Schwann细胞在髓鞘形成过程中的极性调控

周围神经系统髓鞘形成依赖Schwann细胞和神经元之间复杂的相互作用。细胞极性分子蛋白Par-3在Schwann细胞与轴突接触面密集分布,为BDNF/p75NTR介导的启动成髓提供分子支架。然而,Par-3在该界面聚集并呈不对称性分布的机制仍是一个谜。不少研究发现,JAM和nectin等细胞粘附分子与Par-3不对称性分布有关。另外,通过改变轴突信号如神经营养因子和神经素的水平,也能影响Schwann髓鞘的形成。本文综述和阐释在髓鞘形成过程中,Schwann细胞极性是如何被调控的。     

β-1,4半乳糖基转移酶-I在大鼠坐骨神经损伤后的表达变化

目的分析β-1,4半乳糖基转移酶-I(β-1,4-galactosyltransferase I,β-1,4-GalT-I)mRNA在正常和损伤坐骨神经髓鞘上的定位及其表达变化.方法采用RT-PCR方法,分析β-1,4-GalT-I在小鼠坐骨神经中的表达水平.将RT-PCR扩增的β-1,4-GalT-I片段,克隆到pGEM-T载体中.采用体外转录的方法合成地高辛标记的正、反义β-1,4-GalT-I RNA探针.通过原位杂交及图像分析的方法,分析β-1,4-GalT-I mRNA在正常和损伤大鼠坐骨神经的定位及其表达变化.结果检测到β-1,4-GalT-I在大鼠坐骨神经的髓鞘中有表达,并在坐骨神经损伤后1～2d内表达最高,随后表达下降.结论提示β-1,4-GalT-I可能在周围神经最主要的胶质细胞-施万细胞中表达,并在周围神经损伤后发生表达变化,这样为进一步分析β-1,4-GalT-I在周围神经再生中的调控机制奠定基础.     

Tropic 1808基因在大鼠损伤神经组织中的表达

目的观察Tropic 1808基因在大鼠正常和损伤坐骨神经组织中的表达,探讨Tropic 1808基因在周围神经损伤与再生过程中的作用.方法采用地高辛标记的Tropic 1808 cDNA探针、抗大鼠S-100蛋白抗体,以原位杂交和免疫组织化学双重染色法,观察Tropic 1808基因在正常和损伤大鼠坐骨神经组织中的表达.结果免疫组化结果显示,大鼠正常坐骨神经可表达S-100蛋白,但表达量较低;神经损伤后,其远侧端S-100蛋白的表达量明显增加.原位杂交结果显示,大鼠正常坐骨神经组织未见Tropic 1808 mRNA杂交信号;损伤神经的远侧端呈现较强的阳性信号,而且在部分S-100强阳性反应区可见Tropic 1808 mRNA杂交信号.结论 Tropic 1808基因在正常坐骨神经组织中未见表达;坐骨神经损伤后,其远侧端增殖的雪旺氏细胞可表达Tropic 1808 mRNA.提示,Tropic 1808是一种周围神经损伤后特异表达的基因.     

β-1，4半乳糖基转移酶-Ⅰ在大鼠坐骨神经损伤后的表达变化

目的分析β-1,4半乳糖基转移酶-Ⅰ(β-1,4-galactosyltransferaseⅠ,β-1,4-GalT-Ⅰ)mRNA在正常和损伤坐骨神经髓鞘上的定位及其表达变化。方法采用RT-PCR方法,分析β-1,4-GalT-Ⅰ在小鼠坐骨神经中的表达水平。将RT-PCR扩增的β-1,4-GalT_I片段,克隆到pGEM-T载体中。采用体外转录的方法合成地高辛标记的正、反义β-1,4-GalT-ⅠRNA探针。通过原位杂交及图像分析的方法,分析β-1,4-GalT-ⅠmRNA在正常和损伤大鼠坐骨神经的定位及其表达变化。结果检测到β-1,4-GalT-Ⅰ在大鼠坐骨神经的髓鞘中有表达,并在坐骨神经损伤后1～2d内表达最高,随后表达下降。结论提示β-1,4-GalT-Ⅰ可能在周围神经最主要的胶质细胞一施万细胞中表达,并在周围神经损伤后发生表达变化,这样为进一步分析β-1,4-GalT-Ⅰ在周围神经再生中的调控机制奠定基础。     

神经生长因子通过抑制内质网应激促进外周神经损伤后髓鞘碎片的清除

神经生长因子 (never growth factor,NGF）可促进损伤外周神经的修复并加速轴突和髓鞘的再生,但对外周神经损伤前期作用的研究报道较少。本研究主要探究在损伤外周神经前期,NGF能否加速施旺细胞 (Schwann cells, SCs) 对髓鞘碎片的清除及其调控机制。将坐骨神经损伤的Wistar雄性大鼠连续5 d给予NGF治疗,并运用分子生物学检测手段分析损伤坐骨神经内部髓鞘碎片的清除,细胞的凋亡及内质网应激 (endoplasmic reticulum stress, ERS) 的表达和变化。免疫荧光分析结果显示,与模型组相比,NGF给药组显著加速髓鞘碎片的清除,并促进SCs的增殖 (46.33 ± 5.68 vs. 66.69 ± 8.76, P＜ 0.05 for MPZ; 47.58 ± 4.52 vs. 37.69 ± 2.50, P＜ 0.01 for GFAP)。TUNEL免疫组化证实,NGF可有效抑制SCs的凋亡(25 ± 4 vs. 37 ± 6, P＜ 0.05),Western印迹结果显示,模型组坐骨神经内部内质网应激水平被过度激活,给予NGF治疗后,相关蛋白质表达被逆转 (1.03 ± 0.03 vs. 1.24 ± 0.07, P＜ 0.01 for PDI; 1.16 ± 0.16 vs. 1.48 ± 0.10, P＜ 0.05 for GRP-78; 1.33 ± 0.11 vs. 1.76 ± 0.17, P＜ 0.01 for Caspase-12; 1.01 ± 0.05 vs. 1.39 ± 0.16, P＜ 0.01 for CHOP)。上述结果证实,NGF可通过抑制内质网应激减少神经组织内细胞的凋亡,并加速髓鞘碎片的清除,促进外周神经损伤的修复。     

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。     

戊型肝炎病毒ORF1多聚蛋白的不同功能域在细胞内的定位

为了探讨戊型肝炎病毒多聚蛋白ORF1的多个功能域在宿主细胞中的表达和定位情况,我们首先将psk-HEV重组载体上的ORF1各功能域的编码序列克隆到绿色荧光蛋白载体pcDNA3.1-GFP上,构建成融合表达的重组质粒,并测序和酶切鉴定其构建成功。再通过Western-Blot验证各融合蛋白在细胞中正确表达,并用激光扫描共聚焦显微镜观察融合蛋白在细胞内的分布和定位。在Huh7细胞中,RdRp蛋白主要分布于细胞核内,HEL蛋白以囊泡状分布于细胞核周,MET蛋白以颗粒状存在于细胞核和细胞质中,PLP蛋白呈极性分布于细胞核周,X蛋白在细胞核和细胞质中均存在。各融合蛋白在细胞中的不同定位印证了对这些蛋白质的功能预测和体外研究结果,这为进一步研究HEV不同蛋白功能提供了支持。     

Olig2在cuprizone介导的脱髓鞘动物模型中的表达变化

目的探讨Olig2在cuprizone诱导的急性脱髓鞘动物模型中的表达变化规律。方法应用含0.2%cuprizone饲料饲育小鼠,通过调控饲育时间,造成神经脱髓鞘及髓鞘再生,使用免疫荧光染色和实时定量PCR(qRT-PCR)的方法,观察模型髓鞘脱失后及髓鞘再生2周后Olig2、少突胶质细胞碱性髓鞘蛋白(MBP)及星形胶质细胞神经胶质酸性蛋白(GFAP)的表达变化。结果 Cuprizone饲育6周后,动物胼胝体白质内髓鞘脱失严重,在恢复正常饲料后,髓鞘逐渐恢复正常结构。正常小鼠大脑Olig2低水平表达。髓鞘脱失后Olig2、GFAP表达增高,并可见Olig2+/GFAP+细胞,MBP表达明显降低。髓鞘再生2周后Olig2表达降低,MBP、GFAP表达增高。结论 Olig2基因在cuprizone诱导的脱髓鞘模型中的表达变化,提示Olig2可能参与祖细胞向有活性的星形胶质细胞的分化过程,并与胶质瘢痕的形成有关。     

肾素(原)受体在大鼠肾小球系膜细胞和肾脏的表达

近年发现的肾素（原）受体（renin／prorenin receptor,RnR）已被证明具有生物学功能,在心、肾及多种细胞表达。本文旨在观察RnR在体外培养的大鼠肾小球系膜细胞（mesangial cells,MCs）和肾脏中是否表达,及其表达的细胞部位,并用RnR的多肽阻断剂肾素原“柄区肽”（handle region peptide,HRP）与RnR结合后观察受体复合物进入细胞的过程与定位。结果显示,RnR主要存在于大鼠肾脏皮质肾小球系膜区和体外培养的MCs的细胞核周围胞浆和细胞膜。将FITC标记的HRP（FITC-HRP）加入细胞培养液后30S到30min期间,可观察到FITC-HRP由培养液转移到胞浆内并进入细胞核。用免疫荧光和激光共聚焦技术观察到,HRP与RnR的共定位主要位于细胞膜和细胞核周围胞浆;在30min时,一部分HRP已进入细胞核,而RnR没有进入细胞核内,仍主要位于细胞核周围胞浆。上述结果提示,RnR与其配基结合后进入细胞内并发挥生物学效应。     

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.     

Temporal-Spatial Expressions of Spy1 in Rat Sciatic Nerve After Crush

As a novel cell cycle protein, Spy1 enhances cell proliferation, promotes the G1/S transition as well as inhibits apoptosis in response to UV irradiation. Spy1 levels are tightly regulated during mammary development, and overexpression of Spy1 accelerates tumorigenesis in vivo. But little is known about the role of Spy1 in the pathological process of damage and regeneration of the peripheral nervous system. Here we established a rat sciatic nerve crush (SNC) model to examine the spatiotemporal expression of Spy1. Spy1 expression was elevated gradually after sciatic nerve crush and peaked at day 3. The alteration was due to the increased expression of Spy1 in axons and Schwann cells after SNC. Spy1 expression correlated closely with Schwann cells proliferation in sciatic nerve post injury. Furthermore, Spy1 largely localized in axons in the crushed segment, but rarely co-localized with GAP43. These findings suggested that Spy1 participated in the pathological process response to sciatic nerve injury and may be associated with Schwann cells proliferation and axons regeneration.     

Dynamic Change of Prohibitin2 Expression in Rat Sciatic Nerve After Crush

As a novel cell cycle inhibitor, PHB2 controls the G1/S transition in cycling cells in a complex manner. Its aberrant expression is closely related to cell carcinogenesis. While its expression and role in peripheral nervous system lesion and repair were still unknown. Here, we performed an acute sciatic nerve crush (SNC) model in adult rats to examine the dynamic changes of PHB2. Temporally, PHB2 expression was sharply decreased after sciatic nerve crush and reached a valley at day 5. Spatially, PHB2 was widely expressed in the normal sciatic nerve including axons and Schwann cells. While after injury, PHB2 expression decreased predominantly in Schwann cells. The alteration was due to the decreased expression of PHB2 in Schwann cells after SNC. PHB2 expression correlated closely with Schwann cells proliferation in sciatic nerve post injury. Furthermore, PHB2 largely localized with GAP43 in axons in the crushed segment. Collectively, we suggested that PHB2 participated in the pathological process response to sciatic nerve injury and may be associated with Schwann cells proliferation and axons regeneration.     

Changes in CLIP3 expression after sciatic nerve injury in adult rats

CLIP3 (cytoplasmic linker protein 3) is a 547 amino acid residue cytoplasmic protein that localises to Golgi stacks and tubulovesicular elements juxtaposed to Golgi cisternae. Composed of three Ank (ankyrin) repeats and two CAP-Gly (cytoskeleton-associated protein-glycine) domains, CLIP3 may function as a cytoplasmic linker protein that is involved in TGN–endosome dynamics. To define the expression and role of CLIP3 during peripheral nervous system degeneration and regeneration, we created an acute sciatic nerve injury (SNI) model in adult rats. Western blot analyses revealed prominent up-regulation of CLIP3 and PCNA (proliferating cell nuclear antigen) protein levels at 3?days after SNI. Immunohistochemistry displayed that the expression of CLIP3 was noticeably increased in the injured nerve. Immunofluorescence further revealed that the CLIP3 and PCNA proteins colocalised respectively with S100 in the cytoplasm of Schwann cells. The expression profile of the SC/neuron co-cultures demonstrated that CLIP3 and PCNA protein levels were markedly expressed during the early stage of myelination. These results suggest that CLIP3 is likely associated with the myelination of proliferating Schwann cells, and nerve tissue regeneration after peripheral nerve injury. CLIP3 and PCNA expression during early myelination may be related to the direct uptake and transport of lipids and cholesterol, which were derived from the degenerating myelin, by Schwann cells to prepare for the formation of myelin sheath-like structures around regenerated axons after SNI.     

Sulfation of Rat Apolipoprotein E

The synthesis of a 37-kilodalton (kDa) protein which has been shown recently to be identical with apolipoprotein E (apo-E) was increased after sciatic nerve injury of the rat. When regeneration of the nerve was allowed, its synthesis returned to control levels at about 8 weeks post injury. In this report it is shown that similar time-course studies of the protein in the rat optic nerve revealed a delayed increase of the protein but a comparably high level of synthesis at 3 weeks post injury. This level was maintained up to at least 18 weeks after crush. Furthermore, two-dimensional electrophoresis revealed that the characteristic "trailing" of the protein is due to its sialylation, because it was reduced after neuraminidase treatment. This treatment, however, detected a neuraminidase-resistant heterogeneous form in CNS tissue and a homogeneous form in peripheral nervous tissue. The trailing persisted up to 18 days of culture of optic nerve explants, of CNS glial cells, and of peritoneal macrophages, but disappeared during the first culture days of sciatic nerve explants and was not observed in Schwann cell culture media. Incorporation studies with 35SO4 revealed that apo-E was the major sulfated protein in culture media conditioned by CNS glial cells, whereas sulfation of the protein was undetectable in Schwann cell cultures. Because macrophages are likely to be the major source of apo-E in both peripheral and central glial cell cultures as well as in injured optic and sciatic nerves, it is hypothesized that resident cells of sciatic nerves secrete potent sulfatases. As a result, sialic acid residues may be more susceptible to degradation.(ABSTRACT TRUNCATED AT 250 WORDS)     

CAP1 was associated with actin and involved in Schwann cell differentiation and motility after sciatic nerve injury

Adenylate cyclase-associated protein 1 (CAP1), a member of cyclase-associated proteins that regulating actin dynamics, was shown to regulate actin filaments, localize to dynamic actin structures and mediate such processes as establishment of cell polarity, motility, morphogenesis, receptor-mediated endocytosis and mRNA location. But little is known about the role of CAP1 during peripheral nervous system injury. Here, we found the spatiotemporal protein expression of CAP1 after sciatic nerve crush. After crush, CAP1 had an increased protein expression level, reached a peak at about day 5 and then returned to the normal level at 4 weeks, similar to Oct-6. Besides, in 5-day injured tissue, using double immunofluorescent staining we found CAP1 had a colocalization with S100 and Oct-6. In vitro, during the process of cAMP-induced Schwann cells differentiation, we observed enhanced expression of CAP1 and P0. Specially, CAP1-specific siRNA-tranfected SCs did not show significant actin structure which form cellure surface tension and protrusion shape after cAMP treatment. And we observed the interaction of CAP1 with actin and that CAP1-specific siRNA-transfected SCs had a decreased motility and migration. Together, all these data indicated that the change of CAP1 protein expression was associated with Schwann cells motility and differentiation after the crush of sciatic nerve.     

Regulation of ciliary neurotrophic factor expression in myelin-related Schwann cells in vivo.

Adult rat sciatic nerve is known to express high levels of ciliary neurotrophic factor (CNTF) mRNA and protein. Here we examine the cellular localization of CNTF protein and mRNA in peripheral nerve and the regulation of CNTF expression by peripheral axons. In intact nerve, CNTF immunoreactivity is found predominantly in the cytoplasm of myelin-related Schwann cells. After axotomy, CNTF immunoreactivity and mRNA levels fall dramatically and do not recover unless axons regenerate. This behavior is similar to the pattern of myelin gene expression in these nerves. We conclude that the expression of CNTF in Schwann cells depends on axon-Schwann cell interactions.     

Interleukin-6 induces proinflammatory signaling in Schwann cells: A high-throughput analysis

Interleukin-6 plays an important role in peripheral nerve regeneration. We recently reported that IL-6 targets Schwann cells in the peripheral nerve for its function. In this study, we analyzed genes whose expression is regulated by IL-6 in a cell line derived from Schwann cells, the peripheral glia, using the Illumina gene microarray. At measurements 3 and 12 h after IL-6 treatment, 35 genes were found to be upregulated by IL-6. Most upregulated genes were proinflammatory genes that are known to be induced in inflammatory conditions. Interestingly, the expression of immunoproteasome subunits was upregulated by IL-6 in Schwann cells. Treatment with forskolin, an agent that mimics axonal signaling, suppressed the expression of IL-6-inducible genes. Finally, we found for the first time that sciatic nerve injury induced immunoproteasome expression in vivo. These findings indicate that IL-6 is involved in peripheral nerve regeneration by regulating proinflammatory signaling in Schwann cells.     

Connexin43 Is Another Gap Junction Protein in the Peripheral Nervous System

Abstract: That many cells express more than one connexin (Cx) led us to examine whether Cxs other than Cx32 are expressed in the PNS. In addition to Cx32 mRNA, Cx43 and Cx26 mRNAs were detected in rat sciatic nerve by northern blot analysis. Cx43 mRNA, but not Cx26 mRNA, was expressed in both the primary Schwann cell culture and immortalized Schwann cell line (T93). The steady-state levels of the Cx43 mRNA in the primary Schwann cell culture increased 2.0-fold with 100 µ M forskolin, whereas that of P₀ increased 7.0-fold. Immunoreactivity to Cx43 was detected on western blots of cultured Schwann cells, T93 cells, and sciatic nerves but not on blots of PNS myelin. Immunohistochemical study using human peripheral nerves revealed that anti-Cx43 antibody stained cytoplasm around nucleus of Schwann cells but not myelin, confirming western blot results. Although P₀ expression was markedly decreased by crush injury of the sciatic nerves, Cx43 expression showed no apparent change. Developmental profiles showed that Cx43 expression in the sciatic nerve increased rapidly after birth, peaked at about postnatal day 6, and then decreased gradually to a low level. In adult rats, the Cx43 mRNA value was much lower than that of Cx32. These findings suggest that Cx43 is localized in Schwann cell bodies and that, compared with P₀, its expression is less influenced by axonal contact and cyclic AMP levels. The high expression on postnatal day 6 indicates that Cx43 may be related to PNS myelination. Cx43 is another gap junction, but its function appears to differ from that of Cx32, as judged by the differences in their localization and developmental profiles.