【作者】 莫镇涛；

【导师】 方永奇；

【作者基本信息】 广州中医药大学 ， 中医学， 2012， 博士

【摘要】 目的：1.研究PC12细胞在缺糖缺氧（2h）再给氧过程中ATP与Beclin-1的相关性。2.研究自噬在PC12细胞缺糖缺氧（2h）再给氧过程中的的作用。3.研究β-细辛醚对缺糖缺氧再给氧PC12细胞Beclin-1、线粒体膜电位（MMP）、细胞内游离钙离子浓度（[Ca2+]i）、细胞成活率、细胞形态的影响。方法：1.建立缺糖缺氧再给氧PC12细胞损伤模型：将高糖DMEM完全培养液换为无糖的Earle’s平衡盐溶液,并将培养板放入1%O2、94%N2和5%CO2的培养箱培养2h,作为模拟细胞缺糖缺氧的状态,然后弃Earle’s平衡盐溶液并换回高糖DMEM完全培养液,并将培养板放入5%CO2的培养箱培养模拟再给氧状态。2.建立高效液相（HPLC）检测ATP的方法学：通过设置阴性对照组,标准品对照组,样品对照组,考察细胞提取液ATP检测的专属性；精密称取10.0mgATP并用双蒸水溶解定容至10mL。精密量取以上1.00mg/mL ATP标准液0.5mL定容至10mL,浓度即为50.0μg/mL。依次精密量取50.0μg/mL ATP标准液16、32、48、64、80μL并用双蒸水定容至10mL,浓度依次为0.080、0.160、0.240、0.320、0.400ng/mL。进样器精密吸取20μL进样。在0.080-0.400mg/mL范围内,考察ATP浓度与峰面积的线性关系；取0.080、0.240、0.400μg的低、中、高三种不同的ATP对照品溶液各一份,连续进样五次,考察精密度；取再给氧0、10、24h3个不同时点的细胞各5份,分别加入600μL PBS重悬,然后加入80μL50%的HCl04-80℃/室温反复冻融2次,分别加入0.1、0.2、0.4μg/mLATP标准溶液1mL,再用300μL,2mol/LNaOH将细胞提取液的pH值调至中性,4℃12000r/min离心15min,取上清液进行测定,考察平均回收率。将处理后的再给氧24h的细胞提取液于-80℃放置Od、3d、7d检测,考察样品储存的稳定性。3.建立流式细胞仪检测Beclin-1的方法学：包括考察自噬抑制剂3-甲基腺嘌呤（3-methyladenine,3-MA）,2%牛血清的PBS封闭液,一抗的浓度(0.002、0.004、0.006μg/μL孵育温度（25、37℃）,孵育时间（15、30、60min）,保存方法及时间（标本标记制备后立即检测和4℃放置6h、24h上机检测；或用1%多聚甲醛固定后4℃保存放置1、3、7d检测）对Beclin-1蛋白测定的影响。4.研究自噬的作用及自噬与ATP关系。体外培养PC12细胞48h,弃细胞完全培养液,PBS洗一遍后,加入Earle’s平衡盐溶液并放入1%O2、94%N2和5%CO2的培养箱中培养2h,然后弃Earle’s平衡盐溶液并换回细胞完全培养液培养0,4,10,24,,18h,HPLC检测ATP（并检测24h的正常对照组ATP）,BCA法检测蛋白含量,流式细胞仪检测Beclin-1,MTT法检测细胞活力,倒置相差显微镜下观察细胞形态。透射电子显微镜下观察正常培养PC12细胞与再给氧24h PC12细胞的自噬体数量和形态。将同一孔的再给氧0、24h的细胞分别分成两份,一份用于检测ATP含量,另一份用于检测Beclin-1表达,并做两者的相关性分析。13-细辛醚对缺糖缺氧再给氧PC12细胞自噬的影响：体外培养PC12细胞48后,分别用含β-细辛醚（20、30、45μg/mL）,尼莫地平（10μmol/L）的细胞完全培养液预给药1h后,弃药液,PBS洗1遍,分别加入含β-细辛醚（20、30、45μg/mL）、尼莫地平（10μmol/L）的Earle’s平衡盐溶液并放入1%02、94%N2和5%CO2的培养箱中培养2h,然后弃Earle’s平衡盐溶液并换回细胞完全培养液培养24小时,流式细胞仪检测Beclin-1,MMP,[Ca2+]i,MTT法检测细胞活力,倒置相差显微镜观察细胞数量和形态,透射电子显微镜观察自噬体。6.统计分析方法：数据以均数±标准差（x±s）表示,采用独立样本t检测法检测组间的差异性和Pearson相关性分析法分析两指标之间的相关性。结果：1.在0.08-0.40mg/mL范围内,ATP浓度与峰面积有良好的线性关系（r=0.9995）；连续测定5次0.080,0.240,0.400μg/mL ATP的峰面积,RSD分别为2.8、2.1、1.8%,精密度良好；测得再给氧0、10、24h3个不同时点细胞提取液的平均回收率为95.6±2.8%,RSD为2.9%,准确度良好；-80℃冰箱储存0,3,7d,ATP浓度分别为0.331±0.010,0.312±0.012,0.303±0.008μg/mL,RSD为4.5%,储存7天内稳定性良好。2.正常细胞的Beclin-1表达很低（6.49±1.19%）,但缺糖缺氧再给氧后,Beclin-1表达显著升高（33.14±2.16%）。自噬抑制剂3-MA能够显著降低模型组Beclin-1表达（15.27±1.51%vs模型组,P<0.001）。标本标记过程中使用2%BSA/PBS与不使2%BSA/PBS测得的Beclin-1表达率分别是35.40±2.13%和43.99±7.26%,两者有统计学差异（P<0.01）一抗浓度为0.004μg/μL时,测得的Beclin-1表达率最高（35.40±2.13%）,一抗浓度为0.002、0.006时,测得的Beclin-1表达率均所下降（分别为33.54±2.27%,29.95±1.76%）；抗体孵育温度在室温（25℃）和37℃水浴时,测得的Beclin-1表达率无显著性差异（36.27±3.29%vs36.70±2.84%6,P>0.05）；孵育时间30min、60min检测结果无统计学差异（36.38±3.51%vs36.36±3.64%,P>0.05）,但孵育时间15min时,测得的Beclin-1表达率有所降低（31.67±3.76%vs36.38±3.51%,P<0.05）；标本制备后立即检测和放置于4℃保存6h检测结果无显著性差异（36.30±3.29%vs34.93±3.38%,P>0.05）,但4℃保存24h检测结果明显下降（20.81±3.99%vs36.30±3.29%,P<0.001）；标记制备后的标本固定在1%多聚甲醛中并置放于4℃可保存3天其检测结果与当天检测的结果无显著性差异（36.59±3.19%vs37.67±3.38%,P>0.05）,第7天检测结果则明显下降。（24.04±3.83%vs37.67±3.38%,P<0.001）。3.PC12细胞缺糖缺氧2h再给氧期间,细胞成活率、细胞ATP含量先降低后升高,Beclin-1表达先升高后降低,细胞形态的变化是先损伤后改善。PC12细胞缺糖缺氧2h,再给氧0、4、10h,细胞成活率持续下降（64.6±4.6%、51.8±1.6%、36.0±5.6%）,再给氧24、48h细胞成活率持续上升（56.8±3.7%、73.4±4.3%）。再给氧0、4h细胞ATP含量持续下降（3.4±0.5、3.2±0.4mg/g蛋白）,10h有所恢复（4.1±0.7mg/g蛋白）,但是仍低于缺氧前水平（4.6±0.9mg/g蛋白）,再给氧24、48h,细胞ATP含量持续升高（5.4±0.9、6.7±1.2mg/g蛋白）。再给氧24h细胞ATP含量显著低于正常对照组（5.4±0.9mg/g蛋白vs7.7±1.2mg/g蛋白,p<0.001）。再给氧0、4、10、24,48h,Beclin-1蛋白表达上调（10.5±1.4%、12.7±1.4%、16.1±1.8%、28.2±3.1%、20.0±3.2%）,再给氧48h与再给氧24h相比,Beclin-1蛋白表达下调。再给氧0、4、10h,细胞数量持续减少,并在10h时减少至最低值,突起变短,细胞变圆变小,皱缩聚集。再给氧24h,细胞数量增加,胞体变得较为饱满,轴突变长。再给氧48h,细胞数量增加,胞体变得饱满,突起相互交织形成网状。正常培养的细胞较难观察到自噬体,再给氧24h的细胞可明显观察到一些自噬体。再给氧0h,ATP与Beclin-1呈显著负相关关系（r=-0.61,P<0.05）,再给氧24h,ATP与Beclin-1没有显著相关性（r=0.24,P>0.05）。4.体外正常培养48h的PC12细胞缺糖缺氧2h,再给氧24h后,在光镜下可观察到细胞数量减少,胞体变圆,轴突变细长；在电镜下能够明显观察到一些自噬体；Beclin-1表达显著增加（28.5±2.3%,vs正常组（6.3±0.8%）,p<0.001）,OD值显著降低（0.46±0.02,vs正常组（0.79±0.04）,p<0.001）；MMP显著降低（335.2±19.3,vs正常组（542.5±26.6）,p<0.001）；[Ca2+]i显著升高（294.9±42.7,vs正常组（124.7±14.9）,p<0.001）。β-细辛醚（20,30,45μg/mL）和尼莫地平（10μmol/L）都非常显著地降低模型组细胞Beclin-1表达（19.1±2.8%、17.6±2.6%、20.5±2.8%、15.6±2.5%,vs模型组,p<0.001）和[Ca2+]i（137.8±23.5、132.4±25.5、169.7±23.9、139.0±19.0,vs模型组p<0.001）,升高模型组细胞的OD值（0.64±0.07,0.75±0.09,0.56±0.08,0.74±0.04,vs模型组,p<0.01）和MMP（422.4±27.5,478.7±21.6,414.1±25.1,483.5±28.2,vs模型组,p<0.01）,并且不同程度地增加造模细胞数量,改善细胞形态。其中β-细辛醚（30μg/mL）使模型组细胞的活力和[Ca2+]i接近于正常组水平（P>0.05）。结论：1.使用终浓度约为6%的HCLO₄反复冻融细胞2次能够较好的提取细胞内ATP。HPLC能够分离细胞内的复杂成分,并且快速、准确定量分析细胞内ATP含量。2.流式细胞仪和透射电子显微镜的研究结果表明,缺糖缺氧再给氧使PC12细胞自噬表达增加。流式细胞仪能够简便、快速、准确地定量细胞Beclin-1的相对百分含量。筛选分析结果表明,优化的流式细胞仪检测Beclin-1蛋白的参数是：标记过程中使用2%牛血清的PBS封闭液；一抗浓度为0.004μg/μL;孵育温度室温（25℃）,孵育时间30min；标记制备后标本立即检测或置4℃保存6h内检测,否则要固定在1%多聚甲醛并于4℃保存,3天内检测。3.PC12细胞缺糖缺氧期间升高的自噬与能量代谢障碍有关,再给氧期间升高的自噬与[Ca2+]i、降低的MMP有关。自噬在2h缺糖缺氧再给氧期间可能起到细胞保护的作用。4.20、30、45μg/mL的p-细辛醚预给药1h,缺糖缺氧期间给药2h,能不同程度地减轻缺糖缺氧再给氧造成的细胞损伤,其中以30μg/mL的β-细辛醚效果最佳。p-细辛醚可能通过钙离子拮抗作用,改善细胞功能,从而降低自噬,提高细胞成活率。更多还原

【Abstract】 ObjectiveFirst, study the relationship between autophagy and ATP during reoxyg enation.Second, study the role of autophagy in PC12cells subjected to2h of oxygen-glucose deprivation （OGD） with different time-points of reoxygenation.Third, study the effect of β-asarone on Beclin-1, intracellular free calcium concentration ([Ca²⁺] mitochondrial membrane potential （MMP）, cell viability and cellular morphology in PC12cells treated with2h OGD followed by24h reoxygenation.MethodsFirst, establish an oxygen-glucose deprivation and reoxygenation induced injury model of PC12cells:The high glucose Dulbecco’s modifed Eagle’s medium （DMEM） was exchanged for Earle’s balanced salt solution, and the plates were placed in a hypoxia chamber with1%O₂,94%N₂and5%CO₂for2h. Then the full culture medium replaced the Earle’s balanced salt solution, and the plates were placed under an atmosphere of95%air and5%C02for different time-points of reoxygenation.Second, establish a method of high-performance liquid chromatography （HPLC） for detection of ATP. Study the specificity of the ATP in cell extracts by setting negative control group, standard control group, and sample control group.10mg ATP was dissolved with10mL double distilled water, the concentration of which was1. OOmg/mL.0.5mL β-asarone （1.00mg/mL） was diluted with9.5mL double distilled water to achieve the concentration of 50.0μg/mL16,32,48,64,80μLβ-asarone （50.0μg/mL） was diluted with appropriate double distilled water to achieve the concentration of0.080,0.160,0.240,0.320,0.400μg/mL.20μL of each concentration was injected for analysis. Investigate the linear relationship of the ATP concentration and peak area within0.080-0.400mg/mL ATP. For precision and accuracy, five replicate quality control samples at three concentrations (0.080,0.240and0.400（μg/20μL） were continuously assayed.600μL PBS was added to suspense the cells at three time-points of reoxygenation （0,10,24h）.80μL of HClO₄（concentration50%, v/v） was added to the cell suspensions, then-80℃freezed and room temperature thawed for two times.0.1,0.2,0.4μg/mL ATP standard solution was added to the cell suspensions, respectively.300μL of2mol/L NaOH was added to adjust pH value to neutral. The neutral cell suspensions were then spun at12000rpm for15min. The upper] ayer was injected for analysis and for investigation of the recovery. For investigation of the stability of samples, ATP in cell extractions （reoxygenation time-point24h） were detected after storage at-80℃for0d,3d,7d.Third, establish a flow cytometric method for quantitative detection of Beclin-1expression:Investigate the influence of3-methyladenine （3-MA, the autophagic inhibitor）,2%Bovine serum albumin/Phosphate buffer solution （BSA/PBS）, the concentration of primary antibody （0.002,0.004,0.006μg/μL）, incubation temperature （25,37℃）, incubation time （15,30,60min）, preserved time and methods （Sample preparation was determined immediately or determined after6h or24h of storage at4℃respectively. Sample preparation was determined after0,3or7d of storage fixing with1%paraformaldehyde at4℃, respectively.） on analysis of Beclin-1.Forth, study the role of autophagy and the relationship of autophagy with ATP. After growth of PC12cells for48h, the full culture medium wa s discarded, the cells was washed once with PBS and incubated with Earle’ s balanced salt solution in a hypoxia chamber with a compact gas oxygen c ontroller to maintain oxygen concentration at1%by injecting a gas mixtu re of94%N2and5%CO2for2h. After OGD, the cells were transferred bac k to full culture medium with oxygen for0,4,10,24,48h. ATP （includi ng normal control group at24h reoxygenation） was measured using HPLC, c ell protein determined using the BCA assay, cell viability was measured b y3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assa y, cellular morphology was observed under inverted phase contrast microsc ope, and autophagosomes （model control group and normal control group at24h reoxygenation） were observed under transmission electron microscope. Cells grown in the same well were divided into two groups. One was used for ATP detection, and the other was used for Beclin-1analysis. Correlat ion of ATP and Beclin-1was analyzed.Fifth, Protective mechanism of β-asarone on Beclin-1in PC12cells following oxygen-glucose deprivation and reoxygenation induced injury. After growth for48h, PC12cells were incubated with full culture medium containing β-asarone （20,30or45μg/mL） or nimodipine （10μmol/L） under normoxic conditions for1h before OGD. The full culture medium containing drug was discarded. The cells were rinsed once with PBS, and incubated with Earle’s balanced salt solution containing beta-asarone （20,30or45μg/mL） or nimodipine （10μmol/L） for2h OGD. The Earle’s balanced salt solution was discarded, and then PC12cells were incubated with full culture medium free of drug under normoxic conditions for24h. After these treatments, Beclin-1,[Ca²⁺]i and MMP were analyzed by flow cytometry, cell viability was measured by MTT assay, cellular morphology was observed under inverted phase contrast microscope, and autophagosomes were observed under transmission electron microscope.Sixth, the methods of statistical analysis were used in this study. Measurement data were expressed as mean±standard deviation （Mean±SD）. Statistical significance was determined by independent-samples t-test. Correlation analysis was determined by Pearson correlation analysis.ResultsFirst, a good linear relationship （r=0.9995） between ATP concentration and peak area in0.08-0.40mg/mL ATP. For precision and accuracy, the relative standard deviation （RSD） of the three concentrations （0.080,0.240and0.300μg/mL） were2.8%,2.1%and1.8%, respectively （five samples for each concentration level）, indicating the precision and accuracy was good. The average recoveries of three concentrations （0.100,0.200and0.400μg/mL） were95.6±2.8%, respectively, and the RSD were2.9%.（five samples for each concentration level）, indicating the method is accuracy. ATP concentration was0.331±0.010,0.312±0.012,0.303±0.008μg/mL, respectively, after0,3,7d of storage in-80℃refrigerator, indicating the stability of storage within7d is good.Second, Beclin-1（6.49±1.19%） expression was very low in normal control cells. However, after OGD/R treatment, Beclin-1expression （33.14±2.16%） was significantly upregulated. The autophagic inhibitor3-MA significantly reduced Beclin-1expression （15.27±1.51%） in OGD/R treated cells （P<0.001）. Detected Beclin-1was35.40±2.13%or43.99±7.26%in blocked group or non-blocked group, respectively. These two groups were significant statistical difference （P<0.01）. Detected Beclin-1was highest at0.004μg/μL of primary antibody （35.40±2.13%）. Detected Beclin-1was decreased at0.002,0.006μg/μL of primary antibody （33.54±2.27%,29.95±1.76%）. Detected Beclin-1was36.27±3.29%or36.70±2.84%in group incubated at room temperature （25℃） or at37℃, respectively. There was no significant statistical difference （P>0.05）. Detected Beclin-1was36.38±3.51%or36.36±3.64%in sample incubated for30min or60min, respectively. The statistical difference was no significant （P>0.05）. However, detected Beclin-1（31.67±3.76%） in sample incubated for15min was significantly decreased （P<0.05） compared to group incubated for30min or60min. Samples were stored at4℃and were not fixed with1%paraformaldehyde. There was no significant statistical difference （P>0.05） of detected Beclin-1（36.30±3.29%,34.93±3.38%, respectively） in group determined immediately or determined after6h of sample preparation. Compared to group determined immediately, detected Beclin-1（20.81±3.99%） was dramatically decreased in group determined after24h of sample preparation （P<0.001）. Samples were all fixed with1%paraformaldehyde and stored at4℃. Detected Beclin-1was36.59±3.19%in sample after3d of storage and37.67±3.38%in sample in0d of storage. They had no significant statistical difference （P>0.05）. Detected Beclin-1was significantly reduced in group after7d of storage （24.04±3.83%） compared to group in0d of storage （P<0.001）.Third, during reoxygenation, the cell viability and ATP content decreased at first, and then increased, Beclin-1expression was upregulated at first, and then was downregulated, and cellular morphology was injury at first, and then was improved. Cell viability continued to decrease at0,4,10h reoxygenation （64.6±4.6%,51.8±1.6%,36.0±5.6%）, and increase at24,48h reoxygenation （56.8±3.7%、73.4±4.3%）. ATP continued to decrease at0,4h reoxygenation （3.4±0.5,3.2±0.4mg/g protein）. It recovered at10h reoxygenation （4.1±0.7mg/g protein）, but it was still lower than that before OGD （4.6±0.9mg/g protein）. ATP continued to increase at24,48h reoxygenation （5.4±0.9,6.7±1.2mg/g protein）. ATP at24h reoxygenation was lower than normal control （5.4±.09mg/g protein vs7.7±1.2mg/g protein, p<0.001）. Beclin-1expression was upregulated at0,4,10,24,48h reoxygenation （10.5±1.4%,12.7±1.4%,16.1±1.8%,28.2±3.1%,20.0±3.2%）. Beclin-1expression at48h reoxygenation was lower than that at24h reoxygenation. The numbers showed a trend of reduction at0h reoxygenation after2h OGD,, and continued to be reduced as well at4and10h reoxygenation, reaching its minimum at10h reoxygenation, while they were increased significantly at24and48h. Somas were smaller at0,4or10h reoxygenation than that before OGD. The cells exhibited round, gathered, slender and degenerated morphology at0,4and10h reoxygenation. Somas became satiation, and neurite became slender at24h reoxygenation. Somas was satiation, neurite interweave with each other to form a mesh at48h reoxygenation. Autophagosomes cound be hardly observed in the normal cultured cells, but some autophagosomes could be obviously observed in cells treated with OGD/R. At0h reoxygenation after2h OGD, a significant negative correlation was occurred between ATP and Beclin-1expression （r=-0.61, P<0.05）, while there was not a significant correlation between ATP and Beclin-1expression at24h reoxygenation （r=0.24, P>0.05）.Forth, After24h reoxygenation, the number of neurons showed a significant reduction, which was observed under inverted phase contrast microscope. The PC12cells exhibited round, slender and degenerated morphology. Some autophagosomes could observed under transmission electron microscopy. Beclin-1expression was increased （28.5±2.3%, vs normal control6.3±0.8%, p<0.001）. Cell viability （OD value） was decreased （0.46±0.02, vs normal control0.79±0.04, p＜0.001）. MMP was decreased （335.2±19.3, vs normal control （542.5±26.6）, p<0.001）.[Ca⁹2+)] was increased （294.9±42.7, vs normal control （124.7±14.9）, p<0.001）. Beta-asarone （20,30,45g/mL）μand nimodipine (a calcium antagonist,10（μmol/L） both significantly decreased Beclin-1expression （19.1±2.8%,17.6±2.6%,20.5±2.8%,15.6±2.5%, vs model control group, p<0.001） and [Ca²⁺]（137.8±23.5,132.4±25.5,169.7±23.9,139.0±19.0,vs model control group, p<0.001）, but increased MMP （422.4±27.5,478.7±21.6,414.1±25.1,483.5±28.2, vs model control group, p<0.01）, cell viability （0.64±0.07,0.75±0.09,0.56±0.08,0.74±0.04, vs model control group, p<0.01） in PC12cells treated with OGD/R. They also increased the cell numbers and improved the cellular morphology in PC12cells subjected to OGD/R. For cell viability and [Ca²⁺]; there was no significant difference between beta-asarone （30μg/mL） group and normal control group normal control group （P>0.05）.ConelusionsFirst, ATP can be well extracted from the cell using6%HClO₄（final concentration, v/v）. HPLC can isolate complex components in the cells, and analyzed intracellular ATP content quickly, accurately and quantitatively.Second, the results got from flow cytometry and transmission electron microscopy indicates that OGD/R can generate autophagy. Flow cytometry c an easily, quickly, accurately and quantitatively determine Beclin-1. The results of screening analysis indicate that the optimization of multi-pa rameters for Beclin-1protein staining is as follows.2%BSA-PBS was used for sample block. Concentration of primary antibody was0.004μg/μL. Sam ples were incubated at room temperature（25℃） for30min. They were detec ted immediately or in6h storing at4℃or in3days fixing in1%paraf ormaldehyde and storing at4℃after sample preparation.Third, energy metabolism dysfunction can induce autophagy during OGD in PC12cells. Increased [Ca²⁺], and decreased MMP may induce autophagy during reoxygenation in PC12cells. Autophagy may be a protective effect on PC12cells treated with different time-points of reoxygenation after2h OGD.Forth, treatment with β-asarone （20,30or45μg/mL） for1h before OGD and2h during OGD can protect PC12cells against OGD/R induced injury, of which30μg/mL β-asarone is the most effective. It may reduce Beclin-1expression partly by decreasing [Ca²⁺],and increasing MMP in OGD/R treated PC12cells.β-asarone may improve cell function through calcium antagonism, and then reduces autophagy and improves cell viability.更多还原