雪旺氏细胞分泌的神经营养活性物质的提取及体外生物活性研究

雪旺氏细胞（ＳＣ）分泌多种神经营养因子促进周围神经再生，但ＳＣ分泌的各种蛋白质对周围神经再生的影响尚未完全阐明。为此，从成年大鼠瓦勒氏变性坐骨神经中分离出ＳＣ，经培养获得ＳＣ无血清条件培养液（ＳＣ－ＳＦＣＭ）。通过ＰＭ１０超滤浓缩、Ｄｉｓｃ－ＰＡＧＥ和Ｂｉｏｔｒａｐ电洗脱，从ＳＣ－ＳＦＣＭ中分离出Ｄ蛋白带，分子量在４３～６７Ｋｄ之间。经ＭＴＴ检测，Ｄ带蛋白在２５～５０ｎｇ／ｍｌ浓度时对脊髓前角神经元表现出明显的体外成活作用。Ｄ蛋白带可能是一种有别于已知的ＳＣ源神经营养物质，其活性浓度达到了神经营养因子分子检测水平，值得深入研究     

雪旺氏细胞分泌蛋白质及其神经营养活性物质的生化分析

成年大鼠瓦勒氏变性神经来源的雪旺氏细胞（ＳＣ），经体外无血清分离培养，收集ＳＣ无血清条件培养液，用ＳＤＳ－ＰＡＧＥ分析其超滤浓缩液（分子量大于１０ＫＤａ）中ＳＣ分泌蛋白，发现其含有１４条蛋白带，分子量从大于９４ＫＤａ至小于３４ＫＤａ。用免疫印迹技术、抗２．５Ｓ神经生长因子（ＮＧＦ）抗体鉴定证实ＳＣ分泌蛋白中含有ＮＧＦ。用Ｄｉｓｃ－ＰＡＧＥ（４℃）分离ＳＣ分泌蛋白带，分别经电洗脱、浓缩收集１０份蛋白区带，结合有髓腹侧运动神经元细胞培养、ＮＴＦｓ生物学活性鉴定，初步发现其中一组蛋白区带（Ｂ＋）含运动性神经营养因子（ＮＴＦ）活性，其分子量在６５ＫＤａ至３４ＫＤａ。     

雪旺氏细胞分泌的神经营养活性物质的提取及体外生物活…

雪旺氏细胞（ＳＣ）分泌多种神经营养因子促进周围神经再生，但ＳＣ分泌的各种蛋白质对周围神经再生的影响尚未完全阐明。为此，从成年大鼠瓦勒氏变性坐骨神经中分离出ＳＣ，经培养获ＳＣ无血清条件培养液（ＳＣ－ＳＦＣＭ）。通过ＰＭ１０超滤浓缩、Ｄｉｓｃ－ＰＡＧＥ和Ｂｉｏｔｒａｐ电洗脱，从ＳＣ－ＳＦＣＭ中分离出Ｄ蛋白带，分子量在４７－６７Ｋｄ之间，经ＭＴＴ检测，Ｄ带蛋白在２５－５０ｎｇ／ｍｌ浓度时对脊髓产角神经元     

培养中的雪旺氏细胞对脊髓前角神经元的营养作用

通过隔玻片ＳＤ大鼠瓦勒氏变性远段坐骨神经获得的雪旺氏细胞（ＳＣ）与胎龄１４天的ＳＤ大鼠胚胎脊髓前角神经元（ＳＡＨＮ）联合培养，用倒置显微镜、Ｎｉｓｓｌ染色、ＳＡＨＮ免疫学观察，证实培养中的ＳＣ具有维持神经元成活和促进突起生长的作用。在这二种神经营养因子作用下，脊髓前角神经元胞体增大，突起生长可达数倍胞体长度。为认识和研究运用神经营养学说促进神经再生提供了新资料。     

不同时间差速粘附分离`纯化瓦勒变性神经的雪旺细胞

不同时间差速粘附分离、纯化瓦勒变性神经的雪旺细胞顾立强，朱家恺雪旺细胞（ＳｃｈｗａｎｎＣｅｌｌ，ＳＣ）培养是周围神经再生研究深入细胞、分子水平的要求之一，但有关瓦勒变性神经ＳＣ培养报道甚少。本文报道用不同时间差速粘附法分离、纯化成年大鼠瓦勒变性神经的...     

雪旺细胞分泌神经营养蛋白的提纯

目的：寻找出提纯神经营养蛋白的实用而有效的方法。方法：收集ＳＤ乳鼠周围神经的雪旺细胞无血清条件培养液，离心，将上清液超滤浓缩（ＰＭ１０）收集分子量大于１０ＫＤａ浓缩蛋白液，经ＳｅｐｈａｄｅｘＧ－７５凝胶过滤获得三个蛋白峰，经生物活性检测，证明洗脱Ⅱ峰液生物活性最大。将冼脱Ⅱ峰液再经ＰＭ１０超滤膜浓缩收集浓缩蛋白液，分别用ＴＨＫ２４、ＴＴＫ２４微型过滤器去除大于１００ＫＤａ和小于３０ＫＤａ蛋白，过分子筛高效液相色谱柱。结果：得到４个主要蛋白峰，前三峰蛋白分子量均超过１５０ＫＤａ，第四峰分子量为６７ＫＤａ，经ＳＤＳ－ＰＡＧＥ和银染色法进一步证实该蛋白分子量为６７ＫＤａ，基本为单一蛋白带。结论：Ｓｅｐｈａｄｅｘ凝胶过滤、多次超滤和分子筛高效液相色谱技术综合应用是纯化雪旺细胞源神经营养蛋白较好的方法。     

雪旺细胞浆神经营养蛋白高效液相分离纯化及其生物活 …

目的 分离纯化雪旺细胞浆神经营养蛋白并研究其生物活性。方法 取３００只出生后１～３天ＳＤ大鼠双侧坐骨神经,纯化培养,收集雪旺细胞,超声粉碎超速离心,不同孔径分子筛滤膜（ＰＭ１０、３０、５０）超滤浓缩,收集分子量１０～３０ｋｕ,３０～５０ｋｕ、〉５０ｋｕ三种组份乐蛋白浓缩液,分别加入体外无血清培养的Ｅ１４ＳＤ胎鼠脊髓运动神经元培养液中,用ＭＴＴ酶标法检测证实１０～３０ｋｕ,〉５０ｋｕ两组份蛋白有神经     

雪旺细胞胞浆神经营养蛋白的分离纯化和理化性质测定

目的：分离纯化雪旺细胞胞浆神经营养蛋白并测定其等电点。方法：培养收集新生１－３天ＳＤ乳鼠四肢神经雪旺细胞，超声粉碎，超速离心，取上清胞浆成份超滤浓缩，收集分子量＞１０ＫＤａ浓缩蛋白液，通过ＤＥＡＥ－Ｓｅｐｈａｃｅｌ离子交换层析，ＳｅｐｈａｄｅｘＧ－１００凝胶过滤层析和Ｄｉｏｌ－１５０高压液相分子筛层析进行分离纯化，等电聚焦电泳测定纯化的活性蛋白质等电点和ＳＤＳ－聚丙烯酰胺凝胶电泳测定其亚基分子量。结果：成功地分离纯化出一种活性蛋白质，分子量约５８ＫＤａ，等电点４．５５，结论：联合应用超滤浓缩、离子交换层析、凝胶过滤层析和高压液相能较理想地分离纯化雪旺细胞胞浆蛋白     

肝硬变门脉高压症及不同术式对大鼠肝脏储备功能的影响

用四氯化碳／乙醇诱导的大鼠肝硬变模型分为４组：①选择性远端脾腔分流术（ＤＳＣＳ）；②门奇断流术（ＰＡＤ）；③肠腔侧侧分流术（ＭＣＳ）和④肝硬变组。在术后１、３、５周进行口服糖耐量试验（ＯＧＴＴ）、胰高糖素负荷试验（ＧＬＴ），比较各组肝脏储备功能的变化。结果表明：术后第１周ＤＳＣＳ、ＰＡＤ和ＭＣＳ与肝硬变组比较无显著性差异（Ｐ＞０．０５）；而术后第３和５周，ＤＳＣＳ和ＰＡＤ组与肝硬变组比较有显著性差异（Ｐ＜０．０５），ＭＣＳ组与肝硬变组比较无显著性差异（Ｐ＞０．０５）。本研究结果表明：ＤＳＣＳ和ＰＡＤ组大鼠肝脏储备功能明显优于ＭＣＳ组和肝硬变组。     

许旺细胞源神经营养蛋白受体的初步定位

目的探讨脊髓前角运动神经元是否存在许旺细胞源神经营养蛋白的受体。方法采用放射性配体结合分析法,对许旺细胞源神经营养蛋白进行１２５Ｉ标记,以１２５Ｉ标记的许旺细胞源神经营养蛋白（１２５Ｉ－ＳＣＤＮＴＰ）为配体,与ＳＤ胚鼠的脊髓前角运动神经元进行受体结合分析。结果（１）１２５Ｉ－ＳＣＤＮＴＰ特异结合最大结合容量为（６．１±０．４８）ｆｍｏｌ／１０６ｃｅｌｓ,平衡解离常数为（３．８２±０．６０）ｐｍｏｌ／Ｌ;（２）动力学分析显示ｋ１＝（８．０３±０．３２）×１０８ｍｏｌ－１·Ｌ·ｍｉｎ－１,ｋ－１＝（２．６２±０．８２）×１０－３ｍｉｎ－１;（３）特异性分析表明许旺细胞源神经营养蛋白与运动神经元的结合具有高度特异性。结论在ＳＤ胚鼠的脊髓前角运动神元上存在着高亲和力许旺细胞源神经营养蛋白的受体。     

雪旺细胞浆神经营养蛋白高效液相分离纯化及其生物活性研究

目的　分离纯化雪旺细胞浆神经营养蛋白并研究其生物活性。方法　取 30 0只出生后 1～ 3天SD大鼠双侧坐骨神经 ,纯化培养 ,收集雪旺细胞 ,超声粉碎 ,超速离心 ,不同孔径分子筛滤膜 (PM10、30、5 0 )超滤浓缩 ,收集分子量 10～ 30 ku,30～ 5 0 ku和 >5 0 ku三种组份胞浆蛋白浓缩液 ,分别加入体外无血清培养的 E14SD胎鼠脊髓运动神经元培养液中 ,用 MTT酶标法检测证实 10～ 30 ku和 >5 0 ku两组份蛋白有神经营养活性。再通过Diol- 15 0高效液相分子层析进一步从雪旺细胞胞浆中纯化分离出分子量为 2 6 ku和 5 8ku两种胞浆蛋白 ,并对其神经生物活性进行研究。结果　 2 6 ku和 5 8ku雪旺细胞胞浆蛋白能维持体外无血清培养的脊髓运动神经元存活 ,其最佳生物活性浓度为 2 0 ng/孔。结论　雪旺细胞胞浆内含有分子量为 2 6 ku和 5 8ku的运动神经营养蛋白     

Role of Schwann cells in the regeneration of penile and peripheral nerves

Schwann cells (SCs) are the principal glia of the peripheral nervous system. The end point of SC development is the formation of myelinating and nonmyelinating cells which ensheath large and small diameter axons, respectively. They play an important role in axon regeneration after injury, including cavernous nerve injury that leads to erectile dysfunction (ED). Despite improvement in radical prostatectomy surgical techniques, many patients still suffer from ED postoperatively as surgical trauma causes traction injuries and local inflammatory changes in the neuronal microenvironment of the autonomic fibers innervating the penis resulting in pathophysiological alterations in the end organ. The aim of this review is to summarize contemporary evidence regarding: (1) the origin and development of SCs in the peripheral and penile nerve system; (2) Wallerian degeneration and SC plastic change following peripheral and penile nerve injury; (3) how SCs promote peripheral and penile nerve regeneration by secreting neurotrophic factors; (4) and strategies targeting SCs to accelerate peripheral nerve regeneration. We searched PubMed for articles related to these topics in both animal models and human research and found numerous studies suggesting that SCs could be a novel target for treatment of nerve injury-induced ED.     

骨髓基质细胞源性神经活性物质的提取与纯化

目的分离纯化骨髓基质细胞(marrow stromal cells,MSCs)胞浆中神经营养蛋白,并验证其神经活性作用. 方法自小鼠股骨分离培养 MSCs,超声粉碎后离心并收集其上清液,超滤浓缩后收集大于10 ku浓缩蛋白液.Sephadex G-100层析、高效液相色谱分析(high performance liquid chromatography,HPLC)和十二烷基硫酸钠-聚丙烯酰胺凝胶不连续电泳(sodium dodecyl sulfate-polyacrylamide gel electropheresis,SDS-PAGE)等技术分离神经活性蛋白,培养脊髓运动神经元,通过MTT法及观察细胞形态来验证其神经活性作用. 结果 MSCs胞浆上清液经Sephadex G-100层析后发现,Ⅱ峰对神经细胞生长有促进作用.对Ⅱ峰行HPLC分析发现,A峰对神经细胞生长有明显促进作用.对A峰行SDS-PAGE分析,提示为单一蛋白带,分子量为13 ku. 结论 MSCs胞浆中含有神经营养蛋白,其分子量为13 ku.     

游离脂肪酸对大鼠肾小球系膜细胞增殖和细胞周期的影响

目的：探讨游离脂肪酸（FFAs）对体外培养的大鼠肾小球系膜细胞增殖和生长周期的影响。方法：用不同浓度的游离脂肪酸处理大鼠HBZY-1细胞株（即大鼠肾小球系膜细胞）24h～72h。采用噻唑蓝比色（MTT）法检测内皮细胞增殖情况,流式细胞术（FCM）分析法测定细胞周期变化。结果：游离脂肪酸可抑制HBZY-1细胞的生长增殖（与对照组比较,P〈0.01）,且这种抑制作用具有剂量和时间依赖性;游离脂肪酸作用于HBZY-1细胞24h、48h、72h,细胞周期发生明显改变,G1期细胞数增多,S期细胞数减少（与对照组比较,P〈0.01）。结论：游离脂肪酸可通过停滞细胞生长于G1期,抑制大鼠肾小球系膜细胞的生长增殖。     

巨噬细胞在周围神经修复再生中的作用

周围神经在损伤后可以通过瓦勒氏变性反应实现神经的修复和再生,而受损神经发生瓦勒氏变性反应的过程中,巨噬细胞发挥着不可替代的重要作用.在此就周围神经损伤后修复和再生研究领域中,有关巨噬细胞作用的研究进展进行综述.     

Pathology of lumbar nerve root compression. Part 1: Intraradicular inflammatory changes induced by mechanical compression.

STUDY DESIGN: This study is to investigate the intraradicular inflammation induced by mechanical compression using in vivo model. OBJECTIVES: The relationship between the intraradicular edema and nerve fiber degeneration induced by mechanical compression was determined in the nerve root. SUMMARY OF BACKGROUND DATA: Recently some studies reported that mechanical compression increased microvascular permeability of the endoneurial capillaries and resulted in an intraradicular inflammation. These changes may be an important factor of the pathogenesis of radiculopathy. However, the natural courses of the intraradicular inflammation after mechanical compression are still poorly understood. METHODS: In dogs, laminectomy was performed at L7 and the seventh nerve root was exposed to compression at 7.5 gram force (gf) clipping power. The animals were evaluated at 1 and 3 weeks after clipping. After the appropriate period of nerve root compression, Evans blue albumin (EBA) was injected intravenously. The nerve root sections were divided into two groups. The sections were used to investigate the status of the blood-nerve barrier function under the fluorescence microscope. The other sections were used for light and transmission electron microscopic study. RESULTS: After 1 and 3 weeks, intraradicular edema was observed not only at the site of compression but also in the peripheral zone of a compressed anterior root and in the central zone of a compressed posterior root. The evidence of active Wallerian degeneration was also seen in the area of intraradicular edema. In addition, the nerve roots showing Wallerian degeneration were infiltrated by inflammatory cells, such as macrophages and mast cells. CONCLUSIONS: Inflammatory reaction, such as Wallerian degeneration, breakdown of blood-nerve barrier and appearance of macrophage, may be deeply involved in radiculitis arising from mechanical compression, and these factors seem to be important in the manifestation of radiculopathy.     

Effects of Ginsenoside Rb1 on proliferation of Schwann kcells in culture

Objective:To investigate the effects of Ginsenoside Rb1 on the proliferation ofSchwann cells in culture.Methods:Applying MTT assay and Thymidine incorporation assay,the effects of Ginsenoside Rb1 on the proliferation of Schwann cells isolated from the sciatic nerve of adult rat were studied.Results:Ginsenoside Rb1(10μg/ml)significantly induced Schwann cell proliferation,the effect was similar to NGF(50μg/ml).At high concentrations of Ginsenoside Rb1(1 mg/ml) ,the proliferation of Schwann cells was significantly inhibited.Conclusions:Ginsenoside Rb1 at the optimal concentratios is found to be effective in inducing the proliferation of Schwann cells ,but at higher concentrations the drug is cytotoxic for Schwann cells.     

壳聚糖-明胶支架/滑膜干细胞的相容性

目的 检测壳聚糖-明胶支架与人滑膜干细胞(SMSCs)的相容性.方法 接种滑膜干细胞于壳聚糖-明胶支架上,于第3、7和10天进行扫描电镜观察和噻唑蓝(MTT)分析.结果 扫描电镜显示支架上有大量滑膜干细胞生长,且细胞形态与对照组无明显差异,MTT分析显示支架组的A值关系为10d(1.43±0.11)≥7d(1.35±0.08)＞3d(0.43±0.08),且第7、10天组的A值明显大于同期对照组(0.64±0.15、0.59±0.13)(P＜0.05).结论 壳聚糖-明胶支架与滑膜干细胞的相容性良好.

Abstract：

Objective To investigate the compatibility between chitosan-gelatin scaffold and synovium-derived mesenchymal stem cells (SMSCs).Methods The SMSCs were obtained with passage isolation, and then seeded on chitosan-gelatin scaffold. Cytocompatibility was detected by using scanning electron microscopy (SEM) and methyl thiazol tetrazolium (MTT) assay at day 3, 7 and 10 after seeding.Results SEM image of scaffold showed that SMSCs spread well and homogenously distributed throughout the entire scaffold. MTT assay revealed that the cell viability in 3D scaffolds was as follows: (1.43±0.11) at day 10≥ (1.35±0.08) at day 7 > (0.43±0.08) at day 3, and at day 7 and 10, it was significantly higher than that in controls (day 7: 0.64±0.15, and day 10: 0.59±0.13;P＜0.05).Conclusion The chitosan-gelatin scaffold is compatible with SMSCs.     

Sutures or fibrin glue for divided rat nerves: Schwann cell and muscle metabolism

Peripheral nerve anastomoses using either epiperineurial sutures or a fibrinogen adhesive technique have been compared in the rat sciatic nerve model. Evaluation of results was made using radiolabelling of the metabolically active acid-soluble phosphate fractions of both nerve and muscle. In none of the situations tested--traumatic degeneration and regeneration in the sciatic nerve proximal segment, Wallerian degeneration and regeneration in its distal segment, atrophy and regeneration of the fast gastrocnemius muscle, and atrophy and regeneration of the slow soleus muscle--was one repair method significantly superior to the other. A significant degree of cross-reinnervation was shown to take place after anastomosis, altering the characteristics of the regenerating muscles. Both repair methods were equally inferior to the spontaneous repair occurring after a simple nerve crush.