摘要
目的：糖尿病周围神经病变(DPN)是糖尿病足(DF)的重要病理基础，是代谢紊乱、神经营养障碍、血管病变、氧化应激等多种因素综合作用的结果。其中，神经营养因素的改变发挥着重要作用。研究表明，DPN患者周围神经存在明显的病理学改变，而且高血糖引起神经组织雪旺氏细胞(SCs)中小窝蛋白-1(Car-1)和胰岛素样生长因子-1(IGF-1)减少，两者表达水平下降与神经病变程度相关，因此认为两者的下降可能通过不同机制导致了神经营养因素的改变，从而成为DPN发生的重要危险因素。由于目前的研究结果多来源于动物实验，对人组织的研究较少，所以，本实验取DF高位截肢患者的股神经，观察不同病程的患者神经病理改变的特点，以及Cav-1和IGF-1在雪旺氏细胞中表达的改变，探讨它们在DPN中的可能作用。
方法：研究对象选取2003年1月至2005年11月在天津医科大学代谢病医院DF科住院进行高位截肢的患者(均合并有周围神经病变)，共40例(男性22例，女性18例，平均年龄67.83±8.74岁，按照病程分三组：A组(≤5年)，B组(6-10年)和C组(＞10年组)。于高位截肢(大腿中下1／3)手术时留取截肢平面的股神经。1.10％(体积分数)福尔马林固定，常规石蜡包埋切片。通过HE染色观察股神经的形态改变，Weil氏铁明矾苏木素染色法观察髓鞘的形态改变，光镜下计数神经纤维的密度。2.4％戊二醛和1％四氧化锇溶液中进行双重固定后，以HITACHI H7500透射电镜摄片，观察股神经的超微结构变化。3．应用免疫组织化学方法观察股神经SCs中Cav-1和IGF-1的表达，并在显微镜下计算阳性细胞的百分数。4．对数据用SPSS13.0进行统计学处理。
结果：1．三组间患者临床资料及生化指标具有可比性，经方差分析，病程、FBG和HbA1c的差别具有统计学意义(P＜0.01)，其余各指标的差别无统计学意义。2．组织学观察：(1)HE和Weil髓鞘染色观察发现：三组患者股神经均存在明显的病理改变。A组主要表现为神经纤维粗细不均，偶见轴索深染变性，可见再生丛产生，神经束内微小血管数量增加，管壁增厚；B组主要表现为神经纤维稀疏、粗细不均，轴索深染变性多见，可见SCs增生；C组神经纤维明显减少、变细，再生丛密度低，神经束内微小血管数量减少，管壁变形、增厚明显。三组间神经纤维密度差别有统计学意义(P＜0.05)，与HbA1c呈负相关(r=-42.630，P＜0.01)，与Cav-1呈正相关(r=394．930，P＜0.01)；(2)超微病理显示：神经髓鞘增生，部分板层结构融合、消失或内层分层，呈泡状水肿。髓索水肿，微丝微管数量减少。SOs内有大的高密度脂滴，线粒体数量显著减少，水肿、空泡化，大部分或全部嵴融合、消失，粗面内质网有脱颗粒现象。3．免疫组化结果显示：三组患者股神经SOs中均有Cav-1和IGF-1的表达。三组间差别有统计学意义(P＜0.01)，Cav-1与Hbk1c呈负相关(r=-9.010，P＜0.05)，与神经纤维密度呈正相关(r=0.001，P＜0.05)，IGF-1与病程(r=-2.543，P＜0.01)和HbA1c(r=-4.580，P＜0.05)呈负相关，与神经纤维密度呈正相关(r=0.044，P＜0.05)。
结论：1．光镜和电镜下观察的结果均证实，DF合并DPN行高位截肢的患者股神经存在明显的病理学改变，随病程延长病理改变加重。2．神经纤维密度随病程延长和HbA1c水平升高下降，二者可能与神经病变的严重程度相关。另外，神经纤维密度与Cav-1水平呈正相关，Cav-1的减少可能促进了DPN的发展。3．股神经SOs中Cav-1和IGF-1的表达均存在，但随病程延长二者的表达水平均下降。根据多元回归结果分析，Cav-1的下降可能与HbA1c水平升高和神经纤维密度下降有关；IGF-1的下降可能与病程延长、HbA1c水平升高以及神经纤维密度下降有关。总之，长期高血糖导致不同病程DF高位截肢患者股神经不同程度的病理学改变，神经纤维的密度下降，SCs中Cav-1和IGF-1表达减少，它们的减少可能参与了DPN的发生、发展过程。
Objective: Diabetic peripheral neuropathy (DPN) is a crucial patho-foundation of diabetic foot (DF). It's the result of various factors' complex action, such as metabolic disorder, neurotrophy disturbance, vasculopathy and oxidative stress, and so on. Among the total, altered neurotrophism plays an important role in the mechanism of DPN. Researches show that, there are evident pathobiological changes in nerve tissue of DPN patients, at the same time, hyperglycaemia reduces the expression of Caveolin-1(Cav-1) and IGF-1 in Schwann cells. The reduction has correlation with the severity degree of neuropathy. So, it's thought that the reduction of these two factors may alter the neurotrophy by different mechanisms, and this may become an important risk factor in the development of DPN. At present, most of results come from animal, but research based on human tissue is seldom. We obtain the femoral nerve from the DF patients for the observation the pathobiological changes with different course of diabetes, and the expression level of Cav-1 and IGF-1 in Schwann cells, to discuss the relationship between them and the role of these factors in the development of DPN.
Methods: We selected patients who had undertaken high amputation in the Foot department of the Metabolic Disease Hospital of Tianjin Medical University from January 2003 to November 2005 as the research objects. And they all had DPN. There were 40 patients (22 males and 18 females) in our study(mean age, 67.83±8.74 years). We divided them into three groups depending on their course of diabetes: group A (≤5 years), group B (6-10 years) and group C、(＞10 years). We obtained nerve samples when high amputation operation was progressing, l. The samples were putted in the solution of 10% formalin, and then embedded in paraffin, the sections were used to observe the general and the myelin sheath structure of the femoral nerve tissue by HE staining and Weil's staining. And then we counted the nerve fiber's density under light microscope. 2. The samples were putted in the solution of the 4% glutaric dialdehyde and 1% osmium tetroxide to fixate, and then observed the changes of ultramicrostructure with HITACHI H7500 transmission electron microscope. 3. To observe the expressions of caveolin-1 and IGF-1 in femoral nerve by immunohistochemistry and count the percent of positive cells under light microscope.
Results: 1. There's comparability in clinical data and biochemical indicators among three groups, and variances in course, fasting blood glucose(FBG) and glycosylated hemoglobin ( HbAlc ) show statistical significance. And variances in the others have no statistical significance. 2. Observation of histology: (1) HE and Well's staining: we found there's evident pathobiological changes in femoral nerve among the three groups. Group A: Diameters of nerve fiber are not uniformity, axis cylinder anachromasis and degeneration can be seen by chance, regenerate axis cylinders show up, and the numbers of small vessels in nerve tract increase, small vessel walls thicken. Group B: The nerve fiber is sparse, diameters of which are not uniformity. Many axis cylinders display anachromasis and degeneration, Schwann cells proliferation. Group C: The numbers of nerve fiber decrease obviously, and the fibers thin. The density of regeneration clump decrease, the quantity of small vessels in nerve tract decrease, and the deformity and thickening of vessel walls get worse. The variance of fiber's density among the three groups has statistical significance (P＜0.05) , and it has negative correlation with HbAlc (r=-42.630, P＜0.01) and has positive correlation with Cav-1 ( r=394.930, P＜0.01 ). (2) Ultrastructural pathology: Medullary sheaths hyperplasy, the structure of the layers mixes together, vanishes or delaminate. Medullary cord is oedema, and the numbers of microfilament and microtubule decrease. There are a lot of big and high density lipid drop. Chondriosomes, which numbers decrease, display dropsy and vacuolization, and cristaes mix together or vanish. And rough endoplasmic reticulum degranulate. 3. Results of immunohistochemistry: The expression of Cav-1 and IGF-1 are all positive in three groups. The variances have statistical significance(P＜0.01). Cav-1 has negative correlation with HbAlc (r=-9.010, P＜0.05)and has positive correlation with density of nerve fiber(r=0.001, P＜0.05). IGF-1 has negative correlation with course(r=-2.543, P＜0.01) and HbAlc(r=-4.580, P＜0.05),and has positive correlation with density of nerve fiber(r=0.044, P＜0.05).
Conclusions: 1. Results obtained from light microscope and electron microscope all prove that, there are significant pathological changes in DF patients with DPN, and the degree is aggravating along with the course of diabetes. 2. As the lengthen of the course and the increase of HbA1c, the density of nerve fiber decreased. They may have correlation with the severity of DPN. The reduction of Car-1 may promote the development of DPN. 3. The expressions of Car-1 and IGF-1 are all positive in femoral nerve among three groups, but they all decreased along with the course. Depending on the multiple regression results, the reduction of Car-1 may have correlation with HbAlc and the density of nerve fiber. And the reduction of IGF-1 mayhave correlation with the course, HbA1c and the density of nerve fiber. Ina word, long-term hyperglycemia leads to different pathological changes.It decreases the density of nerve fiber and the level of Cav-1 and IGF-1in Schwann cells in DF patients with different course. And the reductionof Cav-1 and IGF-1 may takes part in the development of DPN.
引文
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