摘要
目的：本实验旨在通过：1．研究静脉麻醉药丙泊酚与咪唑安定、氯胺酮联合应用对缺血缺氧及再灌注损伤PC12细胞的保护作用；2．研究单胺递质耗竭剂利血平和对氯苯丙氨酸对丙泊酚保护作用的影响，为临床工作中脑缺血及再灌注损伤的救治提供思路和理论依据。实验分组与方法：1．细胞培养：将分化的PC12细胞用含胎牛、马血清各5％的DMEM细胞完全营养液培养：传代至96孔细胞培养板，用于细胞保护实验；传代至6孔细胞培养板，用于细胞内钙离子浓度测定。2．静脉麻醉药丙泊酚与咪唑安定联合应用对缺血缺氧及再灌注损伤PC12细胞的保护作用。取生长期96孔板培养PC12细胞，将细胞分成缺血缺氧及再灌注损伤对照组、丙泊酚(12.4μmol·L~(-1))组、咪唑安定(浓度分别为3.3、10、30μmol·L~(-1))组及丙泊酚与咪唑安定联合应用组。利用缺血缺氧及再灌注损伤细胞模型，观察各组细胞存活率的变化。取生长期6孔板培养PC12细胞，将细胞分成损伤对照组、丙泊酚(12.4μmol·L~(-1))组、咪唑安定(3.3、10μmol·L~(-1))组及丙泊酚与咪唑安定联合应用组。观察各组对缺血缺氧及再灌注损伤PC12细胞内钙离子浓度的影响。3．静脉麻醉药丙泊酚与氯胺酮联合应用对缺血缺氧及再灌注损伤PC12细胞的保护作用。取生长期96孔板培
军医遗传合院硕士会代价或
养 pC12细胞，将细胞分成损伤对照组、丙泊酚＊．4ifmOI·L－’）组、氯gy酮
o石、18、90llmol工一‘）组及丙泊酚与氯胺酮联合应用组，观察各组细胞存
活率的变化。取生长期 6孔板培养 PC细胞，将细胞分成损伤对照组。
丙泊酚一2．4pmol·L’）组、氯胺酮O6、18pmOI工一’）组及丙泊酚与氯sy酮联
合应用组，观察各组对细胞内钙离子浓度的影响。4．丙泊酚与利血平联
合应用对缺血缺氧及再灌注损伤 PC细胞保护作用。取生长期 96孔板
培养PC细胞，将细胞分成损伤对照组、丙泊酚门．4J7．4＊·卜‘）
组、利血平门石、8、40卜mol工’）组及两者联合应用组，观察各组细胞存
活率的变化。取生长期 6孔板培养 PC细胞，将细胞分成损伤对照组。
丙泊酚（12．4、37．4pmol·L1）组、利血平（40Hmol·L‘）组及丙泊酚与利血平
联合应用组，观察各组细胞内钙离于浓度的变化。5．丙泊酚与对氯苯丙
氨酸…ara－chlorophenylalanine pCM）联合应用对缺血缺氧及再灌注损伤
PC 12细胞保护作用。取生长期96孔极培养PC12细胞，将细胞分成损伤
对照组、丙了酚（12．4、37．4、112pmol工”’）组、对氯苯丙氨酸（．l、3．3、
10HmO·L”’）组及两者联合应用绳，观察各组细胞存活率的变化。取生长
期 6孔板培养 PC细胞，将细胞分成损伤对照组、丙泊酚门．4、
37．4pmol·L”’）组、对氯苯丙氨酸门m＊·L“’）组及两者联合应用组，观察
各组细胞内钙离于浓度的变化。6．实验时用低糖无血清DMEM细胞培
养液在37C及5％COZ、95％NZ混合气体环境下培养细胞模拟缺血缺氧；
14小时后，恢复高糖DMEM细胞完全培养液及37OC、5％COZ空气环境
培养细胞摸拟再灌注4小时。损伤对照组不做任何其它处理，药物处理
组则在实验时将相应浓度的药物加人到细胞培养液中。药物处理含括缺
血缺氧及再灌注的整个过程。测定指标包括，乳酸脱氢酸释放量（LD法）
和活细胞吸光度AS川m值（MTT法＼ 以及细胞内钙离子浓度广ura 2／AM
荧光标记法户 结果：1．咪哩安定O．3、10、30pmol·L’）与内泊酚
（12．4pmol工一’）对缺血缺氧及再灌注损伤 PC细胞均有保护作用
4
军区进修沓院顾十合性价大
o＜0．01卜联合应用后，可使保护作用增强，除咪华安定3．3卜md工一’外，
其它浓度咪哩安定与两泊酚联合应用组与两泊酚单独应用组相比存在显
著性差异（Ino．0肝与对照组相比，咪华安定与丙泊酚均可降低细胞内钙
离子浓度，合用后可使细胞内钙离子浓度进一步降低（WO刀 1卜 2．氯股酮
O石。18、90卜mol·L’）对缺血缺氧及再灌注损伤 PC细胞具有保护作用
沙＜0刀5厂与两泊酚联合应用后，除3石pOOl·L－’外，合用组与两泊酚单
独应用组相比存在显著性差异（WO刀1肝 与对照组相比，氯肢酮与两泊酚
均可降低细胞内钙离子浓度，合用后作用进一步增强（Ino刀5）。3．不同
浓度的利血平对缺血缺氧及再灌注损伤 PC 细胞具有保护作?
Objective 1. To evaluate the protective actions of intravenous anesthetics propofol, midazolam and ketamine, as well as combination of propofol with midazolam or ketamine on cultured PC 12 cells impaired by mimic ischemia and following reperfusion and the possible mechanisms. 2. To evaluate the protective actions of monoamine transmitter inhibitors reserpine and para-chlorophenylalanine (pCPA) on cultured PC 12 cells impaired by mimic ischemia and following reperfusion and the possible mechanisms, as well as the changes of these effects when these inhibitors are combined with propofol. Methods 1. Cellular culture: the nerve growth factor (NGF) differentiated PC 12 cells were planted in 96 or 6 well plates in the DMEM culture complete medium consisted of 5% heat-inactivated fetal calf serum and horse serum independently. Incubation was carried out at 37 in a humidified atmosphere with 5% CO2. 2. To detect the protective actions of propofol and midazolam on PC12 cells impaired by mimic ischemia and reperfusion(I/R), the cells in the 96 wells plate were allocated into control group (impaired by ischemia and reperfusion without any treatment), propofol-treating group (at dose of 12.4 mol-L-1) midazolam-treating groups (at dose of 3.3, 10, 30 mol-L-1), and propofol/midazolam-treating groups. The growth PC 12 cells, which were incubated in the 6 wells plate, were used to detect the changes of [Ca2+]i. The cells were allocated into control group, propofol-treating group (12.4 mol-L"1), midazolam-treating groups (3.3, 10 mol-L"1), and propofol/midazolam-treating group. 3. To detect the protective actions of
propofol and ketamine on PC 12 cells impaired by mimic I/R, the cells in the 96 wells plate were allocated into control group, propofol-treating groups (12.4umol-L~'), ketamine-treating groups (3.6, 18, 90umol-L"1), and propofol/ketamine-treating groups. The growth PC 12 cells, which were incubated in the 6 wells plate, were used to detect the changes of [Ca2+]|. The cells were allocated into control group, propofol-treating group (IZ^umol-L"1), ketamine-treating groups (3.6, ISumol-L-1), and propofol/ketamine-treating group. 4. To detect the protective actions of propofol and reserpine on PC 12 cells impaired by mimic I/R, the cells in the 96 wells plate were allocated into control group, propofol-treating groups (12.4, 37.4, 112 mol-L-1), reserpine-treating groups (2.5, 10, 40umol-L"1), and propofol/reserpine-treating groups. The growth PC 12 cells, which were incubated in the 6 wells plate, were used to detect the changes of [Ca2+]j. The cells were allocated into control group, propofol-treating group (12.4, 37.4 mol-L-'), reserpine-treating group (40 mol-L-1) and propofol/reserpine-treating group. 5 To detect the protective actions of propofol and para-chlorophenylalanine (pCPA) on PC 12 wells impaired by mimic I/R, the cells in 96 wells plate were allocated into control group, propofol-treating groups (12.4, 37.4, 112umol-L~'), pCPA-treating groups (1.1, 3.3, 10 mol-L-1) and propofol/pCPA-treating groups. The growth PC12 cells, which were incubated in the 6 wells plate, were used to detect the changes of [Ca2+]j. The cells were allocated into control group, propofol-treating group (12.4, 37.4 mol-L-1), pCPA-treating group (10 mol-L-1) and propofol/pCPA-treating group. 6 We exposed the PC 12 cells to experimental oxygen-glucose deprivation culture medium in a humidified 95%N2, 5%CO2
mixed gases at 37 to mimic ischemia impairment, and then incubated in DMEM complete culture medium at 37 in a humidified 5% CO2 atmosphere to mimic the following reperfusion. The degree of cellular impairment and the cellular viability were detected using LDH and MTT assays. Meanwhile, the change of [Ca2+]i was detected by Fura-2/AM fluorescence measure. Results 1. Compared with control group, the Asvonm values increased in all of the groups treated with propofol, midazolam and ketamine (p<0.05), which indicated increasing cell survival rates. Also, the protective actions of propofol were enhanced by midazolam and
引文
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