海马脑片氧糖剥夺模型中KCC2-GABA_A受体通路对丙泊酚后处理脑保护作用的调控

发布时间：2018-06-20 09:08

本文选题：丙泊酚 + 后处理　；参考：《天津医科大学》2014年硕士论文

【摘要】：目的：临床工作中神经外科手术、脑动脉瘤切除术以及心跳骤停等可导致脑缺血再灌注损伤,患者的生存质量受到严重影响。对于脑缺血高危患者,如何合理有效的选择麻醉药物是需要麻醉医生慎重思考的问题。丙泊酚是临床常用的静脉麻醉药。能够降低脑氧代谢率、颅内压,且具有抗氧化和抑制细胞凋亡的作用,从而能够减轻脑缺血再灌注损伤。课题组前期研究工作发现,丙泊酚后处理对缺血再灌注损伤具有保护作用,其保护机制与兴奋性谷氨酸受体(a-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor, AMPAR)受体有关。近年来,越来越多的学者认同缺血再灌注损伤以及氧糖剥夺(Oxygen and Glucose Deprivation, OGD)引起的损伤为兴奋性毒性损伤,是机体兴奋-抑制平衡被打破造成。GABAa受体作为成熟哺乳动物中枢神经系统主要的抑制性受体,钾氯共转运体(K+-Cl-cotransporter, KCC2)是保证GABAA受体介导抑制性突触后传递的重要前提条件,但该通路是否参与了丙泊酚后处理的脑保护机制尚不清楚。本研究旨在探讨在离体海马脑片氧糖剥夺损伤中,丙泊酚后处理是否对具有保护作用,并进一步探讨KCC2-GABAA受体通路调控机制,从而为临床上围手术期脑缺血人群麻醉药物的合理选择提供理论依据。方法：本课题采用离体海马脑片建立氧糖剥夺、复糖复氧模型,模拟在体缺血再灌注损伤。实验主要分三部分进行,第一部分,探讨丙泊酚后处理对氧糖剥夺海马脑片的保护作用。首先建立氧糖剥夺模型,筛选合适的氧糖剥夺时间。选取7min,10min,12min,15min作为氧糖剥夺处理时间,分别记做O1-04组,处理相应时间后复糖复氧孵育1h。采用全细胞膜片钳技术诱发记录动作电位(AP),以评价不同时程OGD对CA1区锥体神经元兴奋性的影响。然后取54张海马脑片,采用随机数字表法,将其分为3组(n=12)：对照组(C组)置于正常人工脑脊液中培养7min+1h; OGD组置于持续通以95%N2的无糖人工脑脊液中孵育7min,然后放入正常人工脑脊液中孵育1h；丙泊酚后处理组(P组)给予OGD7min后转入含丙泊酚浓度为1.2μg/ml的人工脑脊液中孵育1h。采用全细胞膜片钳技术记录白发兴奋性突触后电流(spontaneous ExcitatoryPostsynaptic Currents, sEPSC, sEPSC)和自发抑制性突触后电流(spontaneous Inhibitory Postsynaptic Currents, sIPSC)的幅值和频率,探讨丙泊酚后处理对海马OGD后CA1区神经元突触传递的影响。碘化丙啶染色比较各组之间脑片的损伤程度。第二部分,探讨GABAA受体在丙泊酚后处理中的作用。72张脑片随机分为四组(n=18)：对照组C组、OGD组、丙泊酚后处理组P组,GABAA受体抑制剂组P+Bic组。采用全细胞膜片钳技术记录海马CA1区锥体神经元中GABAA受体介导的微小抑制性突触后电流(mininature Inhibitory Postsynaptic Current, mIPSC)幅值及频率的变化,碘化丙啶染色评价海马CA1区细胞损伤情况,以及氯离子染色测定CA1区锥体神经元内氯离子浓度的改变。第三部分,加入KCC2激动剂NEM,主要分为六组：对照组C组、OGD组、丙泊酚后处理组P组,C+NEM组、OGD+NEM组P+NEM组(n=24),以研究KCC2-GABAA受体在丙泊酚后处理脑保护中的调控作用。采用全细胞膜片钳技术记录mIPSC,离子染色测定CA1区锥体神经元内氯离子浓度的改变,Western Blots法测定KCC2在海马细胞的表达情况,RT-PCR法测定KCC2在海马细胞转录水平的改变。结果：第一部分：在探索OGD模型适宜时间过程中,与C1组比较,仅01组诱发动作电位的频率降低(P0.05),02和03组差异无统计学意义(P0.05)；而与01组比较,02、03组频率升高(P0.05),可能是由于OGD造成的膜电位改变更接近于钠离子平衡电位,损伤较为严重。故选取7min作为OGD时间。与Con组相比,OGD组和Pro+OGD组sEPSC的幅值和频率升高,sIPSC的幅值和频率降低,荧光强度增加,细胞凋亡增多(P0.05)；与OGD组比较,Pro+OGD组sEPSC的幅值和频率降低,sIPSC的幅值和频率相对升高细胞凋亡减少(P0.05)。第二部分：与C组比较,其余三组mIPSC幅值和频率均降低,PI荧光强度增加,细胞凋亡增多,细胞内氯离子浓度升高(P0.05)；与OGD组比较,P组mIPSC幅值和频率均升高,PI荧光强度降低,细胞凋亡减少,细胞内氯离子浓度降低(P0.05)；P+Bic组与OGD组比较差异无统计学意义(P0.05)。第三部分：与C组相比,OGD组、P组mIPSC幅值和频率均降低,细胞内氯离子浓度升高,KCC2表达水平均降低(P0.05)；与OGD组相比,P组mIPSC幅值和频率升高,细胞内氯离子浓度降低,KCC2表达水平升高(P0.05)；各组分别加入KCC2激动剂NEM后仍保持上述趋势。结论：丙泊酚后处理对离体海马脑片氧糖剥夺损伤具有保护作用,神经元性钾氯共转运体KCC2的上调以维持GABAA受体的抑制性受体特性在丙泊酚后处理中发挥了重要作用。
[Abstract]:Objective : To study the protective effect of propofol on ischemia - reperfusion injury in patients with high risk of cerebral ischemia - reperfusion injury . It is found that the post - treatment of propofol has protective effect on ischemia - reperfusion injury .

Methods : The oxygen - sugar deprivation and complex - sugar complex oxygen model was established in this study . The protective effects of propofol on brain slices in CA1 pyramidal neurons were studied .
The effect of propofol on synaptic transmission in CA1 region of hippocampal CA1 region was studied by using whole cell patch clamp technique .

Results : In the first part , in the course of exploring OGD model , only the frequency of action potential decreased ( P0.05 ) compared with C1 group , and there was no significant difference between the 02 and 03 groups ( P0.05 ) .
Compared with Con group , the amplitude and frequency of sEPSC increased , the amplitude and frequency of sIPSC decreased , the fluorescence intensity increased and the apoptosis increased ( P0.05 ) .
Compared with the OGD group , the amplitude and frequency of sEPSC in Pro + OGD group decreased , and the amplitude and frequency of sIPSC increased relative to that of OGD group ( P0.05 ) .

The second part : Compared with group C , the amplitude and frequency of the remaining three groups of mIPSC decreased , PI fluorescence intensity increased , cell apoptosis increased , and the intracellular chloride ion concentration increased ( P0.05 ) ;
Compared with OGD group , the amplitude and frequency of mIPSC increased , PI fluorescence intensity decreased , cell apoptosis was decreased , and the intracellular chloride ion concentration decreased ( P0.05 ) .
There was no significant difference between the P + Bic group and the OGD group ( P0.05 ) .

The third part : Compared with group C , the amplitude and frequency of mIPSC in the OGD group and P group decreased , and the concentration of chloride ion in the cells increased , and the expression level of KCC decreased ( P0.05 ) .
Compared with the OGD group , the amplitude and frequency of mIPSC increased in the P group , the concentration of chloride ion in the cells decreased , and the expression level of KCC increased ( P0.05 ) .
The trend was maintained after NEM was added to each group .

Conclusion : Post - treatment of propofol has protective effect on oxygen - glucose deprivation injury in isolated hippocampal slices , and the up - regulation of the neuronal potassium - chlorine co - transporter Kc2 plays an important role in the post - treatment of propofol .
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R-332;R614

【共引文献】