ｌ，对ＭＴ去甲工反应也是抑制的，其Ｋ     

Melatonin protects N2a against ischemia/reperfusion injury through autophagy enhancement

Researches have shown that melatonin is neuroprotectant in ischemia/reperfusion-mediated injury.Although melatonin is known as an effective antioxidant,the mechanism of the protection cannot be explained merely by antioxidation.This study was devoted to explore other existing mechanisms by investigating whether melatonin protects ischemia/reperfusion-injured neurons through elevating autophagy,since autophagy has been frequently suggested to play a crucial role in neuron survival.To find it out,an ischemia/...     

针刺能量的物理学分析

从针刺和次声的角度,回顾近年来腧穴和经络问题的研究现状。从能量的角度对针刺过程进行研究,将针灸中的提插法作为直进式输入能量的方法,捻针法作为扭转式输入能量的方法。认为2种方法的作用均使经络物质（黏弹性体）产生振动,以次声波的频率将能量沿经络线传播,并诱发放大过程产生,使体内其他能量随针刺能量传播。最后从经典物理和近代物理两方面,给出了动力学方程和近代物理理论分析,得出针刺是以次声能量方式进行刺激从而引起神经系统产生的综合作用效果。     

针刺过程能量的输入

从经典物理和近代物理两个角度对针刺过程能量的输入进行了讨论.认为进针和行针中的提插法是一种直进式输入能量的方法,行针中的捻针法是一种扭转式输入能量的方法.它们的作用都是使经络物质(黏弹性体)产生振动,以次声波的频率将能量沿经络线传播,并诱发放大过程产生,使体内其他能量随针刺能量传播.最后给出了针刺过程的动力学方程和能量方程.     

移行上皮反应基因TERE1的结构与功能分析

目的 用生物信息软件分析移行上皮反应基因TERE1蛋白的跨膜结构,并预测分析其重要蛋白结构域功能.检测TERE1在细胞中表达定位.方法 采用不同生物信息学软件预测分析TERE1的蛋白跨膜结构及重要结构域功能.采用分子克隆方法构建绿色荧光蛋白-移行上皮反应基因(pEGFP-TERE1)重组质粒,转染人膀胱癌细胞系T24,激光共聚焦显微镜观察TERE1在细胞中表达定位.结果 TERE1蛋白为多次跨膜的蛋白,保守的区域形成3个环loop 1、loop 2和loop 3;N端和loop 1合有高度保守的位点;以loop 2和loop 3之间的区域采用PROSITE软件分析显示UbiA类异戊烯转移酶家族具有一致性序列.采用菌液PCR、双酶切鉴定和DNA测序验证pEGFP-TERE1重组质粒构建成功.TERE1主要在T24细胞浆中表达.结论 TERE1为多次跨膜的蛋白,保守的区域形成3个环,UbiA类异戊烯转移酶家族具有一致性序列.TERE1主要在T24细胞浆中表达.     

蝙蝠葛酚性碱与褪黑素对缺血再灌注损伤神经元的作用比较

BACKGROUND： Recent studies have demonstrated that phenolic alkaloids from Menispermum dauricum （PAMD） can protect the heart and brain from ischemia/reperfusion injury, and promote neuron survival by inhibiting neuronal Bax and upregulating Bcl-2 expression following ischemia/reperfusion.
OBJECTIVE： To investigate the neuroprotective effects of PAMD versus exogenous melatonin against ischemia/reperfusion injury.
DESIGN, TIME AND SETTING： Observation and comparison experiments at a cellular level were performed at the Department of Biochemistry and Molecular Biology, Tongji Medical College of Huazhong University of Science and Technology between February 2007 and February 2008.
MATERIALS： PAMD （95% purity） was provided by Kunming Institute of Botany, Chinese Academy of Sciences; melatonin was provided by Sigma, USA.
METHODS： N2a mouse neuroblastoma cells were cultured in vitro deprived of glucose, serum and oxygen for 90 minutes, then cultured in normal medium containing different concentrations of PAMD （0.1, 1.0, 10 mg/L） or melatonin （1, 10, and 100 μmol/L）. Cells cultured in normal conditions served as a control.
MAIN OUTCOME MEASURES： The culture solution was collected to determine the content of ex- citatory neurotransmitters such as glutamic acid and aspartic acid; cell viability was detected by MTT methods; reactive oxygen species production was determined by fluorescence spectroscopy; mito- chondrial transmembrane potential （？Ψm） was detected by laser confocal scanning; cytochrome C was measured by western blotting; and caspase-3 activity was determined by visible spectropho- tometry.
RESULTS： Melatonin and PAMD both promoted oxygen-glucose-serum deprivation-mediated N2a cell survival （P 〈 0.01） and inhibited glutamic acid release （P 〈 0.01）, but melatonin did not inhibit aspartic acid production. The protective effects were the strongest using melatonin 100 μmol/L and PAMD 10 mg/L, so subsequent experiments were the performed at those doses. Although PAMD could no longer maintain mitochondrial transmembrane potential 6 hours after reperfusion, its in- hibitory effects on cytochrome C release from mitochondria and scavengers of reactive oxygen species were stronger than those of melatonin （P 〈 0.01）. However, its inhibitory effect on caspase-3 activity was weaker than that of melatonin： PAMD could inhibit caspase-3 activity 12 hours after reperfusion （P 〈 0.01）, but melatonin inhibited caspase-3 activity 28 hours after reperfusion （P 〈 0.01）.
CONCLUSION： The results show that melatonin and PAMD have neuroprotective effects, but that the mechanisms are varied. Melatonin can maintain mitochondrial transmembrane potential, but its inhibitory effects on cytochrome C release, caspase-3 activity, and reactive oxygen species scav-enging are different from those of PAMD.