静电纺丝对RSC96细胞生物学行为的影响
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摘要
研究背景：
周围神经损伤在全世界范围内发生率及致残率都很高。在周围神经损伤众多类型中,神经干的横断性损伤,特别是造成较长神经节段缺损的患者预后不好,为了促进神经再生和功能恢复,往往选用外科的重建手术。因此如何修复损伤的周围神经成为了神经科学研究中的一个重大的挑战。在周围神经发生离断性损伤时,断端远侧部分和近侧1-2个轴突和髓鞘发生一系列的分子和细胞学事件,即我们通常所说的华勒变性,导致轴浆微管和神经纤维管的连续性遭到破坏。24小时后断端远侧的轴突发生变性、解体；48小时后髓鞘开始破坏。然后巨噬细胞和单核细胞迁移到变性神经断端附近清除髓鞘和轴突的残渣,与此同时雪旺细胞增殖形成Bunger带。在雪旺细胞分泌的神经营养因子和细胞外基质的影响下,损伤近端的神经纤维芽生出一个被基底膜围绕的再生单位。新的轴突芽生往往从朗飞氏结开始并被雪旺细胞重新髓鞘化。功能性的支配需要再生的轴突在生长圆锥的引导下延伸至突触的终末器官,在人类,轴突再生速度约2-5mm/天,因此一些重大的损伤的恢复可能需要几个月。为了帮助周围神经损伤的修复,早在17世纪就进行了临床干预。到了19世纪和20世纪,外科技术取得了巨大的进步,被广泛地应用于周围神经损伤的诊疗中。尽管在外周神经损伤的外科修复取得了很大的进步,但自体神经的移植一直被认为是金标准。尽管如此,自体神经移植也有自己的缺陷如：供体神经的有限性,需要进行二次手术等。另外,采用这种方法也仅能恢复原有功能的80%。因此寻找更有前途的方法是周围神经修复一个巨大的挑战。近年来,陆续出现了不同类型的生物或人工移植物,并不乏拿这些移植物与自体神经移植进行比较的研究。组织工程,作为一个新兴的领域,这些年来蓬勃发展。这些给神经科学家和外科医生合作发展组织工程神经移植物提供了契机。与其它组织工程产物类似,组织工程神经移植物也是由物理支架并导入支持细胞和(或)生长因子或其它的生物分子。
随着周围神经修复中组织工程神经移植物的应用,周围神经系统中的主要胶质细胞即雪旺细胞也越来越受到重视。在周围神经系统的发育过程中,雪旺细胞沿着轴突生长的方向迁移并在轴索的前方,首先包裹几个轴突,最终仅仅包绕一个轴突节段。当周围神经损伤时,雪旺细胞增殖,产生生长因子,清除残渣,并且为轴突的再生提供指引和支持。另外,体外实验证实平行排列的活体和离体雪旺细胞都能促进轴突生长。因为在周围神经损伤修复中的重要作用,雪旺细胞在治疗中的作用越来越受到重视。最近已经得到证实,神经组织工程移植物,特别是神经诱导管道需要移植支持细胞如雪旺细胞才能促进神经修复。
雪旺细胞起源于神经嵴,发育最终形成周围神经系统中形成髓鞘的雪旺细胞和无髓鞘形成雪旺细胞,分别包绕粗和细的轴突。在形成雪旺细胞之前,还经过了另外两种细胞类型,一种是雪旺细胞前体,这种细胞是14-15天胚胎鼠中的胶质细胞类型；另一种是不成熟的雪旺细胞,这种细胞是由雪旺细胞前体产生的,是17-18天胚胎鼠(大约是出生时)中的胶质细胞类型。出生后的不成熟雪旺细胞发育主要跟它随机包绕的轴突有关系,只有在包绕粗的轴突时才能形成髓鞘。整个发育过程可以分为三个阶段：从神经嵴干细胞到雪旺细胞前体；从雪旺细胞前体到不成熟的雪旺细胞；从不成熟的雪旺细胞到两种成熟雪旺细胞类型。这一系列的过程都受到与细胞相联系的轴突信号的调控。
体内的细胞生活在一个复杂的微环境中,既受到化学因素也受到物理因素的影响,其中微环境中的拓扑结构往往以物理因素的形式影响细胞行为。尽管在不同细胞系统生物化学的影响已经得到详尽的证实,但我们对于物理因素怎样影响细胞的行为尚处于懵懂状态。1934年,Formalas发明了用静电力制备聚合物纤维的实验装置并申请了专利,其专利公布了聚合物溶液如何在电极间形成射流,这是首次详细描述利用高压静电来制备纤维装置的专利,被公认为是静电纺丝技术制备纤维的开端。但该技术发展较缓慢,科研人员大多集中在静电纺丝装置的研究上,发布了一系列的专利,但是尚未引起广泛的关注。直到近年来,随着纳米技术的发展静电纺丝技术获得了快速发展,通过静电纺丝技术制备纳米纤维材料是近十几年来世界材料科学技术领域的最重要的学术与技术活动之一。静电纺丝并以其制造装置简单、纺丝成本低廉、可纺物质种类繁多、工艺可控等优点,已成为有效制备纳米纤维材料的主要途径之一。静电纺丝因能模拟体内微环境,因此被广泛应用于制造纳米纤维支架。RSC96细胞株是大鼠雪旺细胞经过原代细胞培养自然转化而成,是一种较理想的类Schwann细胞,本实验首先对该细胞系和原代雪旺细胞的生物学性状进行了比较,然后在此研究基础上以RSC96细胞为模型来探讨静电纺丝对RSC96细胞生物学性状的影响。
目的：
1.对比了原代培养的雪旺细胞与RSC96细胞生物学行为。
2.制备不同空间排列的静电纺丝和粗细不同的平行静电纺丝。
3.研究不同空间排列的静电纺丝对RSC96细胞生物学行为的影响。
4.初步探索粗细不同的平行排列静电纺丝对RSC96细胞生物学行为的影响。
方法
1.取1～3天龄SD乳鼠的双侧的坐骨、臂丛神经,D-Hank's液冲洗2次,解剖显微镜下仔细剥除神经外膜,D-Hanks液反复冲洗2次,剪碎至1mm3大小的组织块,双酶消化法进行分离培养,阿糖胞苷抑制成纤维细胞生长,后加入含Forsoklin和牛脑垂体浸提物的培养液来选择性促进雪旺细胞的生长,最后传代进一步纯化,光镜下进行形态学观察。常规复苏培养RSC96细胞。隔天换液,待细胞融合率为80%时用胰蛋白酶消化传代,光镜进行形态学观察。胰蛋白酶消化收集两种细胞,进行以下实验：1)用Trizol法提取细胞总RNA,用Oligo(dT)逆转录酶得到cDNA。经特定引物PCR扩增,取相同量的逆转录产物进行qPCR反应；qPCR反应采用SYBR Green/ROX qPCRMaster Mix(2X),按照说明书在冰上配制PCR反应液,每个样品重复3次。反应结束后确认PCR反应的扩增曲线和溶解曲线,采用2-ΔΔcT法确定特定荧光域值对应循环数的Ct值,对目标基因定量。2)按照细胞总蛋白分离试剂盒说明书进行分离提取各组细胞的蛋白。用Bradford Protein Assay Kit试剂盒测定蛋白含量。取等量样品总蛋白与等体积2×SDS凝胶上样缓冲液混匀,100℃加热煮沸10min,经SDS-PAGE凝胶电泳分离。用湿电转移法将蛋白转移至PVDF膜上,分别采用抗NGF,抗Laminin和抗(3-actin一抗孵育2h,三次洗膜后,加入HRP-偶联二抗,进行免疫反应,化学发光法检测条带。条带信号强度应用Image J图像分析软件进行分析。
2.直径较细的平行排列的静电纺丝纤维制备：将6-氟异丙醇溶解的10%的聚己内酯在电压为10kv的静电纺丝仪上以4m1/h速度纺成纺丝,用2800rpm的取向接收器接收。随机排列的静电纺丝纤维制备：将6-氟异丙醇溶解的10%的聚己内酯在电压为10kv的静电纺丝仪上以3ml/h速度纺成纺丝,用200rpm接收器接收。在制备上述两种不同空间排列的静电纺丝时,注射器喷头和接收器之间的距离分别是15cm和13cm。直径较粗的平行排列的静电纺丝纤维制备：将二氯甲烷和甲醇混合液溶解的15%的聚己内酯在电压为10kv的静电纺丝仪上以3m1/h速度纺成纺丝,用2800rpm的取向接收器接收。注射器喷头和接收器之间的距离是10cm。
3.RSC96细胞常规复苏后24h换液,2-3d传代扩增,扩增培养24小时后接种于PCL材料上,并进行分组,对照组A：平行排列的静电纺丝上接种培养RSC96细胞。实验组B：随机排列的静电纺丝上接种培养RSC96细胞。实验组C：PCL膜直接接种培养RSC96细胞。各组接种细胞数量相同。各组细胞于37℃含5%CO2的培养箱中培养3天(期间换液1次)后,进行以下实验：1)按照细胞总蛋白分离试剂盒说明书进行分离提取各组细胞的蛋白。用Bradford Protein Assay Kit试剂盒测定蛋白含量。取等量样品总蛋白与等体积2×SDS凝胶上样缓冲液混匀,100℃加热煮沸10min,经SDS-PAGE凝胶电泳分离。用湿电转移法将蛋白转移至PVDF膜上,分别采用抗NGF,抗Laminin和抗β-actin一抗孵育2h,三次洗膜后,加入HRP--偶联二抗,进行免疫反应,化学发光法检测条带。条带信号强度应用Image J图像分析软件进行分析。2)用Trizol法提取细胞总RNA,用Oligo(dT)逆转录酶得到cDNA。经特定引物PCR扩增,取相同量的逆转录产物进行qPCR反应；qPCR反应采用S YBR Green/ROX qPCRMaster Mix(2X),按照说明书在冰上配制PCR反应液,每个样品重复3次。反应结束后确认PCR反应的扩增曲线和溶解曲线,采用2-ΔΔCT法确定特定荧光域值对应循环数的Ct值,对目标基因定量。
4.RSC96细胞常规复苏后24h换液,2-3d传代扩增,扩增培养24小时后接种于PCL材料上,并进行分组,对照组A：六孔板空白孔内直接接种培养RSC96细胞。实验组B：直径较细平行静电纺丝上接种培养RSC96细胞。实验组C：直径较粗平行静电纺丝上接种培养RSC96细胞。各组接种细胞数量相同。各组细胞于37℃含5%CO2的培养箱中培养3天(期间换液1次),用Trizol法提取细胞总RNA,用Oligo(dT)逆转录酶得到cDNA。经特定引物PCR扩增,取相同量的逆转录产物进行qPCR反应：qPCR反应采用SYBR Green/ROX qPCRMaster Mix(2X),按照说明书在冰上配制PCR反应液,每个样品重复3次。反应结束后确认PCR反应的扩增曲线和溶解曲线,采用2-ΔΔCT法确定特定荧光域值对应循环数的Ct值,对目标基因定量。
结果
1.光镜下观察,雪旺细胞胞体呈双极纺锤形,周缘有亮带,胞核呈卵圆形或长圆形,细胞两极有长短不等的突起,细胞间常有端对端、肩并肩排列,排列呈放射状或巢状；RSC96细胞呈现典型的双极纺锤形或多角形,绝大多数细胞伸出长突起,其生长形态与原代雪旺细胞非常相似；qPCR和Western blot结果发现体外培养的RSC96细胞也可表达NGF及Laminin,与原代雪旺细胞相似。
2.运用不同静电纺丝接收装置,制备了直径相似但空间排列不同的两组静电纺丝；通过对纺丝溶液浓度和毛细喷丝头与接收仪器间距离的调控,制备了直径不同但同为平行排列的两组静电纺丝。
3.平行、随机静电纺丝及膜上培养RSC96细胞NGF和Laminin基因的表达及蛋白含量：3组细胞培养3天,后两组的RSC96细胞NGF基因的扩增表达量较多,相比第一组有很明显的差异,差异有统计学意义(P<0.05),后两组之间明显差异；3组细胞均有Laminin基因扩增表达,且呈递减趋势,各组之间差异有统计学意义(P<0.05)。各组细胞蛋白条带显示均有NGF和Laminin的表达,其中各组NGF蛋白的表达明显差异,且有统计学意义(P<0.05)；各组Laminin蛋白的表达呈递减趋势,各组之间差异有统计学意义(P<0.05)。
4.培养在粗细不同的平行静电纺丝上及空白对照组的RSC96细胞NGF和Laminin蛋白表达：3组细胞培养3天,后两组的RSC96细胞NGF基因的扩增表达量较少,相比第一组有很明显的差异,差异有统计学意义(P<0.05),后两组之间无明显差异(P>0.05)；3组细胞均有Laminin基因扩增表达,且呈递增趋势,各组之间差异有统计学意义(P<0.05)。
结论
1.发现体外培养的RSC96细胞生长形态与原代雪旺细胞非常相似；也可表达神经营养因子及层粘连蛋白,是一种较理想的原代雪旺细胞替代品,避免了长周期且繁杂的原代雪旺细胞培养及雪旺细胞纯度不高和长期培养雪旺细胞功能不佳等不足。
2.通过控制静电纺丝工艺中的相关因素如：纺丝溶液浓度、接收装置不同及毛细喷丝头与接收仪器间距离分别制备了两种不同空间排列即平行和随机排列的PCL静电纺丝及粗细不同的平行的PCL静电纺丝。
3.将RSC96细胞培养在不同空间排列的静电纺丝上,检测RSC96细胞神经生长因子及层粘连蛋白的表达差异,发现平行排列的静电纺丝纤维抑制神经生长因子的表达,却促进层粘连蛋白的表达,从而进一步说明了平行排列的静电纺丝纤维可能有类似与轴突的作用,并可能促进RSC96细胞向更成熟的形态发育。
4.初步探讨了将RSC96细胞培养在不同直径的平行排列静电纺丝上,检测RSC96细胞NGF及Laminin基因表达差异,发现直径较粗的平行静电纺丝纤维Laminin的表达较多,有统计学意义。
Background:
Peripheral nerve injury remains a high incidence of life-long disability in the world wide. Among various types of peripheral nerve injuries, transection injuries where the nerve trunk is completely interrupted, especially those resulting in large neural gaps, may have a devastating impact on patients' quality of life, and in these cases reconstructive surgery is required as a therapeutic management to achieve nerve regeneration and function restoration. In consequence, peripheral nerve repair represents a unique challenge and opportunity to clinical and translational neurosciences. After peripheral nerves are transected, a series of molecular and cellular events, collectively called Wallerian degeneration, is triggered throughout the distal stump of transected nerves and within a small zone distal to the proximal stump, resulting in the disintegration of axoplasmic microtubules and neurofilaments Within24h most axons along the distal stump of transected nerves are reduced to granular and amorphous debris; by48h the myelin sheath has begun to be transformed toward the short segment. Then macrophages and monocytes migrate into the degenerating nerve stumps to remove myelin and axon debris, while Schwann cells proliferate to form longitudinal cell columns, known as Bands of Bungner. Under the influences of neurotrophic factors and extracellular matrix molecules produced by Schwann cells, the proximal portion of transected nerves sprouts new daughter axons to generate a "regenerating unit" that is surrounded by a common basal lamina. New axonal sprouts usually emanate from the nodes of Ranvier, and undergo remyelination by Schwann cells. Functional reinnervation requires that the regenerating axons elongate under the mediation of growth cones until they reach their synaptic target, and in humans, axon regeneration occurs at a rate of about2-5mm/day; thus significant injuries may take many months to heal. To aid the repair of peripheral nerve injuries, clinical intervention has been attempted for several hundred years since as early as the17th century, when Ferrara first reported a suture technique for repairing a severed nerve. After the significant progress made in the19th and20th centuries, a wide range of surgical techniques has been put into use for the management of peripheral nerve injuries. Although there have been great advancements in the surgical repair of peripheral nerve injuries, autologous nerve grafting remains the gold standard technique to which other treatments are compared. Unfortunately, autologous nerve grafting is limited by the inherent drawbacks, such as limited availability of donor nerves, the need for a second surgery to obtain the donor nerve, donor site morbidity and secondary deformities, as well as mismatch between the injured nerve and the donor nerve. In addition, clinically functional recovery rates typically approach only80%for nerve injuries treated by autologous nerve grafts. It goes without saying that seeking promising alternatives to supplement or even substitute autologous nerve grafts constitutes a major challenge to peripheral nerve repair. In the last few decades, different types of biological or artificial grafts have been developed and investigated as compared with autologous nerve grafts in terms of the outcomes of nerve regeneration and functional recovery. More importantly, tissue engineering, an emerging multidisciplinary field, has grown at a significant rate in recent years. This offers great opportunities to neuroscientists and surgeons who have been collaborating to develop tissue engineered nerve grafts. Just like most of tissue engineered products, tissue engineered nerve grafts are typically composed of a physical scaffold with the introduction of support cells and/or growth factors or other biomolecular components.
Attention has recently focused on the biological behaviors of Schwann cells, the principal glia of the peripheral nervous system. During development, Schwann cells migrate ahead of and along axonal tracts, ensheath several axons and eventually segregate to wrap around a single axonal segment. In response to peripheral nervous system injuries, Schwann cells proliferate, produce growth factors, remove debris, and lay down longitudinal tracks that provide guidance and support for regrowing axons. Additionally, aligned live Schwann cells and isolated Schwann cells topography have the capacity to direct axon growth in vitro. Because of their instrumental role in aiding peripheral nervous system injury repair, Schwann cells have received attention as an important cell type with therapeutic applications. It has been recently suggested that therapies to promote nerve repair, notably nerve guidance channels, will require transplantation of supportive cells such as Schwann cells.
Schwann cells in spinal nerves originate from the neural crest, although the origin of cells in spinal roots is more complex. The end point of Schwann cell development is the formation of myelinating and non-myelinating cells that ensheath large and small diameter axons, respectively, throughout the PNS. Schwann cell formation is preceded by the generation of two other cell types:SCPs, which are the glial cells of embryonic day (E)14-15rat nerves (mouse E12-13), and immature Schwann cells, which are generated from the SCPs from E15to El7(mouse E13-15). The latter are the glial cells found in rat nerves from E17-18to about the time of birth. The postnatal fate of immature Schwann cells is determined by which axons they randomly associate with, with myelination being selectively activated in those cells that happen to envelop single large diameter axons. These events can be viewed as three main transitions, that is, the transition from migrating neural crest stem cells to SCPs, from SCPs to immature Schwann cells and, lastly, the divergence of this population to form the two mature Schwann cell types. These events are strikingly dependent on survival factors, mitogens and differentiation signals from the axons with which SCPs and Schwann cells continuously associate.
Accumulating evidence suggests that cells interact with their environment not only biochemically but also physically. Although the biochemical interactions have been widely investigated in different cells systems, our understanding of how physical interactions affect cell behaviors is still in its infancy. The first patent that described the operation of electrospinning appeared in1934, when Formalas disclosed and apparatus for producing polymer filaments by taking advantage of the electrostatic repulsions between surface charges. Up until1993, this technique had been known as electrostatic spinning, and there were only a few publications dealing with its use in the fabrication of thin fibers. In the early1990s, several research groups revived interest in this technique by demonstrating the fabrication of thin fibers from a broad range of organic polymers. At this time the term electrospinning was coined and is now widely used in the literature. These timely demonstrations triggered a lot of experimental and theoretical studies related to electrospinning. It is notable that the number of publications in this field has been increasing exponentially in the past few years, on account of the remarkable simplicity, versatility, and potential uses of this technique. With the possibility of generating fibers in the nanoscale and the resulting scaffolds with organized architecture, electrospun fibrous meshes may mimic the extracellular matrix. RSC96cell line is naturally transformed from primary cell culture of rat Schwann cells. It could be served as an ideal class of Schwann cell analogues. Our study is mainly composed of two components. One is to compare the biological behaviors of RSC96cells with the ones of primary schwann cells, the other is to study the influence of electrospun fibers on the biologcial behaviors of RSC96cells based on the previous results.
Objective
1. To compare the biological behaviors of RSC96cells with primary culture of schwann cells.
2. To fabricate different topography of electrospun fibers and varying diameter of aligned elecntrospun fibers.
3. To study the influence of different topography of electrospun fibers on the biologcial behaviors of RSC96cells.
4. To explore the influence of varying diameter of aligned electrospun fibers on the biologcial behaviors of RSC96cells.
Methods
1. The bilateral ischial nerves and brachial plexus nerves were taken from SD rats(1to3days). These were washed by D-Hanks solution several times, stripped the epineurium carefully under a dissecting microscope, and washed three times by D-Hanks solution. We cut the nerve tissue into small blocks in size of1mm3and isolated the tissue blocks with the dual-enzyme digestion. Cytarabine was used to inhibit fibroblast growth. Addition of forsoklin and pituitary extract bovine was to optional stimulate the proliferation of schwann cells and the pass was to further purify Schwann cells. Morphology was observed under light microscopy. Culture medium containing10%FBS,100U/mL penicillin,100μg/mL streptomycin, DMEM/F12was used to culture the RSC96cells.Then the culture dishes were placed in the CO2incubator at37℃. The medium was changed every other day. After the cell fusion rate got80%, the cells were digested and passed by0.25%trypsin with0.02%EDTA. The morphology was also observed under light microscopy. The two types of cells were digested by Trypsin and collected, Procedures were performed as follows:1) total RNA were extracted by Trizol method following the protocol. Reverse transcriptase Oligo (dT) was used to synthesize cDNA. Then RNA was expanded by designed primer (Table) and used for q-PCR. The same amount of transcript was used for q-PCR, with SYBR Green/ROX qPCR MasterMix (2X) by following the protocol and repeated for3times. The relative expression levels of genes were analyzed using the2-△△CTmethod by normalizing with (3-actin house-keeping gene expression.2) Proteins of RSC96cells were extracted from two groups by following the protocols of Total Cellular Protein Isolation Kit. Bradford Protein Assay Kit was used to detect the protein level. Equal quantities of total proteins from samples were mixed with equal volume of2×SDS gel loading buffer. After boiling at100℃for10min, samples were separated on SDS-PAGE and transferred onto PVDF membrane. After blocked by primary antibodies of anti-NGF, anti-Laminin and anti-β-actin for2hours, membranes were rinsed3times and incubated with horseradish peroxidase-conjugated secondary antibodies. Films were digitally imaged using Image J software.
2. Firstly, In order to obtain thinner aligned PCL fibers, a solution of10wt%of PCL dissolved by Hexafluoroisopropanol was dispensed at a flow rate of4ml/h and electrospun under a voltage of10kv. The PCL fibers were collected on a grounded target rotating at-2800rpm. For scaffolds comprising randomly oriented PCL fibers,10wt%of PCL dissolved by Hexafluoroisopropanol was dispensed at a flow rate of3ml/h under a voltage of10kv and collected on rotating a target(-200rpm). The polymer supply-to-target distance were set15cm and13cm respectively. Secondly, In order to obtain thicker aligned PCL fibers, a solution of15wt%of PCL dissolved by a mixture of dichloromethane and methanol was dispensed at a flow rate of3ml/h under a voltage of10kv. The polymer supply-to-target distance was set10cm.
3. After the conventional recovery of RSC96cells, proliferation was done for2-3generations. Group A (experimental):RSC96cells were seeded on the parallel electrospinning fibers. Group B (experimental):RSC96cells were seeded on the randomly electrospinning fibers. Group C (control):RSC96cells were seeded on the polycaprolactone film. Each group containing the equal amount of cells was then placed in the incubator under37℃,5%CO2for3days (medium was changed every other day). Procedures were performed as follows:1) Proteins of RSC96cells were extracted from three groups by following the protocols of Total Cellular Protein Isolation Kit. Bradford Protein Assay Kit was used to detect the protein level. Equal quantities of total proteins from samples were mixed with equal volume of2×SDS gel loading buffer. After boiling at100℃for10min, samples were separated on SDS-PAGE and transferred onto PVDF membrane. After blocked by primary antibodies of anti-NGF, anti-Laminin and anti-β-actin for2hours, membranes were rinsed3times and incubated with horseradish peroxidase-conjugated secondary antibodies. Films were digitally imaged using Image J software.2) RNA of RSC96cells were extracted from three groups by Trizol method following the protocol. Reverse transcriptase Oligo (dT) was used to synthesize cDNA. Then RNA was expanded by designed primer (Table) and used for q-PCR. The same amount of transcript was used for q-PCR, with SYBR Green/ROX qPCR MasterMix (2X) by following the protocol and repeated for3times. The relative expression levels of genes were analyzed using the2-△△CTmethod by normalizing with β-actin house-keeping gene expression.
4. After the conventional recovery of RSC96cells, proliferation was done for2-3generations. Group A (control):RSC96were seeded on the plain cell culture plate directly. Group B (experimental):Cells were seeded on the thinner aligned electrospun fibers. Group C (experimental):cells were seeded on the thicker aligned electro spun fibers. Each group containing the equal amount of cells was then placed in the incubator under37℃,5%CCO2for3days (medium was changed every other day). The total RNA of cells were extracted by Trizol method following the protocol. Reverse transcriptase Oligo (dT) was used to synthesize cDNA. Then RNA was expanded by designed primer (Table) and used for q-PCR. The same amount of transcript was used for q-PCR, with SYBR Green/ROX qPCR MasterMix (2X) by following the protocol and repeated for3times. The relative expression levels of genes were analyzed using the2-△△CTmethod by normalizing with β-actin house-keeping gene expression.
Results
1. The shape of the Schwann cells were bipolar spindle with bright edge, oval or oblong nucleis, protuberances around cell biopolar with multi-length ranges, cells growed side by side and arrayed in radial or nests under the light microscope, while RSC96cells were typically bipolar spindle or polygon shape and mostly extended long processes, and the shape was much similar with primary culture of Schwann cells. The results of qPCR and western blot analysis showed that RSC96cells cultured in vitro can also express NGF and Laminin similar with primary culture Schwann cells.
2. Different topography of electrospun fibers were fabricated by different types of collectors and varying diameter of aligned elecntrospun fibers were created by the different intrinsic properties of the solution and distance between spinneret and collector.
3. To detect mRNA expression by qPCR and protein expression by western blot: According to different culture conditions, cultures of RSC96cells were divided into three groups, group A(aligned), group B(randomly) and group C(film), and all the three groups were cultured for three days. NGF mRNA expression of RSC96was lower in group A, compared to group B and C (P<0.05). There was statistical differences between group B and group C (P<0.05). The laminin mRNA was expressed in all the groups, an decrease from group A to group C (P<0.05). What's more, Laminin and NGF protein expressed in all groups. NGF protein expression had statistical differences in all the groups(P<0.05). The laminin protein expression decreased from group A to group C (P<0.05).
4. To detect gene expression by qPCR:According to different culture conditions, cultured RSC96were divided into three groups, group A(control), group B(thinner diameter of aligned electrospun fibers) and group C(thicker diameter of aligned electrospun fibers), and all three groups were cultured for three days. The NGF gene expression of RSC96cells was higher in group A, compared to group B and C (P<0.05). There was no statistical difference between group B and group C (P>0.05). The Laminin gene expression of RSC96cells was lower in A, compared to group B and C (P<0.05). There was some statistical difference between group B and group C.
Conclusion
1. The growth pattern of RSC96cells cultured in vitro was much similar with the primary culture of Schwann cells, the cells also expressed neurotrophic factors and laminin and were an ideal substitute for the primary culture of Schwann cells, which avoided the long cycle and complicated primary schwann cells cultivation and long-term poor schwann cell function, and so on.
2. Different topography of electrospun fibers and varying diameter of aligned elecntrospun fibers were fabricated dependent on a number of processing parameters.
3. The different biological behaviours of RSC96cells were induced by aligned electrospun fibers and RSC96cells may be directed towards the more maturation state by topographic cues from electrospun fibers.
4. Biologcial behaviours of RSC96cells were evaluated on different diameter of aligned electrospun fibers. There is some statistical difference in the expression of Laminin cDNA between the two groups.
周围神经损伤在全世界范围内发生率及致残率都很高。在周围神经损伤众多类型中,神经干的横断性损伤,特别是造成较长神经节段缺损的患者预后不好,为了促进神经再生和功能恢复,往往选用外科的重建手术。因此如何修复损伤的周围神经成为了神经科学研究中的一个重大的挑战。在周围神经发生离断性损伤时,断端远侧部分和近侧1-2个轴突和髓鞘发生一系列的分子和细胞学事件,即我们通常所说的华勒变性,导致轴浆微管和神经纤维管的连续性遭到破坏。24小时后断端远侧的轴突发生变性、解体；48小时后髓鞘开始破坏。然后巨噬细胞和单核细胞迁移到变性神经断端附近清除髓鞘和轴突的残渣,与此同时雪旺细胞增殖形成Bunger带。在雪旺细胞分泌的神经营养因子和细胞外基质的影响下,损伤近端的神经纤维芽生出一个被基底膜围绕的再生单位。新的轴突芽生往往从朗飞氏结开始并被雪旺细胞重新髓鞘化。功能性的支配需要再生的轴突在生长圆锥的引导下延伸至突触的终末器官,在人类,轴突再生速度约2-5mm/天,因此一些重大的损伤的恢复可能需要几个月。为了帮助周围神经损伤的修复,早在17世纪就进行了临床干预。到了19世纪和20世纪,外科技术取得了巨大的进步,被广泛地应用于周围神经损伤的诊疗中。尽管在外周神经损伤的外科修复取得了很大的进步,但自体神经的移植一直被认为是金标准。尽管如此,自体神经移植也有自己的缺陷如：供体神经的有限性,需要进行二次手术等。另外,采用这种方法也仅能恢复原有功能的80%。因此寻找更有前途的方法是周围神经修复一个巨大的挑战。近年来,陆续出现了不同类型的生物或人工移植物,并不乏拿这些移植物与自体神经移植进行比较的研究。组织工程,作为一个新兴的领域,这些年来蓬勃发展。这些给神经科学家和外科医生合作发展组织工程神经移植物提供了契机。与其它组织工程产物类似,组织工程神经移植物也是由物理支架并导入支持细胞和(或)生长因子或其它的生物分子。
随着周围神经修复中组织工程神经移植物的应用,周围神经系统中的主要胶质细胞即雪旺细胞也越来越受到重视。在周围神经系统的发育过程中,雪旺细胞沿着轴突生长的方向迁移并在轴索的前方,首先包裹几个轴突,最终仅仅包绕一个轴突节段。当周围神经损伤时,雪旺细胞增殖,产生生长因子,清除残渣,并且为轴突的再生提供指引和支持。另外,体外实验证实平行排列的活体和离体雪旺细胞都能促进轴突生长。因为在周围神经损伤修复中的重要作用,雪旺细胞在治疗中的作用越来越受到重视。最近已经得到证实,神经组织工程移植物,特别是神经诱导管道需要移植支持细胞如雪旺细胞才能促进神经修复。
雪旺细胞起源于神经嵴,发育最终形成周围神经系统中形成髓鞘的雪旺细胞和无髓鞘形成雪旺细胞,分别包绕粗和细的轴突。在形成雪旺细胞之前,还经过了另外两种细胞类型,一种是雪旺细胞前体,这种细胞是14-15天胚胎鼠中的胶质细胞类型；另一种是不成熟的雪旺细胞,这种细胞是由雪旺细胞前体产生的,是17-18天胚胎鼠(大约是出生时)中的胶质细胞类型。出生后的不成熟雪旺细胞发育主要跟它随机包绕的轴突有关系,只有在包绕粗的轴突时才能形成髓鞘。整个发育过程可以分为三个阶段：从神经嵴干细胞到雪旺细胞前体；从雪旺细胞前体到不成熟的雪旺细胞；从不成熟的雪旺细胞到两种成熟雪旺细胞类型。这一系列的过程都受到与细胞相联系的轴突信号的调控。
体内的细胞生活在一个复杂的微环境中,既受到化学因素也受到物理因素的影响,其中微环境中的拓扑结构往往以物理因素的形式影响细胞行为。尽管在不同细胞系统生物化学的影响已经得到详尽的证实,但我们对于物理因素怎样影响细胞的行为尚处于懵懂状态。1934年,Formalas发明了用静电力制备聚合物纤维的实验装置并申请了专利,其专利公布了聚合物溶液如何在电极间形成射流,这是首次详细描述利用高压静电来制备纤维装置的专利,被公认为是静电纺丝技术制备纤维的开端。但该技术发展较缓慢,科研人员大多集中在静电纺丝装置的研究上,发布了一系列的专利,但是尚未引起广泛的关注。直到近年来,随着纳米技术的发展静电纺丝技术获得了快速发展,通过静电纺丝技术制备纳米纤维材料是近十几年来世界材料科学技术领域的最重要的学术与技术活动之一。静电纺丝并以其制造装置简单、纺丝成本低廉、可纺物质种类繁多、工艺可控等优点,已成为有效制备纳米纤维材料的主要途径之一。静电纺丝因能模拟体内微环境,因此被广泛应用于制造纳米纤维支架。RSC96细胞株是大鼠雪旺细胞经过原代细胞培养自然转化而成,是一种较理想的类Schwann细胞,本实验首先对该细胞系和原代雪旺细胞的生物学性状进行了比较,然后在此研究基础上以RSC96细胞为模型来探讨静电纺丝对RSC96细胞生物学性状的影响。
目的：
1.对比了原代培养的雪旺细胞与RSC96细胞生物学行为。
2.制备不同空间排列的静电纺丝和粗细不同的平行静电纺丝。
3.研究不同空间排列的静电纺丝对RSC96细胞生物学行为的影响。
4.初步探索粗细不同的平行排列静电纺丝对RSC96细胞生物学行为的影响。
方法
1.取1～3天龄SD乳鼠的双侧的坐骨、臂丛神经,D-Hank's液冲洗2次,解剖显微镜下仔细剥除神经外膜,D-Hanks液反复冲洗2次,剪碎至1mm3大小的组织块,双酶消化法进行分离培养,阿糖胞苷抑制成纤维细胞生长,后加入含Forsoklin和牛脑垂体浸提物的培养液来选择性促进雪旺细胞的生长,最后传代进一步纯化,光镜下进行形态学观察。常规复苏培养RSC96细胞。隔天换液,待细胞融合率为80%时用胰蛋白酶消化传代,光镜进行形态学观察。胰蛋白酶消化收集两种细胞,进行以下实验：1)用Trizol法提取细胞总RNA,用Oligo(dT)逆转录酶得到cDNA。经特定引物PCR扩增,取相同量的逆转录产物进行qPCR反应；qPCR反应采用SYBR Green/ROX qPCRMaster Mix(2X),按照说明书在冰上配制PCR反应液,每个样品重复3次。反应结束后确认PCR反应的扩增曲线和溶解曲线,采用2-ΔΔcT法确定特定荧光域值对应循环数的Ct值,对目标基因定量。2)按照细胞总蛋白分离试剂盒说明书进行分离提取各组细胞的蛋白。用Bradford Protein Assay Kit试剂盒测定蛋白含量。取等量样品总蛋白与等体积2×SDS凝胶上样缓冲液混匀,100℃加热煮沸10min,经SDS-PAGE凝胶电泳分离。用湿电转移法将蛋白转移至PVDF膜上,分别采用抗NGF,抗Laminin和抗(3-actin一抗孵育2h,三次洗膜后,加入HRP-偶联二抗,进行免疫反应,化学发光法检测条带。条带信号强度应用Image J图像分析软件进行分析。
2.直径较细的平行排列的静电纺丝纤维制备：将6-氟异丙醇溶解的10%的聚己内酯在电压为10kv的静电纺丝仪上以4m1/h速度纺成纺丝,用2800rpm的取向接收器接收。随机排列的静电纺丝纤维制备：将6-氟异丙醇溶解的10%的聚己内酯在电压为10kv的静电纺丝仪上以3ml/h速度纺成纺丝,用200rpm接收器接收。在制备上述两种不同空间排列的静电纺丝时,注射器喷头和接收器之间的距离分别是15cm和13cm。直径较粗的平行排列的静电纺丝纤维制备：将二氯甲烷和甲醇混合液溶解的15%的聚己内酯在电压为10kv的静电纺丝仪上以3m1/h速度纺成纺丝,用2800rpm的取向接收器接收。注射器喷头和接收器之间的距离是10cm。
3.RSC96细胞常规复苏后24h换液,2-3d传代扩增,扩增培养24小时后接种于PCL材料上,并进行分组,对照组A：平行排列的静电纺丝上接种培养RSC96细胞。实验组B：随机排列的静电纺丝上接种培养RSC96细胞。实验组C：PCL膜直接接种培养RSC96细胞。各组接种细胞数量相同。各组细胞于37℃含5%CO2的培养箱中培养3天(期间换液1次)后,进行以下实验：1)按照细胞总蛋白分离试剂盒说明书进行分离提取各组细胞的蛋白。用Bradford Protein Assay Kit试剂盒测定蛋白含量。取等量样品总蛋白与等体积2×SDS凝胶上样缓冲液混匀,100℃加热煮沸10min,经SDS-PAGE凝胶电泳分离。用湿电转移法将蛋白转移至PVDF膜上,分别采用抗NGF,抗Laminin和抗β-actin一抗孵育2h,三次洗膜后,加入HRP--偶联二抗,进行免疫反应,化学发光法检测条带。条带信号强度应用Image J图像分析软件进行分析。2)用Trizol法提取细胞总RNA,用Oligo(dT)逆转录酶得到cDNA。经特定引物PCR扩增,取相同量的逆转录产物进行qPCR反应；qPCR反应采用S YBR Green/ROX qPCRMaster Mix(2X),按照说明书在冰上配制PCR反应液,每个样品重复3次。反应结束后确认PCR反应的扩增曲线和溶解曲线,采用2-ΔΔCT法确定特定荧光域值对应循环数的Ct值,对目标基因定量。
4.RSC96细胞常规复苏后24h换液,2-3d传代扩增,扩增培养24小时后接种于PCL材料上,并进行分组,对照组A：六孔板空白孔内直接接种培养RSC96细胞。实验组B：直径较细平行静电纺丝上接种培养RSC96细胞。实验组C：直径较粗平行静电纺丝上接种培养RSC96细胞。各组接种细胞数量相同。各组细胞于37℃含5%CO2的培养箱中培养3天(期间换液1次),用Trizol法提取细胞总RNA,用Oligo(dT)逆转录酶得到cDNA。经特定引物PCR扩增,取相同量的逆转录产物进行qPCR反应：qPCR反应采用SYBR Green/ROX qPCRMaster Mix(2X),按照说明书在冰上配制PCR反应液,每个样品重复3次。反应结束后确认PCR反应的扩增曲线和溶解曲线,采用2-ΔΔCT法确定特定荧光域值对应循环数的Ct值,对目标基因定量。
结果
1.光镜下观察,雪旺细胞胞体呈双极纺锤形,周缘有亮带,胞核呈卵圆形或长圆形,细胞两极有长短不等的突起,细胞间常有端对端、肩并肩排列,排列呈放射状或巢状；RSC96细胞呈现典型的双极纺锤形或多角形,绝大多数细胞伸出长突起,其生长形态与原代雪旺细胞非常相似；qPCR和Western blot结果发现体外培养的RSC96细胞也可表达NGF及Laminin,与原代雪旺细胞相似。
2.运用不同静电纺丝接收装置,制备了直径相似但空间排列不同的两组静电纺丝；通过对纺丝溶液浓度和毛细喷丝头与接收仪器间距离的调控,制备了直径不同但同为平行排列的两组静电纺丝。
3.平行、随机静电纺丝及膜上培养RSC96细胞NGF和Laminin基因的表达及蛋白含量：3组细胞培养3天,后两组的RSC96细胞NGF基因的扩增表达量较多,相比第一组有很明显的差异,差异有统计学意义(P<0.05),后两组之间明显差异；3组细胞均有Laminin基因扩增表达,且呈递减趋势,各组之间差异有统计学意义(P<0.05)。各组细胞蛋白条带显示均有NGF和Laminin的表达,其中各组NGF蛋白的表达明显差异,且有统计学意义(P<0.05)；各组Laminin蛋白的表达呈递减趋势,各组之间差异有统计学意义(P<0.05)。
4.培养在粗细不同的平行静电纺丝上及空白对照组的RSC96细胞NGF和Laminin蛋白表达：3组细胞培养3天,后两组的RSC96细胞NGF基因的扩增表达量较少,相比第一组有很明显的差异,差异有统计学意义(P<0.05),后两组之间无明显差异(P>0.05)；3组细胞均有Laminin基因扩增表达,且呈递增趋势,各组之间差异有统计学意义(P<0.05)。
结论
1.发现体外培养的RSC96细胞生长形态与原代雪旺细胞非常相似；也可表达神经营养因子及层粘连蛋白,是一种较理想的原代雪旺细胞替代品,避免了长周期且繁杂的原代雪旺细胞培养及雪旺细胞纯度不高和长期培养雪旺细胞功能不佳等不足。
2.通过控制静电纺丝工艺中的相关因素如：纺丝溶液浓度、接收装置不同及毛细喷丝头与接收仪器间距离分别制备了两种不同空间排列即平行和随机排列的PCL静电纺丝及粗细不同的平行的PCL静电纺丝。
3.将RSC96细胞培养在不同空间排列的静电纺丝上,检测RSC96细胞神经生长因子及层粘连蛋白的表达差异,发现平行排列的静电纺丝纤维抑制神经生长因子的表达,却促进层粘连蛋白的表达,从而进一步说明了平行排列的静电纺丝纤维可能有类似与轴突的作用,并可能促进RSC96细胞向更成熟的形态发育。
4.初步探讨了将RSC96细胞培养在不同直径的平行排列静电纺丝上,检测RSC96细胞NGF及Laminin基因表达差异,发现直径较粗的平行静电纺丝纤维Laminin的表达较多,有统计学意义。
Background:
Peripheral nerve injury remains a high incidence of life-long disability in the world wide. Among various types of peripheral nerve injuries, transection injuries where the nerve trunk is completely interrupted, especially those resulting in large neural gaps, may have a devastating impact on patients' quality of life, and in these cases reconstructive surgery is required as a therapeutic management to achieve nerve regeneration and function restoration. In consequence, peripheral nerve repair represents a unique challenge and opportunity to clinical and translational neurosciences. After peripheral nerves are transected, a series of molecular and cellular events, collectively called Wallerian degeneration, is triggered throughout the distal stump of transected nerves and within a small zone distal to the proximal stump, resulting in the disintegration of axoplasmic microtubules and neurofilaments Within24h most axons along the distal stump of transected nerves are reduced to granular and amorphous debris; by48h the myelin sheath has begun to be transformed toward the short segment. Then macrophages and monocytes migrate into the degenerating nerve stumps to remove myelin and axon debris, while Schwann cells proliferate to form longitudinal cell columns, known as Bands of Bungner. Under the influences of neurotrophic factors and extracellular matrix molecules produced by Schwann cells, the proximal portion of transected nerves sprouts new daughter axons to generate a "regenerating unit" that is surrounded by a common basal lamina. New axonal sprouts usually emanate from the nodes of Ranvier, and undergo remyelination by Schwann cells. Functional reinnervation requires that the regenerating axons elongate under the mediation of growth cones until they reach their synaptic target, and in humans, axon regeneration occurs at a rate of about2-5mm/day; thus significant injuries may take many months to heal. To aid the repair of peripheral nerve injuries, clinical intervention has been attempted for several hundred years since as early as the17th century, when Ferrara first reported a suture technique for repairing a severed nerve. After the significant progress made in the19th and20th centuries, a wide range of surgical techniques has been put into use for the management of peripheral nerve injuries. Although there have been great advancements in the surgical repair of peripheral nerve injuries, autologous nerve grafting remains the gold standard technique to which other treatments are compared. Unfortunately, autologous nerve grafting is limited by the inherent drawbacks, such as limited availability of donor nerves, the need for a second surgery to obtain the donor nerve, donor site morbidity and secondary deformities, as well as mismatch between the injured nerve and the donor nerve. In addition, clinically functional recovery rates typically approach only80%for nerve injuries treated by autologous nerve grafts. It goes without saying that seeking promising alternatives to supplement or even substitute autologous nerve grafts constitutes a major challenge to peripheral nerve repair. In the last few decades, different types of biological or artificial grafts have been developed and investigated as compared with autologous nerve grafts in terms of the outcomes of nerve regeneration and functional recovery. More importantly, tissue engineering, an emerging multidisciplinary field, has grown at a significant rate in recent years. This offers great opportunities to neuroscientists and surgeons who have been collaborating to develop tissue engineered nerve grafts. Just like most of tissue engineered products, tissue engineered nerve grafts are typically composed of a physical scaffold with the introduction of support cells and/or growth factors or other biomolecular components.
Attention has recently focused on the biological behaviors of Schwann cells, the principal glia of the peripheral nervous system. During development, Schwann cells migrate ahead of and along axonal tracts, ensheath several axons and eventually segregate to wrap around a single axonal segment. In response to peripheral nervous system injuries, Schwann cells proliferate, produce growth factors, remove debris, and lay down longitudinal tracks that provide guidance and support for regrowing axons. Additionally, aligned live Schwann cells and isolated Schwann cells topography have the capacity to direct axon growth in vitro. Because of their instrumental role in aiding peripheral nervous system injury repair, Schwann cells have received attention as an important cell type with therapeutic applications. It has been recently suggested that therapies to promote nerve repair, notably nerve guidance channels, will require transplantation of supportive cells such as Schwann cells.
Schwann cells in spinal nerves originate from the neural crest, although the origin of cells in spinal roots is more complex. The end point of Schwann cell development is the formation of myelinating and non-myelinating cells that ensheath large and small diameter axons, respectively, throughout the PNS. Schwann cell formation is preceded by the generation of two other cell types:SCPs, which are the glial cells of embryonic day (E)14-15rat nerves (mouse E12-13), and immature Schwann cells, which are generated from the SCPs from E15to El7(mouse E13-15). The latter are the glial cells found in rat nerves from E17-18to about the time of birth. The postnatal fate of immature Schwann cells is determined by which axons they randomly associate with, with myelination being selectively activated in those cells that happen to envelop single large diameter axons. These events can be viewed as three main transitions, that is, the transition from migrating neural crest stem cells to SCPs, from SCPs to immature Schwann cells and, lastly, the divergence of this population to form the two mature Schwann cell types. These events are strikingly dependent on survival factors, mitogens and differentiation signals from the axons with which SCPs and Schwann cells continuously associate.
Accumulating evidence suggests that cells interact with their environment not only biochemically but also physically. Although the biochemical interactions have been widely investigated in different cells systems, our understanding of how physical interactions affect cell behaviors is still in its infancy. The first patent that described the operation of electrospinning appeared in1934, when Formalas disclosed and apparatus for producing polymer filaments by taking advantage of the electrostatic repulsions between surface charges. Up until1993, this technique had been known as electrostatic spinning, and there were only a few publications dealing with its use in the fabrication of thin fibers. In the early1990s, several research groups revived interest in this technique by demonstrating the fabrication of thin fibers from a broad range of organic polymers. At this time the term electrospinning was coined and is now widely used in the literature. These timely demonstrations triggered a lot of experimental and theoretical studies related to electrospinning. It is notable that the number of publications in this field has been increasing exponentially in the past few years, on account of the remarkable simplicity, versatility, and potential uses of this technique. With the possibility of generating fibers in the nanoscale and the resulting scaffolds with organized architecture, electrospun fibrous meshes may mimic the extracellular matrix. RSC96cell line is naturally transformed from primary cell culture of rat Schwann cells. It could be served as an ideal class of Schwann cell analogues. Our study is mainly composed of two components. One is to compare the biological behaviors of RSC96cells with the ones of primary schwann cells, the other is to study the influence of electrospun fibers on the biologcial behaviors of RSC96cells based on the previous results.
Objective
1. To compare the biological behaviors of RSC96cells with primary culture of schwann cells.
2. To fabricate different topography of electrospun fibers and varying diameter of aligned elecntrospun fibers.
3. To study the influence of different topography of electrospun fibers on the biologcial behaviors of RSC96cells.
4. To explore the influence of varying diameter of aligned electrospun fibers on the biologcial behaviors of RSC96cells.
Methods
1. The bilateral ischial nerves and brachial plexus nerves were taken from SD rats(1to3days). These were washed by D-Hanks solution several times, stripped the epineurium carefully under a dissecting microscope, and washed three times by D-Hanks solution. We cut the nerve tissue into small blocks in size of1mm3and isolated the tissue blocks with the dual-enzyme digestion. Cytarabine was used to inhibit fibroblast growth. Addition of forsoklin and pituitary extract bovine was to optional stimulate the proliferation of schwann cells and the pass was to further purify Schwann cells. Morphology was observed under light microscopy. Culture medium containing10%FBS,100U/mL penicillin,100μg/mL streptomycin, DMEM/F12was used to culture the RSC96cells.Then the culture dishes were placed in the CO2incubator at37℃. The medium was changed every other day. After the cell fusion rate got80%, the cells were digested and passed by0.25%trypsin with0.02%EDTA. The morphology was also observed under light microscopy. The two types of cells were digested by Trypsin and collected, Procedures were performed as follows:1) total RNA were extracted by Trizol method following the protocol. Reverse transcriptase Oligo (dT) was used to synthesize cDNA. Then RNA was expanded by designed primer (Table) and used for q-PCR. The same amount of transcript was used for q-PCR, with SYBR Green/ROX qPCR MasterMix (2X) by following the protocol and repeated for3times. The relative expression levels of genes were analyzed using the2-△△CTmethod by normalizing with (3-actin house-keeping gene expression.2) Proteins of RSC96cells were extracted from two groups by following the protocols of Total Cellular Protein Isolation Kit. Bradford Protein Assay Kit was used to detect the protein level. Equal quantities of total proteins from samples were mixed with equal volume of2×SDS gel loading buffer. After boiling at100℃for10min, samples were separated on SDS-PAGE and transferred onto PVDF membrane. After blocked by primary antibodies of anti-NGF, anti-Laminin and anti-β-actin for2hours, membranes were rinsed3times and incubated with horseradish peroxidase-conjugated secondary antibodies. Films were digitally imaged using Image J software.
2. Firstly, In order to obtain thinner aligned PCL fibers, a solution of10wt%of PCL dissolved by Hexafluoroisopropanol was dispensed at a flow rate of4ml/h and electrospun under a voltage of10kv. The PCL fibers were collected on a grounded target rotating at-2800rpm. For scaffolds comprising randomly oriented PCL fibers,10wt%of PCL dissolved by Hexafluoroisopropanol was dispensed at a flow rate of3ml/h under a voltage of10kv and collected on rotating a target(-200rpm). The polymer supply-to-target distance were set15cm and13cm respectively. Secondly, In order to obtain thicker aligned PCL fibers, a solution of15wt%of PCL dissolved by a mixture of dichloromethane and methanol was dispensed at a flow rate of3ml/h under a voltage of10kv. The polymer supply-to-target distance was set10cm.
3. After the conventional recovery of RSC96cells, proliferation was done for2-3generations. Group A (experimental):RSC96cells were seeded on the parallel electrospinning fibers. Group B (experimental):RSC96cells were seeded on the randomly electrospinning fibers. Group C (control):RSC96cells were seeded on the polycaprolactone film. Each group containing the equal amount of cells was then placed in the incubator under37℃,5%CO2for3days (medium was changed every other day). Procedures were performed as follows:1) Proteins of RSC96cells were extracted from three groups by following the protocols of Total Cellular Protein Isolation Kit. Bradford Protein Assay Kit was used to detect the protein level. Equal quantities of total proteins from samples were mixed with equal volume of2×SDS gel loading buffer. After boiling at100℃for10min, samples were separated on SDS-PAGE and transferred onto PVDF membrane. After blocked by primary antibodies of anti-NGF, anti-Laminin and anti-β-actin for2hours, membranes were rinsed3times and incubated with horseradish peroxidase-conjugated secondary antibodies. Films were digitally imaged using Image J software.2) RNA of RSC96cells were extracted from three groups by Trizol method following the protocol. Reverse transcriptase Oligo (dT) was used to synthesize cDNA. Then RNA was expanded by designed primer (Table) and used for q-PCR. The same amount of transcript was used for q-PCR, with SYBR Green/ROX qPCR MasterMix (2X) by following the protocol and repeated for3times. The relative expression levels of genes were analyzed using the2-△△CTmethod by normalizing with β-actin house-keeping gene expression.
4. After the conventional recovery of RSC96cells, proliferation was done for2-3generations. Group A (control):RSC96were seeded on the plain cell culture plate directly. Group B (experimental):Cells were seeded on the thinner aligned electrospun fibers. Group C (experimental):cells were seeded on the thicker aligned electro spun fibers. Each group containing the equal amount of cells was then placed in the incubator under37℃,5%CCO2for3days (medium was changed every other day). The total RNA of cells were extracted by Trizol method following the protocol. Reverse transcriptase Oligo (dT) was used to synthesize cDNA. Then RNA was expanded by designed primer (Table) and used for q-PCR. The same amount of transcript was used for q-PCR, with SYBR Green/ROX qPCR MasterMix (2X) by following the protocol and repeated for3times. The relative expression levels of genes were analyzed using the2-△△CTmethod by normalizing with β-actin house-keeping gene expression.
Results
1. The shape of the Schwann cells were bipolar spindle with bright edge, oval or oblong nucleis, protuberances around cell biopolar with multi-length ranges, cells growed side by side and arrayed in radial or nests under the light microscope, while RSC96cells were typically bipolar spindle or polygon shape and mostly extended long processes, and the shape was much similar with primary culture of Schwann cells. The results of qPCR and western blot analysis showed that RSC96cells cultured in vitro can also express NGF and Laminin similar with primary culture Schwann cells.
2. Different topography of electrospun fibers were fabricated by different types of collectors and varying diameter of aligned elecntrospun fibers were created by the different intrinsic properties of the solution and distance between spinneret and collector.
3. To detect mRNA expression by qPCR and protein expression by western blot: According to different culture conditions, cultures of RSC96cells were divided into three groups, group A(aligned), group B(randomly) and group C(film), and all the three groups were cultured for three days. NGF mRNA expression of RSC96was lower in group A, compared to group B and C (P<0.05). There was statistical differences between group B and group C (P<0.05). The laminin mRNA was expressed in all the groups, an decrease from group A to group C (P<0.05). What's more, Laminin and NGF protein expressed in all groups. NGF protein expression had statistical differences in all the groups(P<0.05). The laminin protein expression decreased from group A to group C (P<0.05).
4. To detect gene expression by qPCR:According to different culture conditions, cultured RSC96were divided into three groups, group A(control), group B(thinner diameter of aligned electrospun fibers) and group C(thicker diameter of aligned electrospun fibers), and all three groups were cultured for three days. The NGF gene expression of RSC96cells was higher in group A, compared to group B and C (P<0.05). There was no statistical difference between group B and group C (P>0.05). The Laminin gene expression of RSC96cells was lower in A, compared to group B and C (P<0.05). There was some statistical difference between group B and group C.
Conclusion
1. The growth pattern of RSC96cells cultured in vitro was much similar with the primary culture of Schwann cells, the cells also expressed neurotrophic factors and laminin and were an ideal substitute for the primary culture of Schwann cells, which avoided the long cycle and complicated primary schwann cells cultivation and long-term poor schwann cell function, and so on.
2. Different topography of electrospun fibers and varying diameter of aligned elecntrospun fibers were fabricated dependent on a number of processing parameters.
3. The different biological behaviours of RSC96cells were induced by aligned electrospun fibers and RSC96cells may be directed towards the more maturation state by topographic cues from electrospun fibers.
4. Biologcial behaviours of RSC96cells were evaluated on different diameter of aligned electrospun fibers. There is some statistical difference in the expression of Laminin cDNA between the two groups.
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