【作者】 汪海滨；

【导师】 钟世镇；

【作者基本信息】 南方医科大学 ， 人体解剖学， 2013， 博士

【摘要】 研究背景及目的：临床上导致周围神经缺损的原因很多,如肿瘤切除、创伤等均可造成神经缺损。临床上治疗周围神经离断除及时的神经对位缝合外,一般常用自体神经移植来治疗,但是存在造成供区神经功能丧失、遗留手术疤痕及供体有限、供区的附加切口、移植神经支配区的功能障碍等问题,且并不能保证达到确实的修复效果。近年来,周围神经组织工程技术的兴起,为神经缺损的修复提供了新的方法和手段。从生物学观点看,周围神经再生是一个相当复杂的过程,受到多种因素的影响。周围神经再生过程中,再生轴突的生长、定向和成熟受到周围神经再生微环境中各种因素的影响,其中有细胞、细胞外基质和弥散因子等,在再生早期尚有变性的轴突和髓鞘碎屑,其中细胞成份有SC、成纤维细胞、肥大细胞和巨噬细胞等。周围神经损伤后,其远侧端由于失去了与神经元胞体的联系,远侧端神经纤维的全长发生华勒氏变性,在光镜下可见轴突肿胀、外形不规则、断裂和溶解等变化。近侧端由于与神经元胞体的连续性存在,根据不同程度的创伤而出现一个或几个郎飞结的变性改变。周围神经再生过程中,近侧端轴突的再生和向远侧端的长入,是神经再生的必要条件,也是神经再生的首要因素,而SC恰恰是周围神经再生微环中的最重要因素。SC是PNS特有的胶质细胞,具有增生、迁移和分泌多种活性物质等一系列特有的生物学特性,是周围神经纤维唯一的支持细胞。SC具有显著的功能多样性,在周围神经再生微环境中,Sc通过与轴突之间的联系来调节髓鞘化、髓鞘形成,且SC参与周围神经干细胞外基质的形成.周围神经损伤后,SC发生反应性增殖,与血源性巨噬细胞一起清除溃变产物,产生及分泌某些物质影响神经的再生,SC在周围神经系统中促进轴突再生具有重要作用。因此,国内外的学者越来越重视对SC与轴突再生关系的研究。目前,大多数研究多关注于周围神经损伤后远侧端神经再生微环境的变化。周围神经损伤后,其远侧端中断了与神经元胞体的联系,远侧端神经纤维的全长直至其终末都发生溃变,称为华勒氏变性,包括轴突和髓鞘的变性.留下原先包绕着轴突的基底膜管,SC在基底膜管内分裂、增殖,形成Bunger带,同时伴有巨噬细胞的浸润、神经内膜胶原产物的沉积等一系列变化。所有的这些变化都与SC有着广泛的联系。所以,SC是构成周围神经再生微环境的主要成份,是促进再生轴突生长的关键因素,一旦在神经再生微环境内缺少SC,轴突或不能生长,或再生速度大大下降。因此,对周围神经再生微环境的研究多集中于SC,周围神经损伤后,SC的数量、形态学和生物学特性的变化直接影响神经再生的微环境。李奕对成年大鼠周围神经损伤后远侧端SC的变化进行了体外研究,结果表明,周围神经损伤后,SC形态发生改变,1月最明显；细胞活力和增殖能力也发生改变,1周活力和增殖能力最强,随后逐渐变弱。目前,对于周围神经损伤后近侧端变化的研究较少,特别是没有关于周围神经损伤后近侧端SC的数量、形态学和生物学特性变化的研究的报道。我们认为,在周围神经再生过程中,近侧端轴突的再生和向远侧端的长入,是神经再生的必要条件,也是神经再生的首要因素,而SC恰恰是周围神经再生微环中的最重要因素。雪旺细胞在周围神经组织工程修复中充当种子细胞,是构建组织工程化神经的关键。因此快速获得组织工程化人工神经所需要的SC非常重要。目前国内外在此方面有较多的研究,但是SC是一种终末期细胞,直接取材行雪旺细胞培养,细胞很难贴壁,扩增差,很难得到高纯度及大量的SC,所以普遍存在着技术复杂、所需试剂昂贵、SC纯度不高等不足,限制了人工神经在临床上的应用。国内外许多学者研究将干细胞向类雪旺细胞诱导来解决此难题,但诱导率低、诱导条件撤离后再次向干细胞形态转归及致瘤性等缺点,仍限制了其进一步的应用。在积极探索改进雪旺细胞的体外培养条件,寻找一种较好的获取雪旺细胞的方法,以获得足够数量的较纯净的雪旺细胞的实践中,人们发现,神经预变性不失为一种好的方法,而本实验探索的新的体外预变性方法具有独特的优越性。神经预变性的目的是创造一个有利于神经再生的微环境,雪旺细胞的大量增殖是完成这个目的的必要条件。目前研究表明SC在生长发育的不同阶段及损伤修复过程中表达不同的标记,表明SC并不是一个功能结构同质体,在不同阶段有不同的增值生长能力。神经损伤的瓦勒变性过程中,SC发生去分化变化,表达神经营养因子、细胞粘附分子及不成熟SC标记胶质纤维酸性蛋白(GFAP)、p75NTR的上调,而这些变化有利于细胞的增值及粘附,促进神经的再生,可能有利于SC的体外贴壁培养、扩增,从而获得大量的SC满足人工神经的需要。1999年Keilhoff等通过体内预变性(瓦勒变性)一周后体外再培养获得了大量的增殖能力强的SCs,证实通过预变性再行细胞培养可以获得大量的增殖能力强的SC。2009年,Tomita等进行了体内预变性的研究,他们通过观察预变性后雪旺细胞的迁移情况来评价预变性效果,发现2周后雪旺细胞迁移最多。体内预变性可以提供大量的增殖能力强的雪旺细胞,有令人兴奋的临床应用前景。但体内预变性需要两次手术,花费的时间较长,延误了治疗时机,同时由于存在个体差异预变性达到所需效果的时间点难以控制,临床应用受到一定的限制。因此我们的研究中将成年小鼠的坐骨神经取出放在体外培养液中预先培养一段时间即体外预变性模拟体内的发生情况,可以避免体内方法的不足。2010年Armin kram等报道了体外预变性,他将坐骨神经置于DMEM+10%FBS进行预变性,结果预变性一周后细胞培养,获得了大量高纯度的SCs。体内预变性是一个复杂的过程,是多种细胞及因子参与的结果,因此体外预变性要达到体内的效果必须模拟体内环境。本实验中,我们加入了雪旺细胞生长所需要的生长因子(forskolin、heregulin-β-1、碱性成纤维细胞生长因子)以期模拟体内实验的效果。方法：1.实验动物：绿色荧光蛋白(GFP)转基因小鼠。c57小鼠,雌雄不限。2.体外预变性：小鼠颈椎脱臼处死,分离切取坐骨神经,放于含有DMEM. DMEM+10%FBS、SCCM培养液中培养,1周后待检。体内预变性：小鼠10%戊巴比妥腹腔注射麻醉,暴露右侧坐骨神经,在远离脊神经根部切断坐骨神经,并使断端游离。术后1、2、3周取材待检。3.细胞培养及冰冻切片制作：分别将不同培养液中培养的及新鲜的坐骨神经组织漂洗,切碎,消化酶溶解,接种培养,传代。48小时后对SCCM组细胞进行纯化并做冰冻切片。4.组织和细胞免疫荧光检测：取各组的冰冻切片及培养的原代细胞行兔抗S100β及p75NTR免疫荧光染色。激光共聚焦显微镜观察组织切片中绿色荧光蛋白及S100p表达情况,普通荧光显微镜下观察细胞染色情况,随机选取5个视野计算细胞纯度。5.激光共聚焦下观察拍照。6.统计学处理：所有计数值以均数±标准差(x±s)表示,应用SPSS17.0统计软件进行数据分析。组间均数比较单因素方差分析(One-Way ANOVA),多重比较方差齐性下采用LSD方法,方差不齐时采用Dunnett T3方法。P<0.05提示差异具有统计学意义。结果：1.GFP小鼠组中,体外预变性1周后坐骨神经组织切片绿色荧光表达最强,p75荧光表达最强,说明体外预变性的最佳时间是一周；2.GFP小鼠组中,SCCM组中1、2、3周绿色荧光表达和p75荧光表达最强。SCCM组与DMEM组和DMEM+10%FBS组及对照组雪旺细胞数、细胞纯度和细胞密度均具有统计学差异(P<0.05)。3.C57小鼠组中,对于SCCM培养液中预先培养的坐骨神经获取的细胞S100p蛋白的免疫荧光染色,发现几乎所有的双极或三极样细胞均为阳性,随机选取三个视野计算纯度为98%。SCCM组与DMEM+10%FBS组和对照组比较,雪旺细胞数和细胞密度均具有统计学差异(P<0.05)。各组原代细胞培养48小时后,雪旺细胞纯度,DMEM+10%FBS、SCCM中分别为82.83±3.43%,89.67±3.14%。结论：1.坐骨神经体外预变性后再行细胞培养简单,高效,是一种很好的体外培养成年小鼠雪旺细胞的方法。2.预变性一周后雪旺细胞增值最好。3.雪旺细胞在培养介质DMEM、DMEM+10%FBS、SCCM及体内对照组中纯度对比,SCCM最优,DMEM+10%FBS优于DMEM。更多还原

【Abstract】 Background/Objective:PeriPheral nerve injury has many causes, such as trauma and tumor surgery.The regeneration and function recovery of PeriPheral nerve is one of the hottest issues in the field of neuroscience.The clinical treatment of a severed PeriPheral nerve involves the use of an autologous nerve graft to bridge the gap when it is difficult to suture it directly. But nerve autografting Remains many Problems, i.e. sacrificing one or more functional nerves and donor-site sequelaes include lose of sensation, scarring, etc. Inaddition, the limited availability of donor tissue represents a severe Problem. Inrecent years, more attention has been focused on the development of tissue engineering nerve as substitute for the nerve autograft to avoid these Problems.The PeriPheral nerve tissue engineering technology brings new hope to resolve the difficulties in PeriPheral neuroscience.Peripheral nerve regeneration is a very complicated process,effected from a biological perspective.It is effected by mangy kinds of factors.Peripheral nerve regeneration process,the growth of axonal regeneration, directional and mature from surrounding nerve regeneration is effected by various factors of microenvironment,including cells,extracellular matrix and dispersion factor,there is some degenerated axonal and pith scabbard debris in early regeneration,including cell components such as SC,fibroblasts,mast cells and macrophages,etc.After peripheral nerve damage,Wanllerian degeneration happened in the whole length of the nerve fibres of distal ends due to loss the connection to the body of the neuron with visible axon swelling,irregular appearance,fracture and dissolve changes observed by light microscopy.The near side with the continuity of the body of the neuron exists,and according to different degrees of trauma,one or several several "LangFei node"degeneration was observed.The near side axons of regeneration and grow into the far side is necessary in peripheral nerve regeneration process,as well as the first element of nerve regeneration. And SC is ecactly the most important factors around nerve regeneration microenvironment.SC is PNS peculiar oligodendrocytes, which has a series of special biological characteristics such as hyperplasia, migration and the secretion of various active material, and it is the only peripheral nerve fiber support cells. SC has significant function diversity, in microenvironment for peripheral nerve regeneration.Through the contact between the axons, SC adjust the myelinates, myelination, and participate in the formation of the matrix of peripheral neural stem cells. After Peripheral nerve damage, SC begin to prolifera, and with the blood source macrophages together to clear collapse product, and then produce and secret some material to influence nerve regeneration. SC has an important role in promoting the axonal regeneration in peripheral nerve system. Therefore, the domestic and foreign scholars pay more and more attention to SC and axonal regeneration of the relationship.At present, most studies pay much attention to the changes of the microenvironment of distal ends nerve regeneration after peripheral nerve injury. The distal ends lost the contact to perikaryon after Peripheral nerve damage, the full length of the far side of nerve fibers to the final happened to collapse, called Wallerian degeneration, including degeneration of axons and myelin, leaving basement membrane tube around the axon. SC separate, proliferate to form Bunger belt in the basement membrane tube with a series of changes such as macrophage cell infiltrates, nerve lining collagen deposition product. All of these changes have an extensive touches with SC. So, SC is the main ingredients of microenvironment of nerve regeneration, and is the key factors to promote the regeneration of the axon outgrowth.Once lack of SC in the microenvironment in nerve regeneration, axon or can’t grow, or regeneration rate drops greatly. Therefore,the study on the surrounding nerve regeneration of microenvironment focused on the SC.The number of SC, morphology and biological characteristics of the nerve regeneration change has a direct impact on the environment after peripheral nerve damage. LiYi studied the change of the SC in distal ends of peripheral nerve after injury in adult rats and the results showed that after peripheral nerve injury, SC form change was most obviously in one month; the cell vitality and proliferation capacity is strongest in one week and then gradually become weak. At present, the study for the changes of near side of peripheral nerve injury is less, especially less reports about the change of the number of the SC, morphology and biological characteristics of near side of peripheral nerve after injury. We believe, in peripheral nerve regeneration process, the near side axons of regeneration and grow into the far side, is the necessary conditions of nerve regeneration, is also the first element of nerve regeneration, and SC is exactly the most important factors in nerve regeneration microenvironment.Schwann cell plays the part of seed cell in the process of peripheral nerve repair with tissue engineering technique, and it is the key to build tissue-engineered nerve. So how to get SC tissue-engineered artificial neural needed quickly is very important. There are many research in this respect at home and abroad. SC is a kind of end-stage cells, it is difficult to stick wall and expansion,and it is difficult to get high purity and number of SC with direct draw material to do cell culture. The complex technology, expensive reagents, poor SC purity, restrict the application of artificial nerve in clinic. Many scholars at home and abroad do research to induce stem cells to cells like SC to solve the difficult problem.But induction rate is low, it turned over to stem cells after the induction conditions moved once again and tumorigenicity still limits its further applications.In the actively exploration to improve schwann cells culture condition in vitro, looking for a better get schwann cells method to get a sufficient number of a purer schwann cells in the practice, people found that nerve degeneration is a good method, and the exploration of our experiment is a new method of nerve degeneration in vitro and has unique advantages. The purpose of the nerve degeneration is to create a microenvironment conducive to the nerve regeneration. It is the necessary condition to complete the purpose of the large number of proliferation of schwann cells.Previous studies have shown that SC have different proliferative potential under different developmental stages, and are not functionally and structurally homogeneous. During Waller degeneration of nerve injury, SCs dedifferentiate, express neurotrophic factors, cell adhesion molecules and immature SCs marker glial fibrillary acidic protein (GFAP), and upregulate p75NTR. These may facilitate SC proliferation and attachment, and promote nerve regeneration. In1999, Keilhoff etal. obtained large quantities of SC with high proliferative potentials after in vivo pre-degeneration (Waller degeneration) for a week and in vitro recultivation. In2009, Tomita did the research of nerve degeneration in vitro to evaluate the changes after the degeneration through observing migration degeneration of schwann cells, found that schwann cells in most migration two weeks after degeneration. The degeneration in vitro can provide a large amount of schwann cells with strong proliferation ability and has exciting prospect of clinical application. However, in vivo pre-degeneration requires two surgical operations, is time consuming, and causes delay in treatment. The clinical applications of this approach are also limited due to individual differences and the resulting difficulties in estimating the timing of desired pre-degeneration effect. In our study, sciatic nerves of adult mice were pre-degenerated in vitro in media mimicking in vivo conditions. In2010, Kram et al. have reported in vitro pre-degeneration by incubating sciatic nerves in DMEM with10%FBS for one week and obtained large amount of pure SC.In the peripheral nerve repair process, SC prolified and secret some neurotrophic factors.The degeneration in vivo is a complicated process with variety of cells and factors involved, so we must simulate environment in vivo in order to get the similar result of the degeneration in vitro. We added schwann cells growth need growth factor (forskolin, heregulin-beta1, alkaline fibroblast growth factor) in order to simulate the effect of in vivo and did the corresponding detection.Methods:1. Experimental animals:green fluorescent protein (GFP) transgenic mice. C57mice, male and female are welcome.2. The degeneration in vitro:mice to be put to death by cervical dislocated, cut off and separate the sciatic nerve, put in the DMEM, DMEM+10%FBS, SCCM medium for cultivating and one week later for detection. The degeneration in vivo: mice were in anesthesia by10%pentobarbital intraperitoneal injection, the right sciatic nerve were exposed and cut away from the root of spinal nerve sciatic nerve, and make the cut ends free and1、2、3week later for detection.3. Cell culture and frozen section production:the sciatic nerve tissue in different cultures and fresh sciatic nerve were rinsed, chopped, dissolved with the digestive enzymes, inoculation respectively. SCCM cells were purified and made frozen section after48hour.4. tissue and cellular immune fluorescence detection:take S100β and p75NTR immunofluorescence staining for the frozen section and original generation cells. To observate expression of green fluorescent protein and S100β from tissue slices by laser confocal microscopy and detecting dyeing cells by common fluorescent microscope, computing cells purity with randomly selected five vision.5. taking pictures under Confocal laser observed.6. Statistics processing:the experiment was repeated3times. All count value to±standard deviation mean differences, statistics software SPSS13.0for data analysis. Mean differences between groups compares with the t test, P<0.05suggests a statistically significant difference.Results:1. The GFP mice group:green fluorescent and p75fluorescence expression of section of sciatic nerve tissue was the strongest after one week of the degeneration in vitro,suggest that the best time for schwann cells proliferation is one week after degeneration.2. GFP mice group:green fluorescent expression and p75fluorescence express is the strongest in SCCM group in1,2,3weeks. Compared SCCM with DMEM group, the group DMEM+10%FBS group and control group,schwann cells, the cell purity and cell density have a statistics difference (P<0.05).3. C57mice group:The bipolar and tripolar SCs were all stained positive with S100β immunofluorescence staining for sciatic nerve cells in the cultivation of SCCM medium, the purity was98%with computing random selected three vision. Compared SCCM group with the DMEM+10%FBS group and control group,schwann cells, the cell purity and cell density have a statistics difference (P<0.05).48hours after the original generation cell culture, schwann cells purity of DMEM+10%FBS, SCCM is82.83±3.43%and89.67±3.14%.conclusions: 1. To do the cell culture after the sciatic nerve degeneration in vitro is a good method to culture Schwann cells of adult mice.2. The best time for Schwann cell proliferation is one week after sciatic nerve degeneration.3. The purity of Schwann cells in SCCM is the best compared with DMEM,DMEM+10%FBS and the control group in vivo,and DMEM+10%FBS superior to DMEM.更多还原