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The expression and distribution of ciliary neurotrophic factor in laryngeal nerve regeneration
ZHENG Hongliang, ZHOU Shuimiao，YOU Zhendong, et al.
Department of Otorhinolaryngology, Changhai Hospital, Second Military Medical University, Shanghai 200433
AbstractObjectiveTo explore the expression and distribution of ciliary neurotrophic factor（CNTF） mRNA and its protein in laryngeal nerve regeneration. Methods The recurrent laryngeal nerves were sectioned and then sutured in twelve dogs. Both proximal and distal stumps of sutured region were sectioned on different post-operative days and the sections were separately used for CNTF immunohistochemistry and CNTF mRNA in situ hybridization. The area and intensity of reactive product were measured by computer image processing system. Results Strongly reactive product of CNTF mRNA and its protein deposited in myelin-related Schwann cells in normal laryngeal nerves. At week 2 following neurorrhaphy, there was very little hybridization signal and detectable CNTF immunoreactivity in distal stump and no regenerated nerve fibbers were found. At week 3, reactive product of CNTF mRNA and its protein was detected in thin Schwann cell processes ensheathing regenerated axons, while reactive product was not found in the proliferating Schwann cells which didn't ensheathe axons. CNTF immunoreactivity was also detected in the regenerated nerve axons. After longer survival times, hybridization signal and the CNTF immunoreactivity in Schwamm cells became more widespread, and the area and intensity of reactive product significantly increased , but even at the longest survival time, they were still significantly less than those in intact nerve. The same change of CNTF mRNA and its protein was observed in a short segment proximal to the sutured region. Conclusion CNTF expression could depend on Schwann cell-axon regeneration. The level of CNTF expression stands in striking contrast to the up-regulation of nerve growth factor in peripheral nerve degeneration and regeneration. These suggest that the exogenous CNTF might provide a supportive environment for axonal regeneration.
Key wordsVocal cord paralysis Nerve regeneration Nerve growth factors Recurrent laryngeal nerve Ciliary neurotrophic factor
近年来的研究资料提示周围神经再生时需要多种神经营养因子的调控，其影响着轴突的生长和生长方向［1］。周围神经干及靶器官是神经营养因子的主要来源之一，神经损伤修复过程中神经营养因子的表达及表达程度是神经再生研究的热点。本研究采用原位杂交及免疫组织化学方法探讨睫状神经营养因子(ciliary neurotrophic factor,CNTF)在喉返神经再生过程中的表达及分布，为探讨神经再生机制和外源性神经营养因子的应用提供参考。
材料及方法
1.手术及取材：选健康成年犬12只, 体重8.0～15.0 kg。 动物全身麻醉后，于右侧第四气管环水平切断喉返神经, 立即在手术显微镜下以9-0无损伤缝线作喉返神经端端缝合术, 术后2、3、6、12、18、24周取材, 每时间亚组2只犬。暴露右侧喉返神经吻合处，在其以近0.2 cm、以远2 cm及对侧相应部位各取长约0.5 cm的神经干，置于4%多聚甲醛PBS中4℃固定8 h，冰冻切片，片厚30 μm。
2. 地高辛标记的CNTF反义cRNA探针制备：将带CNTF基因的pKS质粒 (美国Yancopoules博士惠赠) 转染大肠杆菌(氯化钙转化法)，按常规方法进行扩增及质粒制备。经Ban HI酶切，使质粒完全线性化。酚氯仿抽提、回收，在T7 RNA聚合酶作用下按宝灵曼公司(美国)方法合成cRNA地高辛探针，以免疫检测法测定cRNA探针浓度为0.1 g/L，-80℃低温保存备用。
3. CNTF mRNA原位杂交：切片经漂洗5 min，0.1 mol/L盐酸孵育10 min，Triton X-100孵育10 min，蛋白酶K(1 mg/L)37℃消化30 min，4%多聚甲醛PBS终止酶消化，42℃预杂交2h后42℃杂交过夜，探针浓度约为0.5 mg/L。用4×SSC～0.1×SSC漂洗后，加入标记碱性磷酸酶的抗地高辛抗体(1∶1 000)，室温2h，系列缓冲液洗，加入显色液NBT/BCIP避光显色4h，常规脱水、二甲苯透明，封片保存。对照一组以正义cRNA代替反义cRNA探针，对照二组加探针前以RNA酶消化切片，其余步骤均同实验组。
4. CNTF免疫组化：切片用0.03%H2O2孵育10 min，以CNTF单克隆抗体(本校神经科学研究所提供)4℃孵育48 h，工作浓度1∶2 000，再以1∶200生物素化的羊抗鼠IgG(Sigma, 美国)室温反应2 h，ABC复合物室温1 h，最后以DAB液显色5 min，每步间用0.1 mol/L PBS洗2次，常规脱水、封片。对照组采用3% BSA缓冲液代替一抗，其余同实验组。实验过程中将近远侧段神经干切片与健侧神经切片裱在同一张载玻片上，所有样品切片同时进行原位杂交或免疫组化反应。
5. 计算机图像分析：在图像采集卡(华东理工大学自动化研究所)、IBM586和Olympus B X 60显微镜组成的图像处理系统上测量CNTF mRNA杂交信号及CNTF阳性反应产物的灰度及其面积占神经索面积的百分率。灰度值：O(黑)～255(白)，无反应产物的样品灰度为255， 同一样品切片灰度测试10个，面积测试5个不同的视野，各时间亚组计平均值，作t检验。
结果
1. 神经再生过程中CNTF mRNA表达：原位杂交显示健侧神经组织CNTF mRNA杂交信号呈紫黑色位于有髓神经纤维周边的雪旺细胞内，呈晕状或瘦月牙状环绕髓鞘及轴突，髓鞘及轴突无杂交信号。在同一切面的无髓神经纤维束内仅少数有髓小纤维着色，无髓纤维无杂交信号(图1)。神经修复术后2周时，远段神经髓鞘降解，仅见散在微弱的杂交信号。术后3周可见再生的髓鞘及轴突，其周围的杂交信号强弱不均(图2)。术后6周杂交信号明显增多，但强弱分布仍不均匀(图3)。术后12周杂交信号形态与正常相似呈晕状、瘦月牙状，但信号强弱仍不均匀，有较多区域无杂交信号，术后18、24周与12周相似(图4)。神经再生时轴突、髓鞘及神经纤维外无杂交信号。近段神经干出现类似远段的改变，但术后2周即可见杂交信号环绕髓鞘及轴突，术后6周即与远段神经干12周相似。
图1 正常喉返神经CNTF mRNA原位杂交×400 图2术后3周远段神经干CNTF mRNA原位杂交。×400 图3术后6周远段神经干CNTF mRNA原位杂交。×400 图4术后24周远段神经干CNTF mRNA原位杂交。图1～4中，CNTF mRNA杂交信号呈紫黑色位于有髓神经纤维周边的雪旺细胞内，呈晕状或瘦月牙状环绕未着色的髓鞘及轴突，无髓纤维无杂交信号。×400
2. 神经再生过程中CNTF免疫反应物质的改变:健侧神经组织CNTF阳性反应产物呈黄色、棕黄色位于有髓神经纤维周边的雪旺细胞内，与杂交信号相似呈晕状或瘦月牙形环绕髓鞘，髓鞘呈阴性反应。部分轴突也着色，但反应弱、面积小(图5)。神经修复后2周，远段神经纤维降解，代之以散在点状反应物。术后3周散在反应物增多，可见少量呈晕状的反应物环绕着色较浅的轴突。术后6周，纵切面显示较多反应产物呈长细丝状包绕着色浅淡的轴突及未着色的髓鞘，此外可见散在片状反应物。术后12周，反应物形态与正常相似呈晕状、瘦月牙状，但轴突区内及神经纤维外仍可见较多阳性反应物质（图6）。术后18及24周，除轴突区阳性反应物质减少外，其余与12周相似。近段神经干在神经再生时也出现类似远段的改变，但术后2周即出现较多的阳性反应物质，6周时情形与远段12周相似。
