【作者】 屈岭；

【导师】 梁晓春； 郭赛珊； 孙华； 田国庆；

【作者基本信息】 中国协和医科大学 ， 中西医结合临床， 2009， 博士

【摘要】 【目的】1、观察筋脉通对糖尿病大鼠坐骨神经病理形态以及NF-κB蛋白表达的影响。2、观察筋脉通含药血清对高糖培养雪旺细胞超微结构以及NF-κB蛋白及其mRNA表达的影响。【方法】1、在体实验:采用链脲佐菌素(STZ,60mg/kg)一次性腹腔注射Wistar大鼠的方法造模,随机分为模型对照(DM)组、筋脉通小剂量(J小)、中剂量(J中)和大剂量(J大)组及神经妥乐平(Ntp)组,每组各10只。以体重、鼠龄及性别相匹配的正常大鼠10只作为正常对照(Con)组。灌胃16w处死,检测各组大鼠干预前及干预后4w、8w、12w和16w血糖和体重变化,通过光学显微镜和透射电子显微镜观察大鼠坐骨神经病理改变并行形态测量学分析,免疫组织化学染色法测定坐骨神经NF-κB蛋白的表达。2、离体实验:从Wistar乳鼠坐骨神经取材,原代培养并纯化雪旺细胞,S-100蛋白免疫组织化学法进行鉴定。取第3代雪旺细胞,加入不同条件培养基,分为正常对照(Con)组、高糖(Glu)组、筋脉通(JMT)组和神经妥乐平(Ntp)组进行培养,48h后行透射电子显微镜观察雪旺细胞超微结构,采用激光扫描共聚焦显微技术检测NF-κB蛋白表达水平的变化,实时荧光定量PCR技术检测雪旺细胞NF-κB mRNA的表达。【结果】1、在体实验:(1)血糖体重:糖尿病大鼠血糖值均显著高于Con组(P＜0.01),各治疗组血糖在各时间点与DM组相比无明显差异(P＞0.05);治疗组间血糖在各时间点均无明显差异(P＞0.05);干预前后,相同时间点各组大鼠之间的体重无明显差异(P＞0.05)。(2)病理形态:①形态学观察:Con组大鼠坐骨神经形态正常,排列整齐,轴索、髓鞘及雪旺细胞结构完整清晰。DM组大鼠坐骨神经有髓神经减少,排列紊乱,轴索肿胀或皱缩,髓鞘密度不均匀,部分神经髓鞘脱失,雪旺细胞变性,间质胶原纤维增多,血管基底膜增厚。各治疗组大鼠坐骨神经病变程度较DM组减轻。②形态测量学分析:与Con组相比DM组大鼠有髓神经纤维密度、小纤维密度减少,小纤维比率下降,有髓神经纤维平均横截面积、轴索平均横截面积增大,差异显著(P＜0.01);大纤维密度和髓鞘平均横截面积在各组动物间比较无明显差异(P＞0.05)。与DM组比较,各治疗组上述各值均有改善,其中J中组改善显著(P＜0.05)。各治疗组间比较,J大组较J中组有髓神经纤维密度、小纤维比率下降,轴索平均横截面积增大,差异显著(P＜0.05,P＜0.01)。(3)坐骨神经NF-κB蛋白的表达:NF-κB主要表达于雪旺细胞浆及细胞核、部分轴索和血管内皮细胞中,髓鞘表达较弱。与Con组相比,DM组的阳性区域平均面积、积分光密度及平均光密度值显著升高(P＜0.01)。各治疗组上述各值均较DM组降低,其中J中组差异显著(P＜0.05)。J小组平均光密度值较J中组显著升高(P＜0.01);其余各值在各治疗组间比较无明显差异(P＞0.05)。2、离体实验:(1)超微结构:Con组雪旺细胞形态规则,无基底膜包被。Glu组部分细胞无细胞核、剩余细胞核形态不规则,细胞器较少,糖原、脂滴和空泡变性多见;另有一些细胞呈气球样改变,细胞膜不完整。JMT组细胞病理改变较Glu组稍有减轻。Ntp组细胞形态异常较Glu组改善不明显。(2)SC中NF-κB蛋白的表达:Con组NF-κB绿色荧光强度较弱,主要表达于胞浆区;Glu组荧光强度于胞浆区和胞核区表达都较Con组增强;JMT组和Nip组表达都较Glu组减弱,主要表达于胞浆区。与Con组相比,Glu组NF-κB的荧光强度值显著升高(P＜0.01)。JMT组和Nip组均较Glu组显著降低(P＜0.01)。两治疗组间比较无明显差异(P＞0.05)。(3)SC中NF-κB mRNA的表达:与Con组相比,Glu组及各治疗组NF-κB mRNA值均显著升高(P＜0.01)。与Glu组相比,JMT组和Ntp组的NF-κB mRNA值均显著下降(P＜0.01)。两治疗组间比较无明显差异(P＞0.05)。【结论】1、在体实验:空腹单次腹腔注射STZ(60mg/kg)16w,糖尿病大鼠坐骨神经可出现形态结构的病理改变,包括轴索、髓鞘病变等主质成分病变和血管、胶原纤维等间质成分病变,存在有髓神经纤维缺失现象。坐骨神经组织中NF-κB表达水平显著升高。筋脉通可改善糖尿病大鼠坐骨神经病理形态、减少坐骨神经中NF-κB的表达,中剂量组效果最好。2、离体实验:采用组织贴块法,经差速贴壁法结合低浓度胰蛋白酶消化法和G-418进行纯化可成功建立坐骨神经雪旺细胞原代培养模型。高糖环境培养可造成雪旺细胞超微结构的改变,上调雪旺细胞中NF-κB蛋白和mRNA的表达水平。筋脉通含药血清可有效抑制高糖环境培养的雪旺细胞中NF-κB蛋白和mRNA表达,并对其超微结构具有一定的改善作用。更多还原

【Abstract】 [Objective]1.To study the effects of Jinmaitong capsule(JMT) on morphology and expression of nuclear factor kappa B(NF-κB) in sciatic nerves of STZ-induced diabetic rats.2.To study the effects of medicated serum of JMT on ultrastructure, transcription and expression of NF-κB in Schwann cells(SC) cultured in high glucose Medium.[Methods]1.In vivo:Fifty SYZ-induced diabetic rats(single intraperitoneal injection, 60mg/kg) were randomly divided into 5 groups including model group(DM), low-dose JMT group(JL),middle-dose JMT group(JM),high-dose JMT group (JH) and Neurotropin group(Ntp).Ten normal rats matching with weight,age and sex served as normal control group(Con).All rats were given intragastric administration for 16 weeks and then killed.Body weight and blood glucose were detected before and at the 4th,8th,12th,16th week after treatment.The morphologic and morphometric studies of sciatic nerves were investigated by light and transmission electron microscopic techniques.The expression of NF-κB in sciatic nerves were detected by SABC immunohistochemical method.2.In vitro:SC were isolated from the sciatic nerves of newborn Wistar rats,then cultivated and purified by methods of repeated explanation,differential velocity adherent technique,low density trypsin digestion and application of G-418.And SABC immunohistochemical method with S-100 protein antibody was used to identify them.The 3rd passage SC were cultured respectively in different conditions including control gtoup(Con),high glucose group (Glu),JMT group(JMT) and Neutrophin group(Ntp).After 48h cultures,the ultrastructure of SC was investigated by transmission electron microscopy. Furthermore,the expression of NF-κB mRNA and NF-κB in SC were detected by real-time fluorogenetic quantitative PCR and confocal laser scanning microscope respectively.[Results]1.In vivo:(1) Blood glucose and body weight:The blood glucose levels of STZ-DM rats were much higher than those of normal rats(P＜0.01).In all the treated groups,there were no significant differences among them compared each other or compared with the DM group (P＞0.05).And it got the same result when concerning about body weight no matter how the rats were dealt with(P＞0.05).(2) Pathological morphology:①Morphology:The morphology of sciatic nerves in the Con group was normal.The DM group showed the loss of mylelinated nerve fibers,axons swelling or atrophy,segmental demyelination,glycogen wthin axons, collagen fiber increasing and basement membrane thickening.All the treated groups improved compared with the DM group.②Morphometric analysis:To compared with the Con group,the density of myelinated fibers and small myelinated fibers,the rate of small myelinated fibers,the size of myelinated fibers and axon cross sections in the DM group were significantly reduced(P＜0.01).However,the density of big myelinated fibers and the size of myelin cross sections were insignificant changes among them compared each other(P＞0.05).And the index above in all the treated groups decreased compared with the DM group, the JM group more significantly improved than the DM group(P＜0.05).The density of myelinated fibers and small myelinated fibers as well as the size of axon cross sections in the JM group were more significantly improved than the JL group(P＜0.05,P＜0.01) (3) NF-κB expression of sciatic nerve:NF-κB expressed in cytoplasm and nuclei of SC,axon and endothelial cells,but not in myelin sheaths.The average area,average optical density and integrated optical density of NF-κB expression in the DM group were much higher than the Con group(P＜0.01).And the levels of NF-κB in all the treated groups decreased compared with the DM group,the JM group notably lower than the DM group(P＜0.05),and average optical density in the JM group decreased compaired with the JS group(P＜0.01).2.in vitro:(1) Ultrastructure:The morphology of SC in the Con group was normal and there was not basement membrane.Some non-nuclear cells were in the Glu group,remained nuclei showed the irregular form.Few organelles and many glycogen,and vacuolar degeneration were in the cells.Other SC showed the ballooning degeneration and broken plasmalemma.Pathological morphology changed in the JMT group,but not notably changed in the Ntp group compared with the Glu group.(2) NF-κB expression of SC:The green fluorescence intensities of NF-κB expressed weekly in cytoplasm of SC in the Con group,but much stronger in cytoplasm and nuclei of SC in the Glu group than it of the Con group(P＜0.01).All treated group showed the weeker green fluorescence intensities in cytoplasm of SC,which compared with the Glu group(P＜0.01).There was no statistical difference between the JMT and Ntp groups(P＞0.05).(3) NF-κB mRNA transcription of SC:The levels of NF-κB mRNA transcription in the Glu group were much higher than those of the Con group(P＜0.01).Compared with the Glu group,NF-κB mRNA expression of the JMT group and the Ntp group down-regulated noticeably(P＜0.01).Between two treated groups showed no significant changes(P＞0.05). [Conclusion]1.In viyo:STZ-induced diabetic rats(single intraperitoneal injection,60mg/kg) had morphology changes at 16w,which included the changes of axon,myelin sheat,vascular and collagen fiber.Mylelinated nerve fibers were lost. Furthermore,the expression of NF-κB significant increased.Traditional Chinese Medicine JMT could improve the pathological morphology of DPN rats and down-regulate the expression of NF-κB in sciatic nerves.The middle dose of JMT showed the best effectiveness.And its efficacy was independent of hypoglycemia and insulin.2.In vitro:SC isolated from the sciatic nerves of newborn Wistar rats and cultivated and purified by methods of repeated explanation,differential velocity adherent technique,low density trypsin digestion and application of G-418. High glucose could change the ultrastructure and up-regulate the transcription and expression of NF-κB in SC.The medicated serum of JMT could down-regulate the transcription and expression of NF-κB and improved the ultrastructure of SC.更多还原