【作者】 陈静；

【导师】 杨金蓉；

【作者基本信息】 成都中医药大学 ， 中西医结合基础， 2020， 硕士

【摘要】 目的:通过观察管灸对面神经损伤家兔PI3K/Akt与PLC/PKC通路中相关信号转导分子表达的影响,从而探讨管灸治疗周围性面瘫的分子机制。方法:选用日本大耳白兔建立面神经压榨损伤动物模型。实验分两批次进行,每批次采用家兔32只,除取材检测外,其余操作两批次完全相同。用随机数字表法从32只家兔中抽取8只,纳入假手术组,余下家兔经压榨损伤法造模后,按体重随机分为:模型组、管灸组、热疗组。造模成功5天后开始干预治疗,管灸组和热疗组分别在管灸器中加入艾绒、木炭进行灸治,假手术组和模型组不施加干预治疗。治疗结束后次日取材,第一批家兔采取灌注取材,用于免疫组化检测面神经及面神经核Bax、Bcl-2、GAP-43的表达。第二批家兔采取直接取材,用于ELISA、Western Blot检测家兔面神经PI3Kp85、Akt1/2/3、p-Akt1、PLC、p-PLC、DAG、PKCα、p-PKCα的表达。结果:1.对PI3K/Akt通路中信号转导分子的影响:与模型组相比,假手术组家兔面神经PI3Kp85、Akt1/2/3、p-Akt1蛋白表达较低(P<0.05或P<0.01);管灸组家兔面神经PI3Kp85、Akt1/2/3、p-Akt1蛋白表达增高(P<0.01);热疗组家兔面神经PI3Kp85、Akt1/2/3、p-Akt1蛋白表达增高(P<0.05或P<0.01)。2.对PLC/PKC通路中信号转导分子的影响:与模型组相比,假手术组家兔面神经DAG浓度较低(P<0.05),p-PKCα蛋白表达较高(P<0.01);PLC、p-PLC、PKCα蛋白表达无明显差异(P>0.05);管灸组家兔面神经p-PLC、PKC、p-PKCα蛋白表达及DAG浓度增高(P<0.05或P<0.01),PLC蛋白表达无明显差异(P>0.05);热疗组家兔面神经PKCα蛋白表达增高(P<0.01),其余上述指标表达无明显差异(P>0.05)。3.对神经修复再生相关效应分子的影响:与模型组相比,假手术组家兔面神经Bax蛋白表达较低(P<0.05),面神经核Bax、Bcl-2、GAP-43及面神经Bcl-2、GAP-43蛋白表达无明显差异(P>0.05);管灸组家兔面神经核及面神经Bcl-2、GAP-43蛋白表达增高(P<0.05),面神经核及面神经Bax蛋白表达无明显差异(P>0.05);热疗组家兔面神经核及面神经上述指标表达无明显差异(P>0.05)。结论:1.模型组PI3Kp85、Akt1/2/3、p-Akt1蛋白表达及DAG浓度较假手术组增高,提示家兔面神经损伤对PI3K/Akt及PLC/PKC通路的信号转导有一定增强作用,从而促进机体的自我修复。2.热疗组PI3Kp85、Akt1/2/3、p-Akt1、PKCα蛋白表达较模型组增高,提示热疗可通过增强PI3K/Akt及PLC/PKC通路信号转导,从而促进损伤面神经的修复再生。3.管灸组PI3Kp85、Akt1/2/3、p-Akt1、p-PLC、DAG、PKCα、p-PKCα及效应因子Bcl-2、GAP-43的表达较模型组增高,提示管灸治疗可通过增强PI3K/Akt及PLC/PKC通路信号转导,从而促进损伤面神经的修复再生。4.管灸较热疗的作用更广泛,趋势更强。因此可认为管灸的治疗作用优于热疗。更多还原

【Abstract】 Objective:To observe the effect of tube moxibustion on the expression of related signal transduction molecules in PI3K/Akt and PLC/PKC pathway in rabbits with facial nerve injury,so as to explore the molecular mechanism of tube moxibustion in the treatment of peripheral facial paralysis.Methods:The model of facial nerve crush injury in rabbits was established.The experimen t was divided into two stages with 32 rabbits each experiment.Except for sampling and testing,the other operation were exactly same.Eight rabbits were selected from32 rabbits by random number table method and included in sham operation group.The remaining rabbits were randomly divided into model group,tube moxibustion group and thermotherapy group according to their body weight.Intervention treatm ent began after 5 days of successful modeling.Moxa velvet and charcoal were added to the tube moxibustion apparatus in the tube moxibustion group and thermotherapy group respectively,while no intervention treatment was given in the sham operation group and the model group.The samples were taken the next day after treatment,and the first batch of perfusion samples were used to detect the expression of Bax,Bcl-2and GAP-43 in facial nerve and facial nucleus by immunohistochemistry.The second batch of samples were taken directly for ELISA and Western Blot to detect the expre ssion of PI3Kp85,Akt1/2/3,p-Akt1,PLC,p-PLC,DAG,PKCα and p-PKCα in rabbit facial nerve.Results:1.Effect on signal transduction molecules in PI3K/Akt pathway: Compared with the model group,the expression of PI3Kp85,Akt1/2/3 and p-Akt1 protein in facial nerve in sham operation group was lower(P<0.05 or P<0.01).PI3Kp85,Akt1/2/3and p-Akt1 protein expression in facial nerve in tube moxibustion group was higher than that in model group(P<0.01),and the protein expression of PI3 K p85,Akt1/2/3and p-Akt1 in facial nerve in hyperthermia group was higher than that in model group(P<0.05 or P<0.01).2.Effect on signal transduction molecules in PLC/PKC pathway: Compared with the model group,the expression of DAG in facial nerve was lower(P < 0.05),the expression of p-PKCα protein was higher than that in sham operation group(P<0.01),and there was no significant difference in the expression of PLC,p-PLC and PKCαprotein in sham operation group(P>0.05).The expression of p-PLC,DAG,PKCαand p-PKCα protein in facial nerve in tube moxibustion group and the expression of PKCα protein in facial nerve of rabbits in hyperthermia group was higher than that in sham operation group(P<0.05 or P<0.01),and there was no significant difference in other above mentioned indexes(P>0.05).3.Effects on effector molecules related to nerve repair and regeneration: Com pared with the model group,the expression of Bax protein in facial nerve in sham operation group was lower(P<0.05),but there was no significant difference in Bax,Bcl-2,GAP-43,Bcl-2 and GAP-43 protein expression in facial nucleus(P>0.05).The expression of Bcl-2 and GAP-43 protein in facial nucleus and facial nerve washigher in the tube moxibustion group(P<0.05),but there was no significant different ce in the expression of Bax protein in the facial nucleus and facial nerve in the tube moxibustion group(P>0.05),and there was no significant difference in the express ion of the above indexes in the facial nucleus and facial nerve in the hyperthermia group(P>0.05).Conclusion:1.The protein expression of PI3Kp85,Akt1/2/3,p-Akt1 and DAG in the model group was higher than that in sham operation group,suggesting that facial nerve injury can enhance the signal transduction of PI3K/Akt and PLC/PKC pathway,so as to promote the self-repair of the body.2.The protein expression of PI3Kp85,Akt1/2/3,p-Akt1 and PKCα in the hyper thermia group was higher than that in the model group,suggesting that hyperthermia can promote the repair and regeneration of injured facial nerve by enhancing the signal transduction of PI3K/Akt and PLC/PKC pathway.3.The expressions of PI3Kp85,Akt1/2/3,p-Akt1,p-PLC,DAG,PKCα,p-PKCαand effect factors Bcl-2 and GAP-43 in the tube moxibustion group were higher than those in the model group,suggesting that tube moxibustion can promote the repair and regeneration of injured facial nerve by enhancing the signal transduction of PI3K/Akt and PLC/PKC pathway.4.The effect of tube moxibustion is more extensive and the trend is stronger than that of hyperthermia.Therefore,it can be considered that the therapeutic effect of tube moxibustion is better than that of thermotherapy.更多还原