【作者】 郑小敏；

【导师】 范薇；

【作者基本信息】 复旦大学 ， 神经病学， 2009， 硕士

【摘要】 目的应用谷氨酸(glutamate, Glu)细胞损伤模型来研究施普善(Cerebrolysin)是否可以有效地保护细胞,并探讨其保护机制。方法应用SH-SY5Y神经细胞系来建立可靠的Glu损伤模型。采用噻唑兰(methyl-thiazole-tetrazolium, MTT)细胞活性的测定、乳酸脱氢酶(lactatedehydrogenase, LDH)检测的方法研究Cerebrolysin是否具有细胞保护作用。应用细胞内钙离子测定、细胞内活性氧(reactive oxygen species, ROS)测定、细胞培养基超氧化物岐化酶(superoxide dismutase, SOD)、丙二醛(malonaldehyde, MDA)含量测定和细胞线粒体膜电位(mitochondrion membrane potential, MMP)测定等方法进一步探讨Cerebrolysin细胞保护作用的机制。结果1.80mM Glu处理细胞24h后MTT测定数据表明细胞存活率为51.233±3.019%；单独给予细胞4、8、10和20m1／L的Cerebrolysin对细胞存活没有显著影响。2.4、8、10和20ml/L Cerebrolysin和80mM Glu共同处理细胞24h后细胞的存活率分别为48.739±5.786%、71.863±16.821%、75.767±12.454%和71.814±11.788%,其中l0ml／L浓度Cerebrolysin细胞保护作用最好。3.与正常对照组比较(544.670±5.126 U／L),80mM Glu组细胞培养基LDH含量显著增加(835.552±51.530U／L)；与80mM Glu组比较,10ml/L Cerebrolysin加80mM Glu组细胞培养基LDH含量显著降低(680.222±66.688 U／L,P<0.05)。4.与正常对照组比较(28.328±5.286),10ml/L Cerebrolysin对细胞内钙离子荧光没有显著影响(32.523±7.514)；80mM Glu引起细胞内钙离子荧光显著增加(72.434±12.735,P<0.001)；10ml/L Cerebrolysin可以抑制Glu引起的细胞内钙离子荧光增加(53.309±12.626,P<0.001)。5.与正常对照组(94.356±13.417)比较,单独给予细胞10ml/L Cerebrolysin24h可以显著提高线粒体荧光(115.856±9.425,P＜0.001),同时可见细胞线粒体明显增多,分支增加；而80mM Glu处理细胞24h线粒体荧光显著降低(90.281±9.438,P<0.05)；10ml/L Cerebrolysin和80mM Glu共同处理细胞24h后线粒体荧光显著增加(102.847±13.338,P<0.001),同时可见细胞线粒体明显增多,分支增加。6.与正常对照组(20.436±1.762 U／m1)比较,80mM Glu处理细胞24h后培养基中SOD酶的含量显著降低(5.816±1.154 U／ml,P<0.001)；10ml／L的Cerebrolysin对细胞培养基SOD酶的含量没有显著影响(21.588±1.051 U／m1)；10ml／L的Cerebrolysin可以显著抑制80mM Glu引起的细胞培养基SOD酶含量的降低(14.810±1.955 U／ml,P<0.001)。7.与正常对照组(0.989±0.224μmol／L)比较,80mM Glu处理细胞24h后培养基中MDA的含量显著增加(11.138±0.942μmol／L,P<0.001)；10ml／L的Cerebrolysin对细胞培养基MDA的含量没有显著影响(1.071±0.337μmol／L)；l0ml／L的Cerebrolysin可以显著抑制80mM Glu引起的细胞培养基MDA含量的增加(4.424±0.772μmol／L,P<0.001)。8.与正常对照组细胞ROS荧光(39.112±15.283)比较,80mM Glu处理组显著增高(123.058±33.007,P<0.001)；与80mM Glu处理组比较,l0ml／LCerebrolysin和Glu共同处理组细胞ROS荧光显著降低(95.707±28.703,P<0.001)。结论1.单独应用4、8、10、20m1／L浓度的Cerebrolysin对SH-SY5Y细胞存活率没有显著影响。2.10ml／L的Cerebrolysin可以通过抑制80mM Glu引起的SH-SY5Y细胞内钙离子、ROS和MDA含量的增加,提高SH-SY5Y细胞SOD酶的含量,降低SH-SY5Y细胞过氧化反应来抑制Glu引起的细胞损伤。3. Cerebrolysin可以显著提高SH-SY5Y细胞MMP,同时线粒体形态发生改变,这一变化是否参与了Cerebrolysin抑制Glu引起的细胞损伤及其机制依然不祥。更多还原

【Abstract】 Objective To observe the protective effects of Cerebrolysin on SH-SY5Y cells damage induced by gluatmate, and to explore its possible molecular mechanisms.Methods Cell damage model was established by treating SH-SY5Y Cells with 80mM glutamate. Cell viability and lactatedehydrogenase(LDH) concentration were used to estimate the cell protective effects of Cerebrolysin. Its mechanisms were researched by examine cell calcium fluorescent, mitochondria membrane potential(MMP), reactive oxygen species(ROS), malonaldehyde(MDA) and superoxide dismutase(SOD) contents.Result The cell viability of SH-SY5Y cells decreased to 51.233±3.019% after being treated with 80mM gluatmate for 24h and the normal cell morphous were also disappeared, which suggesting that cell damage model was successfully established. 4、8、10 and 20ml/L Cerebrolysin had not effect on cell viability. Co-treated cell with 4、8、10、20ml/L Cerebrolysin and 80mM glutamate for 24h cell viability were 48.739±5.786%、71.863±16.821、75.767±12.454% and 71.814±11.788%. Group of 80mM glutamate cell medium LDH content was 835.552±51.530U/L; Co-treated group of 10ml/L Cerebrolysin and 80mM glutamate cell medium LDH content was 680.222±66.688 U/L. Contrast with control(28.328±5.286),80mM Glutamate could increase cell calcium fluorescent(72.434±12.735); 10ml/L Cerebrolysin had not effect on cell calcium fluorescent(32.523±7.514); Co-treated group of 10ml/L Cerebrolysin and 80mM glutamate cell calcium fluorescent increased to 53.309±12.626. Contrast with control(94.356±13.417), 10ml/L Cerebrolysin could significant increase cell MMP (115.856±9.425) and the mitochondria morphous was changed; 80mM glutamate cell had decreased MMP(90.281±9.438); Co-treated group of 10ml/L Cerebrolysin and 80mM glutamate cell MMP were significant increased (102.847±13.338) and the mitochondria morphous was changed too. Contrast with control(20.436±1.762 U/ml),80mM glutamate could significant decrease cell medium SOD contents(5.816±1.154 U/ml); 10ml/L Cerebrolysin had not effect on cell medium SOD contents(32.523±7.514); Co-treated group of 10ml/L Cerebrolysin and 80mM glutamate cell medium SOD contents decreased to 14.810±1.955 U/ml.80mM Glutamate could significant increase cell medium MDA contents to 11.138±0.942μmol/L, and 10ml/L Cerebrolysin could inhibit it to 4.424±0.772μmol/L. Control cells had a low ROS fluorescent(39.112±15.283), while 80mM glutamate could significant increase ROS fluorescent to 123.058±33.007; 10ml/L Cerebrolysin could inhibit glutamate induced ROS fluorescent to 95.707±28.703.Conclusion Cerebrolysin(10ml/L) can reduce glutamate(80mM) induced SH-SY5Y cells damage by decrease intra-cellular calcium, MDA and ROS increasing, increase cell SOD contents, inhibit cell peroxidatic reaction. Cerebrolysin can significantly increase cell MMP and change the mitochondria morphous. While it’s mechanisms is still unclear. We have no data to support that the increase of cell MMP is in favor of the cell viability.更多还原