姜黄素衍生物减缓大鼠糖尿病周围神经病变进展的作用及其机制研究

摘要 目的：探讨姜黄素衍生物减缓大鼠糖尿病周围神经病变（DPN）进展的作用及其机制。方法：30只健康成年雄性SD大鼠随机分为3组,每组10只;构建链脲佐菌素（STZ）诱导的DPN大鼠模型;研究组1按10 mg/kg体重喂养10 μM的姜黄素衍生物;研究组2按10 mg/kg体重喂养100 μM的姜黄素衍生物;对照组喂养同等量的生理盐水。利用Von Frey电子测痛仪检测各组大鼠痛觉机械戒断阈值（MWT）。采用实时定量聚合酶链反应（qRT-PCR）和蛋白免疫印迹试验检测AMPK、mTOR mRNA和蛋白表达水平。最后测定活化剂AICAR处理的RSC96细胞中mTOR的表达水平。结果：喂养姜黄素衍生物大鼠的痛觉机械戒断阈值（MWT）随时间推移逐渐降低,与对照组相比,研究组1大鼠的MWT显著降低,而研究组2大鼠的MWT也显著低于研究组1,差异均有统计学意义（P＜0.05）。与对照组相比,研究组1的AMPK mRNA和蛋白表达水平显著升高,mTOR mRNA和蛋白表达水平表达显著降低（P＜0.05）;而研究组2的AMPK mRNA和蛋白表达水平显著高于研究组1,mTOR mRNA和蛋白表达水平显著低于研究组1,差异均有统计学意义（P＜0.05）。使用活化剂AICAR处理RSC96细胞后,活化剂AICAR组的mTOR mRNA和mTOR蛋白表达水平明显低于非活化剂组,差异有统计学意义（P＜0.05）。结论：姜黄素衍生物可能通过AMPK调节mTOR信号通路改善DPN,其可能具有潜在的治疗DPN的能力。     

低温保存许旺细胞对周围神经再生的作用

目的：比较原代培养许旺细胞（Schwann cells,SCs)和冷冻保存的SCs移植对损伤后坐骨神经再生的作用。方法：原代培养和液氮保存的SCs分别移植到桥接缺损坐骨神经的硅胶管内。在移植后不同时间（第6和8周末）,硅胶管远端神经干内注射HRP,逆行追踪背根神经节和脊髓前角的标记神经元数量;测量再生神经纤维的复合动作电位传导速度;电镜观察再生神经纤维的髓鞘形成。结果：原代培养和冷冻保存SCs在移植后不同时间其背根神经节和脊髓前角神经元HRP标记细胞数量、再生神经纤维的复合动作电位传导速度基本一致,再生神经纤维髓鞘的形成未见明显差别。结论：冷冻保存的SCs仍具有促进损伤后周围神经再生的能力。     

HRP法对异种神经移植后再生纤维恢复的形态学研究

目的用辣根过氧化酶(HRP)逆行追踪技术探讨异种神经移植后神经纤维的再生.方法将多次冻融处理后的兔胫神经移植于大鼠坐骨神经,术后第2、4、6、8和10周,将HRP注人大鼠坐骨神经吻合部远侧端.结果移植术后第4周起在L4～5脊神经节见到HRP标记细胞,从第6周在腰段脊髓前角内见到标记细胞,其数量随术后存活期延长而增多.术后4周在移植神经内见少量再生神经纤维,6周后再生神经纤维穿过异种移植神经进入大鼠坐骨神经远侧端.结论自移植术后4周起,移植神经内已有再生纤维并部分恢复了轴浆流,证实了用HRP法可反映移植后神经纤维的再生情况.     

黄板对菜地烟粉虱的诱集作用研究

应用黄板诱集花菜和菜豆田中烟粉虱成虫。结果表明：花菜田中东西向放置的黄板诱集效果优于南北向,搭架的菜豆田中顺行向优于垂直行向;黄板放置高度花菜田以黄板下端略高于菜叶顶部为宜,搭架的菜豆田以架中部为宜;诱集时间以11时至15时为佳。     

周围神经再生早期过程中内源性和外源性巨噬细胞数量变化的实验研究

目的：周围神经再生过程中巨噬细胞发挥了重要的作用,然而目前对于神经内内源性和外源性巨噬细胞的具体作用了解的却很少,因此本实验研究了小鼠坐骨神经损伤后早期再生过程中内源性和外源性巨噬细胞数量比例变化的情况,探索周围神经再生的规律。方法：移植CAG-EGFP转基因小鼠的全骨髓有核细胞到骨髓灭活野生型C5781／6小鼠体内建立嵌合体小鼠模型。待移植成功3个月后夹伤小鼠一侧坐骨神经,并在损伤后第2、7、14和28天取材、切片,使用巨噬细胞特异性抗体cD68进行免疫荧光染色,分析损伤神经段中内源性巨噬细胞（CD68＋／EGFP-）、外源性巨噬细胞（CD68＋／EGFP＋）的数量及其比例变化情况。结果：①夹伤骨髓移植模型小鼠坐骨神经后,参与坐骨神经损伤修复的巨噬细胞可分为两类,即内源性巨噬细胞（CD68＋／EGFP-）和外源性巨噬细胞（CD68＋／EGFP＋）;②夹伤坐骨神经后,浸润的总巨噬细胞数量从第2天开始逐渐增加,到第14天达到高峰,约为正常情况下的60倍,随后逐渐减少;③起初外、内源性巨噬细胞间的比例是1：1,差值最大出现在损伤后第14天为4：l。结论：小鼠坐骨神经夹伤后,内外源性巨噬细胞共同参与了受损神经组织远心段的修复和再生过程,损伤初期发挥作用的主要是内源性巨噬细胞,随后大量浸润的外源性巨噬细胞占主导作用。本实验首次连续观察并定量分析了神经损伤后早期内源性和外源性巨噬细胞的数量改变,证实了瓦勒氏变性过程中内源性和外源性巨噬细胞在不同阶段对巨噬细胞总量的贡献作用。     

碱性成纤维细胞生长因子对大鼠晚期周围神经再生作用的实验研究

目的探讨外源性碱性成纤维细胞生长因子(bFGF)对晚期周围神经再生的作用.方法50只SD大鼠随机分治疗组、对照组各25只,切断右侧坐骨神经,12周后予以修复,修复术后每日分别给予bFGF和生理盐水,行神经电生理和组织学检查.结果治疗组和对照组修复处远段神经均有不同程度再生,4周时已可见到再生轴突,且治疗组多见.计量分析治疗组运动神经传导速度、神经肌肉动作电位幅值、髓鞘厚度、再生轴突直径和截面积明显优于对照组.治疗组与对照组相比,差异有显著性.结论bFGF能促进晚期周围神经再生.     

蛇瓜提取物的抗病毒感染作用和诱生干扰素的活性

本实验证明,在流行性乙型脑炎病毒皮下感染前,给小白鼠注射蛇瓜提取物(简称E.T.a.)有明显的保护作用;在感染后给药,则小白鼠存活天数可明显延长,但其保护作用低。给家兔静脉注射E.T.a.,血清中可测到Ⅰ型干扰素,高峰出现在注射后2小时。经初步鉴定,蛇瓜提取物的有效成份似为某种双链RNA,但需进一步确证。     

神经再生过程中细胞骨架蛋白的轴浆转运与再生神经构筑的变化

神经再生是长期未能解决的问题,原因之一是对再生的规律和调控缺乏足够的认识。近代神经生物学的进展表明,神经再生的特点之一是轴突缺乏合成蛋白的能力,其再生所需的     

棉铃虫性诱剂对棉铃虫成虫行为的影响及田间控制作用分析

通过田间试验明确了性诱剂诱杀防治不仅影响棉铃虫成虫的求偶、交配行为 ,而且还干扰取食、扩散和隐蔽过程中的飞行行为 .大面积田间诱杀防治的试验结果表明 ,性诱剂诱杀防治区与对照区相比较 ,雄性个体减少一半 ,平均雌性个体交配次数降低 0 .6次 ,室内孵化率降低 1 9% ,田间孵化率降低 1 3.5% .     

灵长类动物胫神经和腓总神经损伤再生规律的研究

目的:研究灵长类动物胫神经和腓总神经再生能力差异。方法:健康成年恒河猴16只,分为A、B两组,每组8只,使用刀片切割完全损伤胫神经和腓总神经,后立即予神经外膜缝合,在术后3周、8周分别取A、B组胫神经和腓总神经吻合口远、近端神经组织行Luxol Fast Blue染色,观察胫神经和腓总神经远端、近端轴突数目,计算轴突密度,远端轴突密度/近端轴突密度为神经再生通过率。结果:术后3周和8周时,胫神经和腓总神经相比,胫神经在远端轴突密度、神经通过率等指标上,胫神经愈后优于腓总神经(P0.05)。结论:坐骨神经神经损伤修复后,胫神经轴突通过吻合口的通过率较腓总神经高,吻合口远端有更多的神经轴突,其靶器官有更多的神经纤维支配,这是导致坐骨神经损伤修复后胫神经功能恢复较腓总神经功能恢复好的重要原因之一。     

Changes in Rapid Transport of Phospholipids in the Rat Sciatic Nerve During Axonal Regeneration

Abstract: Axonal transport of phospholipids in normal and regenerating sciatic nerve of the rat was studied. At various intervals after axotomy of the right sciatic nerve in the midthigh region and subsequent perineurial sutures of the transected fascicles, a mixture of 60 μCi [Me-^HC]choline and 15 μCi [2-³H]glycerol in the region of the spinal motor neurons of the L5 and L6 segments was injected bilaterally. The amount of radioactive lipid (and in certain cases its distribution in various lipid classes) along the nerve was determined as a function of time. Three days after fascicular suture and 6 h after spinal cord injection of precursors, there was an accumulation of labeled phospholipids and sphingolipids in the transected sciatic nerve in the region immediately proximal to the site of suture. Nine days after, there was a marked increase in the accumulation of radioactivity in the distal segments of the injured nerve, which increased up to 14 days after cutting and disappeared as regeneration proceeded (21–45 days). In all segments of both normal and regenerating nerve fibers, as well as in L5 and L6 spinal cord segments, only phosphatidylcholine and sphingomyelin were labeled with [¹⁴C]choline. These results suggest that the regeneration process in a distal segment of a peripheral neuron, following cutting and fascicular repairing by surgical sutures, is sustained in the first 3 weeks by changes in the amount of phospholipids rapidly transported along the axon towards the site of nerve fiber outgrowth.     

Early structural changes in the axoplasmic cytoskeleton after axotomy studied by cryofixation

Summary Alterations in the cytoskeleton were studied in the axoplasm of neurites at the tips of proximal stumps of transected chicken sciatic nerves. The studies were carried out using cryofixation with a nitrogen-cooled propane jet. The most immediate effect is the almost complete disassembly of axoplasmic microtubules. This consequently causes the axonal transport of membrane-bounded organelles to cease and results in an accumulation of mitochondria and vesicles of the smooth endoplasmic reticulum. The neurofilament network is partially disorganized. Neurofilaments become shorter and fragmented, and are linked by a large number of anastomosed cross-linkers. The neurofilaments become newly aligned to the axis of the axoplasm and are of normal length 48–72 h after the transsection. At this stage the newly formed neurofilament bundles are in close proximity to the anastomosed cisternae and profiles of the smooth endoplasmic reticulum. The axonal sprouts always show a normally organized cytoskeletal network. These studies support the idea that the rapid remodelling of the neurofilament network is apparently a local event, not dependent on the slow transport of cytoskeletal materials to the tip of the proximal stump. The repair of the degraded cytoskeleton may be in accordance with the function of the endoplasmic reticulum as Ca²⁺-sequestering membrane system, which may be involved in restoring the physiological conditions of the axoplasm.     

Cryopreserved muscle basal lamina grafts retain their grafting potential for nerve repair

The development of alternatives to nerve autografts for nerve repair remains a goal of surgeons. Muscle basal lamina grafts have a potential use as bioprostheses, but it is not known whether such grafts retain their ability to support axonal regeneration following storage. In this study, we examined the effect of cryopreservation on the ability of muscle basal lamina grafts to repair nerve lesions. Basal lamina grafts were prepared and cryopreserved for different times and at different temperatures. Their grafting potential was evaluated by examining axonal regeneration after autografting to lesions in rat sciatic nerves. Muscle basal lamina grafts cryopreserved for up to 30 weeks at -20 and -40 degrees C were successfully used. There were no significant differences in the parameters of axonal regeneration between cryopreserved and non-cryopreserved grafts. In conclusion, muscle basal lamina autografts retain their potential usefulness for nerve repair after cryopreservation, providing a basis for the development of a bioprostheses from muscle basal lamina.     

Okadaic Acid and Cultured Frog Sciatic Nerves: Potent Inhibition of Axonal Regeneration in Spite of Unaffected Schwann Cell Proliferation and Ganglionic Protein Synthesis

Abstract: Okadaic acid (OA) is a frequently used phosphatase inhibitor that by inhibiting dephosphorylation increases the net phosphorylation level in various systems. In the present study OA was used to assess the role of balanced phosphorylation-dephosphorylation reactions for successful regeneration of peripheral nerves. To achieve this, the effects of OA on phosphorylation levels, neurite outgrowth, injury-induced support cell proliferation, and neurofilament stability, respectively, were investigated in the in vitro regenerating, adult frog sciatic sensory nerve. OA at a moderate concentration (20 n M ) increased phosphorylation levels and almost completely inhibited the in vitro regeneration in a reversible way. The effect on regeneration was not due to induced neurofilament instability and was only seen when the drug was applied in the outgrowth region. The latter and the absence of effects on support cell proliferation indicate that OA acts locally at the level of newly formed axons. However, the inhibition of regeneration was not a consequence of reduced delivery of proteins by axonal transport, because this process in fact was increased by OA. Altogether, the study suggests that properly balanced phosphorylating-dephosphorylating reactions are critical for regeneration of peripheral nerves.     

Investigations on the Incorporation of a Tritiated Ganglioside, GM1, in the Injured and Intact Sciatic Nerves of the Rat

Following injury of their left sciatic nerves by means of a standardized procedure, male rats received intravenous injections of a tritiated ganglioside. GM1, on different days during the process of regeneration. The rats were killed at two different times after the injection and the concentrations of the total radioactivity, nonvolatile radioactivity, and labelled GM1 were estimated in six segments of the crushed and intact sciatic nerves. The segments of the damaged nerves showed higher concentrations of radioactivity and a higher content of GM1 than the corresponding segments of the contralateral nerves. Within the immediate area of the lesion the highest levels were found on the 3rd and 6th days after the injury; the segments distal from the lesion showed the highest levels of activity on days 9 and 12. The nerve segments proximal to the site of the injury showed a low rate of radioactivity incorporation. The higher concentrations of [3H]GM1 in damaged nerves as well as the rate of incorporation as a function of time indicate that exogenous gangliosides may be involved in the processes of regeneration and have a bearing on the latter.     

补阳还五汤口服和药浴对损伤的大鼠坐骨神经传导速度的影响

目的探讨补阳还五汤口服加药浴对坐骨神经传导速度的影响。方法60只SD大鼠暴露左侧坐骨神经。对照组只钳夹;实验组钳夹并加用补阳还五汤口服及药浴治疗。观察钳夹前和钳夹切除后大鼠坐骨神经传导速度（SNCV）。结果于2、4、6周分别测对照组、实验组的坐骨神经传导速度（SNCV）。各时间段实验组坐骨神经传导速度恢复快于对照组,P〈0．01。结论补阳还五汤口服加药浴对坐骨神经传导速度有明显的促进作用。     

游离锌离子轴突运输的实验研究

目的：研究大鼠坐骨神经结扎后游离锌离子在轴突内的定位分布,探讨锌离子在含锌神经元内的轴突运输。方法：应用轴流阻滞／神经结扎术结合光、电镜锌金属自显影技术,检测含锌神经元轴突内的游离锌离子子。结果：锌阳性反应产物主要分布在靠近结扎点的坐骨神经近端和远端轴突内,并且随着结扎时间的延长锌离子在轴突近端和远端的积累逐渐增加。此外,电镜结果表明锌离子主要定位于无髓神经纤维以及薄髓鞘的有髓神经纤维轴突内。结论：游离锌离子在含锌神经元轴突内进行双向轴突运输,即顺行运输和逆行运输。