【作者】 陈哲；

【导师】 陈正形； 陈海啸；

【作者基本信息】 浙江大学 ， 外科学， 2002， 硕士

【摘要】 周围神经损伤是临床常见病。严重的神经损伤，伤口处和远侧段全长神经纤维发生华勒氏变性，部分相应神经元凋亡，加上局部疤痕增生，吻合口处感觉、运动轴交易相互错长，长期失神经支配后终末器官萎缩、变性，故治疗后功能恢复多不理想。 雪旺细胞是周围神经系统特有的胶质细胞，不但在周围神经的发生、发育、形态和功能维持方面起重要作用，在周围神经损伤、再生和修复过程中，也起关键作用。因此，雪旺细胞近年来成为临床和基础研究的重点和热点。 周围神经华勒氏变性中雪旺细胞的变化，多数学者认为，创伤后雪旺细胞受诸多因素激发，直接进入有丝分裂状态，大量增殖。也有学者提出雪旺细胞存在坏死或凋亡现象。本研究通过比较SD大鼠各组形态学改变、凋亡基因表达变化及原位末端标记法检测结果，探讨华勒氏变性中雪旺细胞凋亡的存在及其时相性变化特点。 材料与方法 本实验利用成年雌性SD大鼠坐骨神经横断建立华勒氏变性典型模型。 将 95只 SD大鼠（麻醉死亡 6只，中途退出实验及备用共 15只）随机分为 12组。l个正常空白组（8只）、11个实验组（66只）。每只实验组大鼠双 后肢再随机分为试验肢（坐骨神经于上 1／3段横断）和对照肢（对侧正常坐 骨神经〕。所有的“试验肢”构成试验组，所有的“对照肢”构成对照组。 各实验组大鼠分别于术后11、6h、12h、24h、Zd、3d、4d、sd、14d、Zld、 30d共 11 个时相点，切取坐骨神经中下段标本。练合分析、比较正常空白 组、对照组、试验组神经纤维形态学改变，雪旺细胞 S—100蛋白和凋亡基 因相关产物B。l－2、FAs、B3X表达水平变化，并运用TUNEL法检测雪旺细 胞凋亡指数。 结 果 1、形态学分析显示，试验组发生典型华勒氏变性。雪旺细胞数量术 后Z4h开始减少，第3d、4CI明显稀少，术后sd开始增多，14d 后形成Bungner带；对照组在创伤早期出现轻度坤经肿胀，炎症 细胞浸润。 2、S—100检测结果与既往文献报道明显不同，各组雪旺细胞 S一］00 普遍呈低水平表达：对照组与正常空白组比较，无统计差异 （P＞0刀5）；试验组术后lh增高，术后6h汗始明显下降，持续到 第 4d才开始回升，第 ZId达高峰。术后 lh、术后 ZId与正常空 门组单独作I检验比较，有统计意义o＜o05）。 3、正常空白组BCI－2低水平表达，士侧肢与右侧肢比较无统计差异 （P＞0．05）；对照组术后 6h明显升高，术后 24h、4d、21d轻度 增高，与．卜常空白组相比有统计学显著差异（P＜0刀1）：试验组在 术后6h、24h、sd、14d、30d进行性升高，与正常空白组相比有 统计学显著差异（P＜0刀1）。 4、正常空白组Bax低水平表达，左侧肢与右侧肢比较无统计差异 （P＞0．05）；对照组术后 6h、第 3d至第 ZId持续高表达，与正常 空白组相比有统汁学显著差异（P＜0刀1）；试验组第1七开始进行 性上升，第 3 cd达高峰，与正常空白组相比有绞计学显著意义 （P＜0刀1〕。 5、正常主白组Fa。表达极低，上侧肢与右侧肢比较无统计差异 （．P＞0．05），对助组分别于木后 24h、3d、sd。Zld轻度增高，｛H 与正常空臼组相比无统计意义（P＞0．05）；试验组从术后 12h开始 波浪式进行性升高，在变性中、晚期高表达，与正常空白组相比 有统计学显著差异o＜0．01．）。 6、TUNEL检测显示，正常空白组雪旺细胞凋亡发生率极低，左侧肢 与右侧肢比较无统计差异 J＞0刀引；试验组分别在术后6h、川、 4d、ZId 四个时相点上出现大量凋亡细胞核。与；厂常空白组相比， 有统计学显著差异（P＜0刀1＼ 结 论l、大鼠坐骨神经横断后远侧段华勒氏变性中，确实存在雪旺细胞凋亡现象；2。雪旺细胞的凋亡与增殖始终贯穿华勒氏变性的全过程，早期以周亡为主， 中晚期以增殖为主；3、雪旺细胞凋亡具有一定的时相性变化特点；4、一侧坐骨神经严重损伤后，对侧正常坐骨神经内雪旺细胞的凋亡相关基 因将活跃表达，并具有一定的时相性特点：5、正常成毕大鼠坐骨神经内极少有雪旺细胞凋亡存在。更多还原

【Abstract】 IntroductionPeripheral nerve injury is one of the most common clinical diseases. Serious nerve injury is always accompanied by Wallerian degeneration in the wound and distal regions of the nerve, apoptosis of some related neurons, wrong growth of motor and sensory neurites with each other in regeneration.More over, it will cause fibroplasias in nerve gap, and atrophy of axon terminals after a long period of denervation. So the treatment result is always dissatisfactory.Schwann cell is special glial cell in the peripheral nerve system. It plays an important role not only in the development, morphology and function of normal nerve, but also during the critical period of nerve injury, regeneration and healing. Hitherto, Schwann cell has become focus in clinical and basic research.As for the changes of Schwann cell during Wallerian degeneration in the peripheral nervous system, most scholars assume that being stimulated by numerous factors Schwann cell comes into mitogenic phase directly and proliferates massively,while a few researchers declare that Schwann cell has necrosis or apoptosis phase. The purpose of this study is to systematically investigate Schwann cell apoptosis in Wallerian degeneration, and evaluate itstime-related feature, by examining the morphologic changes, the different expression of apoptosis genes and TUNEL detection.Materials and MethodsThis study used a typical Wallerian degeneration model of adult female rat in response to sciatic nerve transection in vivo. Ninety-five SD rats were divided randomly into one normal group (8 rats) and eleven experimental groups (66 rats). Secondly, for experimental groups, the both hind legs of each rat were randomly divided into test leg (with sciatic nerve transected on upper 1/3 fraction) and control leg (nerve uninjured). So all test legs constitute a test group and ?11 control legs constitute a control group. After operation, all rats in experimental groups were respectively sacrificed at Ih, 6h, 12h, 24h, 2d, 3d, 4d, 8d, 14d, 21d and 30d. The specimens of mid distal sciatic nerve were collected. Then we examined the morphological changes of the nerve, the different expression level of S-100 protein and apoptosis genes yielding proteins such as Bcl-2, Bax, Fas in Schwann cell. We also use TUNEL method to detect Schwann cell positive rate.Results1. The test group showed Wallerian degeneration. The number of Schwann cell began to decrease at 24h, surprisingly rare at Day3 and Day4, then began to increase from Day8 and formed Bungner belt after 14 days. The control group merely showed slight changes in morphology in early trauma period.2. Schwann cell generally expressed S-100 at low level in all groups, which had significant difference from previous articles. The control group had no statistical significance (P>0.05), compared with the normal group; The S-100 positive rate of the test group began to elevate at Ih, and thendecreased obviously at 6h. Till Day4, it began to increase, arid reached the peak at Day21. It had statistical difference between the test group and the normal group at Ih and Day21 (PO.05).3. The normal group expressed Bcl-2 at low level, and the left legs had no statistical significance (P>0.05), compared with the right legs; Bcl-2 level in the control group apparently elevated at 6h, then slightly elevated at 24h, Day4 and Day21, it had significant difference (PO.01) compared with the normal group; Bcl-2 level in the test group was stepping up at 6h, 24h, 8d,14d and 30d, it had highly statistical significance (PO.01), compared with the normal group.4. The normal group expressed Bax at low level, and the left legs had no statistical significance (P>0.05) compared with the right legs;The control group continuously expressed Bax at high level at 6h, and from the 3rd to the 21st day. It had highly statistical significance (P<0.01) comoared with the normal group. Bax levels in the test group elevated progressively since Dayl4, and reached the top at Day30 . It had highly statistical sign更多还原