脊髓损伤后神经修复过程中的细胞微环境北大核心

背景:以往的研究显示单一改变脊髓损伤区域某一基因表达或者某一细胞的状态,对脊髓损伤后功能恢复无显著影响,而大量证据表明调控脊髓损伤后紊乱的细胞微环境是神经功能恢复的关键因素。目的:对脊髓损伤前后细胞微环境的生物学特性,包括多种细胞之间的相互调控以及细胞外组分对损伤神经修复的作用和机制进行综述。方法:由第一作者检索PubMed及Web of Science数据库,英文检索词为“spinal cord injury,glial cell,neuron,immune cell,neural stem cell,extracellular matrix,cytokine,extracellular vesicle,regeneration”。文献检索的时间范围为2000年1月至2021年12月,最终筛选出64篇文献进行分析。结果与结论:①脊髓损伤后,在细胞微环境的细胞组分中,占比最高的胶质细胞间的相互作用,以及与神经元的相互调控作用最为关键。②在脊髓损伤后的细胞外组分中,利用生物相容性良好的水凝胶模仿天然细胞外基质,可有效模拟和重建损伤区域内的细胞微环境,促进轴突伸长。③在脊髓损伤后的细胞外调节因子中,促炎因子如肿瘤坏死因子α和白细胞介素1β等加剧了细胞微环境的炎症反应,应用受体抑制剂或阻断相关通路抑制上述促炎因子的表达是一种有效的治疗方法,同时在脊髓微环境中增加白细胞介素10等抗炎因子的表达,抑制损伤区域炎症发展的研究也陆续出现。④最近被重视起来的细胞外囊泡作为传递信息的载体在细胞微环境中也发挥了重要作用。⑤文章揭示了脊髓损伤后细胞微环境中的包括细胞组分和细胞外组分之间的多组相互调控关系,证实了细胞微环境中各组分之间所发挥的神经修复作用并不是孤立的。     

Evaluation and application of denaturing HPLC for mutation detection in Marfan syndrome: Identification of 20 novel mutations and two novel polymorphisms in the FBN1 gene

Mutations in the human fibrillin 1 gene (FBN1) cause the Marfan syndrome (MFS), an autosomal dominant connective tissue disorder. Knowledge about FBN1 mutations is important for early diagnosis, management, and genetic counseling. However, mutation detection in FBN1 is a challenge because the gene is very large in size ( approximately 200 kb) and the approximately 350 mutations detected so far are scattered over 65 exons. Conventional methods for large-scale detection of mutations are expensive, technically demanding, or time consuming. Recently, a high-capacity low-cost mutation detection method was introduced based on denaturing high-performance liquid chromatography (DHPLC). To assess the sensitivity and specificity of this method, we blindly screened 64 DNA samples of known FBN1 genotype exon-by-exon using exon-specific DHPLC conditions. Analysis of 682 PCR amplicons correctly identified 62 out of 64 known sequence variants. In three MFS patients of unknown FBN1 genotype, we detected two mutations and eight polymorphisms. Overall, 20 mutations and two polymorphisms are described here for the first time. Our results demonstrate 1) that DHPLC is a highly sensitive (89-99%, P = 0.05) method for FBN1 mutation detection; but 2) that chromatograms with moderate and weak pattern abnormalities also show false positive signals (in all 45-59%, P = 0.05); 3) that the difference in the chromatograms of heterozygous and homozygous amplicons is mostly independent of the type of sequence change; and 4) that DHPLC column conditions, additional base changes, and the amounts of injected PCR products influence significantly the shape of chromatograms. A strategy for FBN1 mutation screening is discussed.     

Ⅰ型胶原膜、胞外基质提取物膜体外构建人工真皮能力比较

比较Ⅰ型胶原与胞外基质(extracellularmatrix,EM)提取物冻干后形成的膜(EM膜)在体外构建人工真皮的能力,以选择一种更有利于人工真皮形成的支架材料。方法分别使用小牛真皮部分提取的Ⅰ型胶原蛋白和胞外基质提取物冻干后制备基质网架,植入皮肤成纤维细胞,形成人工真皮。用扫描电镜观察I型胶原膜和EM膜的表面形态结构;选取不同的时间点,对种植于两种支架材料上的细胞进行计数,并利用间接ELISA方法检测人工真皮复合物中的细胞Ⅰ型胶原分泌情况,通过统计学的分析手段,对细胞的生长情况做出判断;用组织学方法观察人工真皮的形成情况。结果扫描电镜观察结果,两种膜在外观形态上无明显差异;细胞在EM膜上的增殖速度较Ⅰ型胶原膜快;ELISA分析显示,EM-细胞复合物中的成纤维细胞能够分泌更多的Ⅰ型胶原;与Ⅰ型胶原膜相比,EM在培养液中的降解更为缓慢,种植后的成纤维细胞在EM膜上生长旺盛,细胞层次明显多于前者,形成了较为明显的真皮样组织。结论EM膜适于成纤维细胞的生长,体外降解速率慢,是一种较Ⅰ型胶原膜更为理想的真皮支架材料。     

Effect of NO-generating compound NaNO<Subscript>2</Subscript> on ultrastructure of synaptic vesicles of glutamatergic synapses

Ultrastructure of synaptic vesicles in axon terminals of granule cells from isolated cerebellum of Rana temporaria frogs under the influence of NO-generating compound NaNO₂ in various concentrations and electrical stimulation was evaluated by the method of electron microscopy. NO-generating compound in low concentration induced translocation of synaptic vesicles and formation of small clusters. The size and structure of synaptic vesicles remained unchanged under these conditions. Increasing the concentration of NaNO₂ led to swelling of synaptic vesicles, formation of arranged heaps from individual vesicles or fusion of their content. Electrical stimulation of the cerebellum in the presence of NaNO₂ increased damage to synaptic vesicles. These experimental data model some stages observed in stroke. The formation of clusters from synaptic vesicles is a compensatory and adaptive response maintaining the structure of synaptic vesicles and protecting neurons from high concentrations of glutamate. Glutamate produces a toxic effect on nerve cells and glial cells of the cerebellum under pathological conditions, which is accompanied by impairment of signal transduction from presynaptic to postsynaptic neurons. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 146, No. 7, pp. 13–17, July, 2008     

嗅鞘细胞/细胞外基质夹层支架的制备和形态学观察

目的研究嗅粘膜嗅鞘细胞在细胞外基质夹层支架内的生长特性,为治疗神经系统损伤寻找新的移植供体。方法嗅鞘细胞/细胞外基质夹层支架由四层毯状细胞外基质支架和三层嗅粘膜嗅鞘细胞相间叠加构成。纤维蛋白原、层粘连蛋白和纤粘连蛋白按一定比例混合后,在新鲜大鼠血浆促凝作用下,构建单层毯状细胞外基质支架,在支架表面种植嗅粘膜来源的嗅鞘细胞。于倒置显微镜下观察嗅鞘细胞在支架上/内的生长过程,培养3d后,在细胞表面再次滴加上述细胞外基质混合胶以构建第二层毯状支架,依次重复两次。夹层支架制成组织切片和超薄切片后,用光镜和电镜观察和分析夹层支架的内部结构以及嗅鞘细胞在支架内的生长情况。结果由顶层至底层,支架内的嗅鞘细胞数量逐渐增多,细胞突起逐渐延长,细胞内分泌颗粒逐渐增多及其电子密度逐渐增高;细胞可在支架之间迁移,其水平排列方向具有一致性;嗅鞘细胞的细胞膜可与支架紧密粘附,支架内局部区域出现组织间隙。结论嗅粘膜嗅鞘细胞可在细胞外基质夹层支架内正常增殖和分化,细胞与支架具有良好的组织相容性。该夹层支架可作为嗅鞘细胞三维生长的载体,用于移植治疗神经损伤。     

Inhibition of tumor invasion and extracellular matrix degradation by ubenimex (bestatin)

We have investigated the effect of the immunomodulator ubenimex (hereafter referred to as bestatin) on the enzymatic degradation of the extracellular matrix by human renal cell carcinoma SN12M cells during the invasive process. The invasion of SN12M cells into reconstituted basement membrane (Matrigel) was inhibited by the presence of bestatin in a concentration-dependent manner. However, bestatin did not have any effect on tumor cell adhesion and migration to the extracellular matrices which may be involved in tumor cell invasion. Bestatin inhibited the degradation of type IV collagen by tumor cells, but not by tumor-conditioned medium (TCM), in a concentration-dependent manner. We also found that bestatin inhibited hydrolysing activities towards substrates of aminopeptidases in SN12M cells. Since bestatin was found to inhibit aminopeptidase activity, the inhibition of tumor invasion by bestatin is likely to be associated with its action as an enzyme inhibitor. Bestatin only slightly inhibited tumor cell plasmin activity, which can lead to the conversion of the latent collagenase to the active form, but this slight effect was not significant. The zymography of TCM from SN12M cells showed that the treatment of tumor cells with bestatin resulted in the disappearance of the 68 kDa type IV collagenase-enzyme level (active form) and slight reduction of the 72 kDa type IV collagenase-enzyme level (latent form). These results indicated that bestatin may inhibit tumor cell invasion through a mechanism involving its inhibitory action on aminopeptidases in tumor cells, suggesting that the aminopeptidase may partly be associated with the conversion of a latent form of type IV procollagenase to an active form or the secretion of the collagenases from tumor cells.     

Excitatory amino acids and potassium release in the frog spinal cord

Synaptic release of excitatory amino acids such as L-glutamate and/or L-aspartate and subsequent activation of specific receptors by these putative transmitters appears necessary for the release of K+ by afferent stimulation in the isolated frog spinal cord. This conclusion is based on the findings that (-)baclofen, which is thought to reduce the presynaptic release of putative excitatory amino acid transmitters, and some amino dicarboxylic amino acids (D, L-alpha-aminoadipic acid, 2-amino-4-phosphonobutyric acid, and D, L-alpha, epsilon-diaminopimelic acid), which are believed to interfere with the activation of receptors by these same excitatory amino acids, significantly attenuate the increment in extracellular K+ evoked by tetanic dorsal root stimulation.     

In vitro biosynthesis and core glycosylation of the histidine-rich protein of Plasmodium lophurae

We have characterized the early biosynthetic forms of the histidine-rich protein (HisRP), a major, granule-bound protein (Mr 58 000) of the avian malarial parasite Plasmodium lophurae. We have translated poly(A)-containing, size-selected parasite mRNA in the wheat germ cell-free system in the presence of [3H]histidine. HisRP was synthesized as a larger precursor (Mr 63 000). When dog pancreas microsomal membranes were present in the cell-free system during translation, a still larger form of HisRP (Mr 66 000) was detected. This larger form was segregated into the dog pancreas microsomal vesicles and was core glycosylated. Presumably, it corresponds to an intermediate form located in the parasite rough endoplasmic reticulum (RER). The difference in the Mr of approx. 8 000 between this RER associated 'pro' form and the granule-bound, mature form of HisRP suggests that proteolytic processing occurs upon transport from the RER to the granule. Segregation and core glycosylation were strictly coupled to translation and were not observed upon posttranslational addition of microsomal membranes. Thus, the early events in the biosynthesis of HisRP are similar to those established for secretory and lysosomal proteins.     

Ultrastructural Features of Neuroendocrine Differentiated Carcinomas of the Breast

The ultrastructural patterns of neuroendocrine (NE) differentiated breast carcinomas are analyzed and discussed. Reports in the literature describe wide variations in the size of observed dense-core membrane-bound granules and discrepancies in their interpretation. In the present study 24 cases of breast carcinoma with recognized morphologic, histochemical, and immunocytochemical features of NE tumors were investigated. Five different types of dense-core granules of neurosecretory (NS) type (confirmed by the ultrastructural localization of chromogranin A) and five different cell types were recognized. Some amphicrine cells were found to contain both mucin and NS granules. Another notable ultrastructural feature of breast NE carcinomas was the presence of clear vesicles of presynaptic type, which correlated with expression of synaptophysin.     

Over-expression of tenascin-C in malignant pleural mesothelioma

Aims:  Tenascin-C is an extracellular matrix glycoprotein known to have anti-adhesive characteristics and to be expressed in various human malignant neoplasms. We hypothesized that the expression of tenascin-C would be increased in human malignant pleural mesothelioma, and its accumulation associated with the prognosis of the patients with this disease.
Methods and results:  Thirty-seven cases of mesothelioma were studied by immunohistochemically using a monoclonal antibody against tenascin-C, and with a semiquantitative scoring system for tenascin-C in different areas of the tumours. In 10 selected cases tenascin-C mRNA in-situ hybridization was also analysed. Since transforming growth factor-beta (TGF-β) is known to induce both the synthesis of tenascin-C and the growth of mesotheliomas, an immunohistochemical analysis of TGF-β1, -β2 and -β3 was also performed. Normal pleura ( n  = 7) and metastatic pleural adenocarcinomas ( n  = 7) were used as controls. Tenascin-C protein was expressed in every histological subtype of malignant mesothelioma, being most prominent in the fibrotic stroma of a tumour, around tumour cells and at the invasive border, whereas tenascin-C mRNA was scarce in tumour cells. The patients with less immunohistochemical expression for tenascin-C tended to live longer ( P  = 0.028 by Fishers' exact probability test). All mesotheliomas showed positivity for at least one isoform of TGF-β.
Conclusions:  In conclusion, high expression of tenascin-C protein in malignant pleural mesotheliomas may play a role in its invasive growth, and might serve as a prognostic marker of the disease.