糖尿病病理性神经痛大鼠坐骨神经中miR-133a的上调对疼痛阈值的影响及机制

摘要 目的：探究糖尿病病理性神经痛大鼠坐骨神经中miR-133a的上调对疼痛阈值的影响及机制。方法：使用NGS测序和定量PCR分析糖尿病病理性神经痛大鼠坐骨神经中的相关 miRNA表达,预测潜在下游目标。使用miR-133a-3p转染的 RSC96细胞进行体外实验。使用微蛋白质印迹和蛋白质印迹以及免疫荧光分析验证miR-133a-3p的作用。使用miR-133a-3p拮抗剂分析miR-133a-3p与DNP之间的关联。结果：miR-133a-3p模拟物增加了RSC96细胞中VEGFR-2、p38αMAPK、TRAF-6和PIAS3的表达,并降低了NFκB p50和MKP3的表达（P＜0.05）。在正常大鼠中,AAV-miR-133a-3p通过神经内注射到坐骨神经中可诱导机械性异常性疼痛和p-p38 MAPK激活（P＜0.05）。在DM大鼠中,miR-133a-3p 拮抗剂给药可减轻DNP并下调p-p38磷酸化（P＜0.05）。结论：坐骨神经中miR-133a-3p的过度表达会引起这种疼痛,miR-133a-3p可能是患有神经性疼痛的患者的有用治疗靶点。     

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.     

P物质在再生视网膜节细胞中的表达及IBMX和CPT-cAMP 对其表达的影响

采用金黄地鼠视神经切断并缝接坐骨神经的再生实验模型,玻璃体内注射IBMX或/和CPT-cAMP,荧光金逆行标记再生的RGCs结合P物质免疫荧光组化双标法,研究外周神经缝接于视神经断端能否促进P物质阳性的视网膜节细胞(RGCs)再生及IBMX或/和CPT-cAMP处理对其再生的影响。实验结果:①术后四周,对照AG组每个视网膜 再生RGCs数1329±104,双标细胞平均数为45±5,占再生RGCs总数的3.4％;②AG+IBMX组每个视网膜再生RGCs数为2099±419,再生P物质阳性节细胞平均数为119±22,占再生RGCs总数的6.55％;③AG+cAMP组每个视网膜再生RGCs数为2048±133,再生P物质阳性节细胞平均数为127±37,占再生RGCs总数的6.15％;④AG+IB-MX+cAMP组每个视网膜再生RGCs数为4370±487,再生P物质阳性节细胞平均数为339±72,占再生RGCs总数的7.98％,与对照组的差异具有统计学意义。表明成年哺乳动物P物质阳性RGCs能再生,玻璃体内注射IBMX或/和CPT-cAMP可以促进该类RGCs再生。     

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.     

Regulation of Hemopexin Synthesis in Degenerating and Regenerating Rat Sciatic Nerve

Abstract : In injured peripheral nerves, hemopexin mRNA is expressed by fibroblasts, Schwann cells, and invading blood macrophages, and the protein accumulates in the extracellular matrix. This and its absence of regulation in injured central optic nerve suggest that hemopexin could play a positive role in peripheral nerve repair. Here, we studied the regulation of hemopexin expression in degenerating and regenerating nerves. After a sciatic nerve injury, both the synthesis of hemopexin and the level of its mRNA increase sharply during the first 2 days, leading to an accumulation of hemopexin in the nerve. Afterward, hemopexin expression decreases progressively in regenerating nerves. In permanently degenerated nerves, it is again transiently increased and then strongly decreased, whereas hemopexin from blood origin is accumulating. As part of the elucidation of the complex regulation of hemopexin expression in injured nerves, we demonstrate that interleukin-6 increases hemopexin synthesis in intact nerves, whereas adult rat serum, but not purified hemopexin, inhibits it in degenerated nerves. Hemopexin, known as acute-phase protein, is therefore one of the molecules rapidly and specifically up-regulated in injured peripheral nerves. More generally, our findings suggest that the acute phase could be not only a systemic liverspecific response but also a reaction of injured tissues themselves.     

Non-canonical functions of the peripheral nerve

The peripheral nervous system (PNS) is complex and omnipresent. The PNS targets all parts of the body starting from early stages of embryonic development, and in large part, is derived from multipotent migratory neural crest stem cells. Current opinion mostly perceives the PNS as a means of communication and information exchange between the central nervous system, the rest of the body and the environment. Additionally, the PNS is largely associated with autonomic control. Being an “alternative brain” it provides local regulation of processes in organs. However, it has become evident in recent years that in addition to these main canonical functions the PNS possesses a number of other important roles in development and homeostasis of targeted tissues, for instance, in nerve-dependent regeneration. The PNS represents a niche that hosts neural crest-derived peripheral glial cells, or, in other words, neural crest-like multipotent cells throughout the entire body. These multipotent nerve-adjacent cells can be reprogrammed in vivo and play a number of roles from creating pigmentation to controlling regeneration of a limb in amphibians or skin in rodents. In the current review we outline newly emerged, non-canonical functions of the PNS and briefly describe cellular and molecular aspects of these alternative functions.     

Identification of G Protein Subtypes in Peripheral Nerve and Cultured Schwann Cells

In this study, we investigated the expression of various G proteins in whole sciatic nerves, in myelin and nonmyelin fractions from these nerves, and in membranes of immortalized Schwann cells. In myelin, nonmyelin, and Schwann cell membranes we detected two 39-40-kDa pertussis toxin substrates that were resolved on separation on urea-gradient gels. Two cholera toxin substrates with apparent molecular masses of 42 and 47 kDa were present in nerve and brain myelin and in Schwann cell membranes. In these membranes, a third 45-kDa cholera toxin substrate, which displayed the highest labeling, was also present. Immunoblotting with specific antisera allowed the identification of G(o) alpha, Gi1 alpha, Gi2 alpha, Gi3 alpha, Gq/G11 alpha, and the two isoforms of Gs alpha in nerve homogenates, nerve, and brain myelin fractions. In Schwann cell membranes we identified G(o) alpha, Gi2 alpha, Gi3 alpha, and proteins from the Gq family, but no immunoreactivity toward anti-Gi1 alpha antiserum was detected. In these membranes, anti-Gs alpha antibody recognized the three cholera toxin substrates mentioned above, with the 45-kDa band displaying the highest immunoreactivity. Relative to sciatic nerve myelin, the Schwann cell membranes revealed a significantly higher expression of Gi3 alpha and the absence of Gi1 alpha. The different distribution of G proteins among the different nerve compartments might reflect the very specialized function of Schwann cells and myelin within the nerve.     

Prevention of endothelial cell cytokine induction by a Staphylococcus aureus lipoprotein

Staphylococcal strain 8325-4, unlike other staphylococcal strains, fails to induce cytokine IL-1 and IL-6 gene expression in human endothelial cells. In the present investigation, this strain was shown to release a product that inhibited cytokine gene expression in endothelial cells infected with another staphylococcal strain. This inhibition was due to prevention of internalization, but not adherence, of bacteria by endothelial cells. Induction of endothelial cell cytokine gene expression by lipopolysaccharide was not affected by the staphylococcal supernatant. In contrast to endothelial cells, 8325-4 did not inhibit Wb-induced cytokine gene expression in monocytes. Further characterization of the inhibitory factor suggests that it is a lipoprotein and that both protein and lipid components play a role in its inhibitory function.     

细胞因子对中枢神经系统海马脑区的作用

细胞因子是一组多肽蛋白,一般认为其主要功能是介导非特异性免疫反应、促进未成熟白细胞增殖、分化和生长等。但近年来的研究表明,这些在免疫系统中起重要作用的调节因子及其受体也存在于中枢神经系统（ＣＮＳ）中,并发现它们对ＣＮＳ中某些神经元和胶质细胞的生理功能有调控作用。本综述细胞因子白细胞介素１、白细胞介素２和白细胞介素６对ＣＮＳ海马脑区作用的研究进展。     

Associations of interleukin-6 with interleukin-1beta, interleukin-8 and macrophage inflammatory protein-1beta in midlife women

Objective: The aim of the present study was to determine the associations of interleukin (IL)-6 with other cytokines and chemokines and to compare these associations in peri- and postmenopausal women. Methods: Ninety-nine perimenopausal and 92 postmenopausal women were enrolled in this study. Serum concentrations of IL-6, IL-1β, IL-2, IL-4, IL-5, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, tumor necrosis factor (TNF)-α, interferon γ, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage (GM)-CSF, macrophage inflammatory protein (MIP)-1β and monocyte chemotactic protein (MCP)-1 were measured simultaneously using a multiplexed cytokine assay. Results: Among the 17 cytokines, IL-6, IL-1β, IL-5, IL-7, IL-8, IL-10, MCP-1 and MIP-1β were detected in serum in more than 50% of the women. Serum levels of IL-4 and MCP-1 in postmenopausal women were significantly higher than those in perimenopausal women. Serum IL-6 concentrations showed significant and positive correlations with serum concentrations of IL-1β, IL-8, MIP-1β, IL-7 and MCP-1 in women regardless of menopausal status, and these correlations were still significant after adjustment for age and body mass index. Conclusion: Serum IL-6 concentration was found to be closely associated with serum concentrations of IL-1β, IL-8, MIP-1β, IL-7 and MCP-1 in women regardless of menopausal status, suggesting that these cytokines act in concert with the progression of several symptoms and various diseases.     

Rat brain Na channel mRNAs in non-excitable Schwann cells

The expression of rat brain voltage-sensitive Na⁺ channel mRNAs in Schwann cells was examined using in situ hybridization cytochemistry and RT-PCR. The mRNAs of rat brain Na⁺ channel subtype II and III, but not subtype I, were detected in cultured Schwann cells from sciatic nerve and in intact sciatic nerve, which contains Schwann cells but not neuronal cell bodies. These results indicate that rat brain Na⁺ channel mRNAs, which have been considered as mainly neuronal-type messages, are also expressed in glial cells in vitro and in vivo.     

Laminin A, B1, and B2 Chain Gene Expression in Transected and Regenerating Nerves: Regulation by Axonal Signals

Abstract: Laminin A, B1, and B2 chain mRNA levels in degenerating and regenerating mouse sciatic nerves were examined using northern blot analysis. In normal intact nerves, B1 and B2 mRNA steady-state levels were high, but when the nerves were crushed, the steady-state levels of B1 and B2 mRNA per milligram wet tissue weight of the distal segments of the nerves increased five- to eightfold over that of control levels as the total RNA and β-actin mRNA levels increased, suggesting that these increases were the consequence of Schwann cell proliferation after axotomy. When the steady-state levels of B1 and B2 mRNA were normalized as the ratio to total RNA or β-actin mRNA levels, however, they drastically decreased to about 20% of the normal nerve levels in the nerve segments distal to both the crush and transaction sites 1 day after injury. In the crushed nerves, B1 and B2 mRNA levels gradually increased as the regenerating nerves arrived at the distal segments and reestablished normal axon–Schwann cell contact, and then returned to normal levels on the 21 st day. In the transected nerves, where Schwann cells continued to be disconnected from axons, both B1 and B2 mRNA levels remained low. Cultured Schwann cells expressed detectable levels of B1 and B2 chain mRNA which significantly increased when the cells were cocultured with sensory neurons. However, mRNA for A chain was not detectable in the normal, axotomized nerves or in cultured Schwann cells. These data indicate that Schwann cells express laminin B1 and B2 chain mRNA that are up-regulated by axonal or neuronal contact, but they do not express A chain mRNA.     

神经退变和再生的构筑变化

将夹伤的大鼠坐骨神经分离成单根纤维,观察98d内轴突和许旺细胞的构筑变化过程发现,损伤既使轴浆转运阻断、积累的细胞器退变,也使髓鞘板层,特别是斯兰氏切迹撕裂、变形或侵入轴突。轴突或髓鞘虽可各呈单一的退变,但以两者并存多见。伤后1d即出现富含微管的再生芽,它被增殖的许旺细胞突起及其基底膜包绕,并逐步发育成熟。根据再生的特征性构筑变化,提出了再生芽、无髓和有髓纤维、斯兰氏切迹、朗氏结与神经小束的初见、发育和成熟高峰期的时间顺序。无髓纤维的发育成熟早于有髓纤维。     

胶质细胞源神经营养因子cDNA全序列的克隆及其生物学功能的研究

为了研究ＧＤＮＦ在神经系统中的生物学功能,通过ＲＴ－ＰＣＲ方法从大鼠睾丸总ＲＮＡ中扩增出ＧＤＮＦｃＤＮＡ全序列,序列分析表明与ＧｅｎＢａｎｋ中的顺序完全相同．将ＧＤＮＦｃＤＮＡ以非融合方式连接在真核表达载体ｐＥＧＦＰ－ＮⅠ的绿色荧光蛋白的上游,在ＣＭＶ启动子控制下表达．通过绿色荧光蛋白报告基因的表达表明ＧＤＮＦｃＤＮＡ能在真核细胞ＨｅＬａ中很好表达．采用裸ＤＮＡ转染方法研究ＧＤＮＦ对损伤的坐骨神经的修复作用,在雏鸡出生后３ｈ切断其右侧坐骨神经,将ｐＥＧＦＰ－ＧＤＮＦ与Ｌｉｐｏｆｅｃｔｉｎ的混合物注射到坐骨神经切断位点附近肌肉内,５ｄ后追补一次．２０ｄ后进行实验检测,观察到ＧＤＮＦｃＤＮＡ的转染阻止了切断神经侧的腰脊髓内［Ｌ４－Ｌ６］运动神经元的大量死亡,并显著促进了切断坐骨神经的再生．     

Peptide inhibition of cytokine-stimulated aromatase activity in breast tissue fibroblasts

The cytokine interleukin-6 (IL-6) and its soluble receptor (IL-6sR) can act synergistically to stimulate aromatase activity in cultured stromal fibroblasts derived from breast tissues. In this study, a 16 amino acid peptide, AROHIB, has been used in an attempt to block the ability of IL-6 plus IL-6sR to stimulate aromatase activity in stromal fibroblasts. Pre-incubation of cells with AROHIB for a 3-h period before the addition of IL-6 and IL-6sR resulted in a marked (67%) reduction in the ability of these factors to stimulate aromatase activity. AROHIB was found to be rapidly degraded when exposed to MCF-7 breast cancer cells or fibroblasts. Analysis by FAB-MS was used to identify the site of peptide cleavage. Subsequently, a series of 10 amino acid peptides, DP1-DP4, were designed, synthesised and tested for their ability to resist proteolytic degradation and to inhibit IL-6 plus IL-6sR-stimulated aromatase activity. Peptide DP2, a modified version of the active fragment of AROHIB, had N-acetyl and C-amino terminal protection and an internal D-amino acid (instead of L form) at the site of proteolytic cleavage. Using cells cultured in the presence of 2% stripped foetal calf serum, peptide DP2 resulted in a 74% reduction in cytokine-stimulated aromatase activity. Under serum-free conditions, peptides DP1-DP3 showed modest inhibitory properties. Results from this study suggest that it may be possible to develop small peptides to inhibit cytokine-stimulated aromatase activity in a tissue-specific manner.     

Cytokine interactions in mesenchymal stem cells from cord blood

We used cytokine protein array to analyze the expression of cytokines from human cord blood-derived mesenchymal stem cells (CB-MSCs). Several cytokines, interleukins (IL), and growth factors, including ENA-78, GM-CSF, GRO, IL-1β, IL-6, IL-8, MCP-1, OSM, VEGF, FGF-4, FGF-7, FGF-9, GCP-2, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IP-10, LIF, MIF, MIP-3α, osteoprotegerin, PARC, PIGF, TGF-β2, TGF-β3, TIMP-1, as well as TIMP-2, were secreted by CB-MSCs, while IL-4, IL-5, IL-7, IL-13, TGF-β1, TNF-α, and TNF-β were not expressed under normal growth conditions. IL-6, IL-8, TIMP-1, and TIMP-2 were the most abundant interleukins expressed by CB-MSCs. A set of growth factors were selected to evaluate their stimulatory effects on the IL6 secretion for CB-MSCs. IL-1β was the most important factor inducing CB-MSC to secret IL-6. The mechanism by which IL-1β promoted IL-6 expression in CB-MSCs was studied. By using various inhibitors of signal transduction, we found that activation of p38 mitogen-activated protein kinases (MAPK) and MAPK kinase (MEK) is essential in the IL-1β stimulated signaling cascade which leads to the increase in IL-6 synthesis. Additionally, continuous supplement of IL-1β in the CB-MSCs culture will facilitate adipogenic maturation of CB-MSCs as evidenced by the presence of oil drops in the CB-MSCs and secretion of leptin, a molecule marker of adipocytes. These results strongly suggest that cytokine induction and signal transduction are important for the differentiation of CB-MSCs.     

体外培养过程中许旺细胞S100表达规律研究

目的：目前研究发现,周围神经中许旺细胞的标志物有很多,S100是其中之一。S100在体内表达的变化规律已有比较深入的研究,但是其在体外培养的许旺细胞的表达规律尚不清楚。因此,本课题研究小鼠许旺细胞在体外培养过程中S100蛋白的表达变化规律。方法：取新生（出生5-7 d）C57BL/6小鼠的坐骨神经,酶消化分离获取细胞后,培养纯化扩增3次。用S100免疫荧光法及RT-PCR技术研究许旺细胞在体外培养过程中S100的表达规律。结果：从坐骨神经消化所得到的许旺细胞,早期并不都表达S100,阳性率约为43.48%,随着培养时间延长（培养8天）,所有许旺细胞均表达S100,能够达到阳性率95.66%。结论：体外培养的许旺细胞,其标志物S100阳性率表达随培养时间延长而增加。并且我们发现,S100并不能作为一个可靠的标志物来单独应用鉴定体外培养早期的许旺细胞。