【作者】 甄雪克；

【导师】 于炎冰；

【作者基本信息】 北京协和医学院 ， 神经外科学， 2014， 博士

【摘要】 糖尿病周围神经病变(Diabetic Peripheral Neuropathy, DPN)是最常见的糖尿病神经系统并发症,致残率高,但缺乏特异性的治疗方法。目前内科治疗DPN的方法以控制血糖、营养神经等对症治疗为主,故特异性、有效性较差,而外科治疗DPN的方法更是乏善可陈。1988年Dellon率先提出可以应用周围神经减压术治疗DPN,并于1992报道了首批采用周围神经减压术治疗的四肢DPN的病例,疗效优良,从此开启了外科手术治疗DPN的新纪元。本课题第一部分总结了我科自2004年3月至2012年8月采用周围神经显微减压术治疗的DPN患者42例(50侧),分别阐述了外科诊治DPN的诊断标准、不同部位DPN的临床表现、手术指征的把握、手术方法的选择及术中要点、术后注意事项、有效率、复发率、并发症等。结果显示：①、上肢28例(32侧)术后麻木、疼痛症状缓解率是100%；50%以上的患者手部力弱、运动笨拙症状得到缓解；80%-90%患者手指两点辨别觉恢复正常；术后电生理检测显示100%患者均有改善；术后总的复发率小于7%。②、下肢14例(18侧)患者术后麻木、疼痛症状缓解率为77.7%(14/18),肢体平衡问题术后73.3%(11/15)缓解。所有患者随访5—113个月,平均74个月,随访期间无一例发生顽固性溃疡或截肢。对于DPN患者术后疗效的评价目前普遍依据患者的主观感受、临床体格检查及神经电生理监测,未来还需要寻求更为科学、客观、准确的指标来进行评估,如观察病变神经的显微结构甚至超微结构等。本课题第二部分通过采用腹腔注射小剂量链脲佐菌素(Streptozotocin, STZ)的方法制备Ⅰ型糖尿病(Diabetes mellitus, DM)的大鼠模型,持续观察从DM发病到出现DPN的自然过程。DPN大鼠造模成功后,对其足迹步态、神经电生理监测指标、表皮内神经纤维密度以及神经组织病理学检查进行了综合分析,结果显示：腹腔一次性注射60mg/kg剂量的STZ后约8周DPN大鼠动物模型制备成功；模型组与对照组相比：①、足迹步态参数中：后足跟至前足趾之间距离(Print length,PL)、第二足趾至第四足趾之间的距离(Intermediarytoespread, ITS)的测量值的差异具有统计学意义(P<0.05)；②、90%(9/10)DPN大鼠右后肢跗管胫神经入管后较入管前动作电位波幅下降,神经传导速度下降；③、DPN大鼠右后肢外踝上方lcm处表皮内神经纤维数量明显减少、甚至无神经纤维显示,走形更加不规则,更为纤细；④、神经组织严重水肿,髓鞘肿胀明显,局部有空泡变性,轴索水肿,部分轴索分离、变性。通过以上临床和基础研究可以得出周围神经卡压是DPN的重要致病因素,周围神经显微减压手术正是针对此种病因来确切地治疗DPN,然而针对导致DPN的血管因素和代谢因素的治疗则非手术所及,同时我们在临床实践中发现,部分DPN患者在显微减压术的基础上联合应用丹红注射液(Dan Hong injection, DH)可使症状缓解的更为明显,从而推测DH可以促进病变神经的恢复,因此我们在现有的研究基础之上通过分子生物学实验初步阐明了其直接作用于周围神经雪旺氏细胞(schwann cell, SC)的机理。本课题第三部分将SC分为正常对照组、加入糖基化终末产物(Advanced Glycation End Products, AGEs)组、DH+AGEs组,培养48h后对同倍视野下SC细胞计数进行SPSS统计分析；在测定SC表达脑源性神经因子(Brain-derived neurotrophic factor, BDNF)的实验中,运用实时定量逆转录聚合酶链反应(real-time RT-PCR)和Western blot技术检测BDNF mRNA和蛋白含量：在测定DH促进SC表达BDNF信号传导通路实验中运用RT-PCR技术对BDNF mRNA进行检测。结果表明：①、DH可直接作用于周围神经的SC细胞；②、DH能明显抑制AGEs导致的SC的凋亡；③、DH能促进SC表达BDNF的mRNA和蛋白；④、PKC及下游的MEK、ERKs和p38蛋白通路可能参与介导了DH促进BDNF mRNA表达的上调。更多还原

【Abstract】 Diabetic Peripheral Neuropathy is the primary Diabetic Mellitus complication in nervous system.but lacked of effective treatments.Blood surgar level control,neurotrophy intervention and symptomatic treatment may be the main methods from medical,however,specific surgical managements for DPN is less.In1988,Dellon firstly introduced the surgical treatment for DPN with microsurgical decompression,and then reported the excellent therapeutic efficacy in1992.It became an effective surgical method for DPN from then on.In part I, retrospectively analysed42(50sides)DPN patients underwent microsurgical decompression from3,2004to8,2012. Diagnostic criteria,clinical manifestation,operative indication,operative methods selection,key points during operation,effective rate and recurrence rate were analysed respectively.Results demonstrated that:①.all the28(32sides)patients alleviated their pain and numbness in upper limbs, more than50%patients improved their hands motor function,80%-90%patients recovered their two point discrimination, electrophysiologic monitoring showed the improvement rate was100%,recurrence rate under7%.②.77.7%patients(14/18sides) released their pain and numbness in lower limbs,73.3%(11/15) cases resolved their balance dysfunction.No one get amputation because of DPN during follow-up period.Postopertion evaluation at present depends only on patient self felling,physical examination and electrophysiologic monitoring results.So it is necessary to explore more scientific,objective and precise index to judgement operative effection, for example the nervous microstructure,and animal experiment become the first selection.In prat Ⅱ, we first established the type I diabetic rat models by intraperitoneal injection of STZ (60mg/kg) in male Wistar rats, After successful modeling, the footprint and gait analysis,electrophysiologic examination,nerve fibers density observation in epidermis and right hind tibial nerve tissue staining were performed.Results showing: DPN model preparation time was at least8weeks. Compared with control group:The footprint and gait parameters (PL and ITS) were abnormal in the model group (P<0.05), but there was no significant difference(P>0.05) in TS; The tibial nerve conduction velocity in tarsal tunnel was decreased significantly in the model group (90%rats), compared to0in the control group. The density of nerve fibers of calf epidermal was decreased in the model group. The histopathological examination showed obvious nerve edema,demyelination and axon degeneration.During clinical practice.we fount that the treatment result was better combined microsurgical decompression with DH compared to single surgical treatment.Therefore.in part Ⅲ, we carried out molecular biology experiment to explore the effective mechanism of DH for peripheral nerve. In SC apoptosis experiment,SC were divided into control group、AGEs group and DH+AGEs group, SPSS was used for statistical analysis of cells number. RT-PCR and western blot was used to detect the BDNF mRNA and protein expression. PKC inhibitor PI3K inhibitor、PKA inhibitor、 MEK inhibitor、ERK inhibitor and p38inhibitor were respectively added into the experiment to determine signaling pathways. The Results demonstrated:SC was decreased significantly in AGEs group than control one (P=0.001),but increased in DH+AGEs group than AGEs group (P=0.0006);DH could promote the expression of BDNF mRNA and protein than control one (P=0.00004); compared to control group, BDNF mRNA expression was decreased in DH+Calphostin C grop (P=0.001); all of the BDNF mRNA expression was significantly decreased in DH+U0126. DH+FR180204and DH+SB203580group(P<0.05). The Conclusion was:DH could effectively inhibit SC apoptosis induced by AGEs and significantly promote BDNF expression; PKC and EK/ERK/p38may be the important signaling transconduction pathway for BDNF expression.更多还原