【作者】 杨新玲；

【导师】 张晨； 蒋雨平；

【作者基本信息】 新疆医科大学 ， 劳动卫生与环境卫生学， 2008， 博士

【摘要】 目的:研究胰岛素对百草枯（PQ）诱导的PC12细胞的保护作用及其机制的体内和体外试验。1)观察培养的PC12细胞中多巴胺D2受体（dopamine D2 Receptor,DA D2R）蛋白的表达;胰岛素对PQ诱导的PC12细胞形态学、生存率和DA含量的影响。2)探讨胰岛素对PQ诱导的PC12多巴胺能神经元细胞保护的可能机制。3)研究胰岛素联合骨髓基质干细胞（bone marrow derived stroma cell,BMSCs）治疗PD模型鼠的疗效。方法:1)应用免疫沉淀Western印迹分析技术对培养的PC12细胞中DAD2R表达的检测;应用二甲基噻唑二苯基四唑溴盐[3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetra-zoliumbromide,MTT]法,观察PC12细胞暴露于不同浓度PQ组和胰岛素预先干预后再暴露于PQ组后细胞形态学和生存率的改变;应用ELISA方法检测空白对照PC12细胞组、PQ干预组、胰岛素组和胰岛素加PQ组PC12细胞上清液DA的浓度。2)在前一部分实验的基础上,应用免疫沉淀Western印迹分析技术测定空白对照PC12细胞组、PQ干预24小时PC12细胞组、胰岛素干预24小时PC12细胞组和PQ与胰岛素共同干预24小时PC12细胞组INR Tyr^1162/1163和Akt^Ser473磷酸化蛋白的表达。3)①BMSCs的培养。采用贴壁法原代培养BMSCs,经反复传代细胞逐渐纯化,流式细胞仪（luorescence activated cell sorting,FACS）检测第3代BMSCs细胞的表面标志CD29,CD34,CD44,CD45表达情况;②建造PD动物模型。用6-OHDA单侧损毁法制作PD大鼠模型,对其行为学进行检测（旋转实验）;高效液相色谱法（High-performance liquid chromatography,HPLC）检测脑内双侧黑质-纹状体区DA含量;③胰岛素联合BMSCs干预PD动物模型鼠的疗效观察。将6-OHDA单侧损毁成功的PD模型大鼠随机分为3组,分别移植BMSCs（每只鼠共移植1.2×10⁶个细胞）、胰岛素50nM+BMSCs联合组和生理盐水（12μl）于模型鼠右侧纹状体。在干预后4周分别观察模型大鼠阿朴吗啡（apomorphine,APO）诱导的旋转行为的变化,运用免疫荧光观察模型大鼠脑内BMSCs细胞存活情况以及HPLC法检测模型大鼠脑内双侧黑质-纹状体区DA含量的变化。结果:1)①免疫沉淀Western印迹分析技术检测到培养的PC12细胞中有DA D2R蛋白的表达:②空白对照组PC12细胞形态胞体呈梭形,细胞突触完整;PC12细胞暴露于600μM PQ组24小时,多数细胞胞体变圆、空泡变性、突触变短或消失。预加100nM胰岛素预处理20分钟后、再暴露于600μM PQ组24小时,与空白对照组相比细胞形态略有改变,细胞形态呈梭形或不规则但非圆形,细胞突触又有生长;③PC12细胞生存率与PQ干预的浓度呈反向变化趋势;而胰岛素可以减少600μM PQ24小时内对PC12细胞生存率的毒性损伤;④胰岛素可以提高正常PC12细胞和600μMPQ干预的PC12细胞上清液中的DA浓度,但未达到统计学意义（P＞0.05）。2)胰岛素干预组INR Tyr^1162/1163和Akt^Ser473磷酸化程度均有不同幅度增加。3)①第三代的BMSCs表面标志物CD29、CD34、CD44和CD45表达分别为97.1%、0.0%、99.0%和0.5%;②细胞移植治疗4周后联合组旋转次数较BMSCs组（P＜0.05）和生理盐水组（P＜0.01）明显减少;③免疫荧光观察模型鼠脑内BMSCs细胞存活情况:Brdu阳性细胞数量胰岛素+BMSCs组干预4周后Brdu阳性细胞数比BMSCs组明显增多（P＜0.01）;④HPLC结果示:胰岛素联合组模型鼠脑内黑质-纹状体区DA含量虽较其它两组增加,但未达到统计学意义（P＞0.05）。结论:1)胰岛素对PQ损害的PC12多巴胺能神经元细胞有保护作用。2)胰岛素受体INR Tyr^1162/1163和受体后信号传导通路Akt^Ser473磷酸化增加可能是胰岛素保护PC12细胞的机制之一。3)胰岛素联合BMSCs移植治疗PD模型的疗效总体好于BMSCs组和生理盐水组,进一步为胰岛素具有多巴胺能神经元细胞保护作用提供了动物体内实验依据。更多还原

【Abstract】 Objective:Protective efficacy and mechanism of the insulin against paraquat-induced by using PC12 cells,were investigated in vivo and in vitro studies.1) To observe dopamine DA D2R protein expression on PC 12 cells and its effects of insulin on the survival rate,morphology,and dopamine contents of PQ-induced PC12 ceils.2) To investigate the mechanism that insulin may protect the dopaminergic neuron against paraqua-induce PC12 cells.3) To observe the curative effect on the PD rat models with both insulin and BMSCs interfered.Methods:1) Immunoprecipitate Western blotting method was performed to observe DA D2R protein expression on PC 12 cells, 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetra-zolium bromide（MTT） assay was performed to analyze the alteration of viability and cells morphology in PC 12 cells were exposed to the different PQ and insulin concentrations.2) Based on the PartⅠ, Immunoprecipitate Western Blotting method was performed to observe the expression of INR Tyr^1162/1163 and Akt^Ser473 in the group of normal PC12 cells,insulin interfered PC 12 cells,and PQ-induced and PQ combined insulin interfered in PC 12 cells respectively.3)①From BMSCs cultivating pints of view,Adherent method was used to raise and repeatedly purify BMSCs.In addition flow cytornetry was exploited to detect expression of CD29,CD34,CD44 and CD45,which all the markers existed on the cell’s surface of the BMSCs in the third generation;②The rotation test was taken to detect the efficacy rat models of whose striaturn were half destructed by 6-OHDA and induced hemiparkinsonian.Morever high-performance liquid chromatography（HPLC） was adopted for the detection of the DA contents in the brains of PD rat models.③The succeed PD rat models were randomizely divided to three groups,which were employed as injected BMSCs,insulin associates BMSCs and normal saline group,respectively. Four weeks later,the rotating frequency was calculated and the cell’s survival was tested by the immunofluorescent method.The DA level in the brain（substantia nigra and striatum） was detected by HPLC.Results:1)①DA D2R protein in quality was expression on PC 12 cells.②Normal PC12 cells bodies showed fusiform shape and the synapsis were integrity.The cells exposed to the 600μM concentration of PQ became ball-like,vacuolar degeneration,and the synapse shorten or disappeared.But the cell’s morphology showed a little alteration between the normal PC12 and the insulin groups, except to the cells with different morphology of the fusiform shape or abnomalism,not round shape and the synapsis longer again.③With the increase of the concentration of PQ,the viability of the cells decreased.Insulin increased the viability of the cell which was exposed to the 600μM concentrations of PQ.Insulin elevated dopamine concentration both in the normal PC12 and the cells were exposed to 600μM concentrations of PQ,but there was not the statistical significance（P＞0.05）.2) The insulin groups showed the expression in different phosphorylation degree on of INR Tyr^1162/1163 and Akt^Ser473.①CD29,CD34,CD44 and CD45 BMSCs in the third generation were expressed 97.1%,0.0%,99.0%,0.5%.②The rotating frequencies in the insulin group combined BMSCs was much more decreased in comparson with the BMSCs（P＜0.05） and the normal saline group（P＜0.01）.③the cell’survival was to observe.Brdu positive cells in the group interfered by insulin combined BMSCs were much more than the BMSCs group（P＜0.01）.④DA concentration in the group of insulin combined BMSCs were increased compared to the other 2 groups,however it did not have the statistical significance（P＞0.05）.Conclusion:1) Insulin could protect the dopaminergic neuron against PQ-induced PC 12 cells.2) The increased expression on the phosphorylation degree of INR Tyr^1162/1163 and Akt^Ser473 in the insulin groups showed might be one of the clues to prove insulin may have the protective efficacy to PC 12 cells. 3) The curative effect of the insulin combined BMSCs interfered the rat model was better than BMSCs treated group alone.It is essential to provide further evidence in vivo on insulin protect dopaminergic neurons.更多还原