【作者】 张月杰；

【导师】 吉爱国；

【作者基本信息】 山东大学 ， 微生物与生化药学， 2010， 博士

【摘要】 刺参(Stichopus japonicus)，归属于无脊椎动物棘皮动物门(Echinodermata)海参纲(Holothurioider)刺参科(Stichopodidae)。自古以来,海参不仅被冠为海八珍之首,而且历来被认为是一种药食两用的滋补品。刺参多糖类物质具有广泛的生物学活性,诸如抗凝血、增强免疫力、抗肿瘤、抗病毒、调节脂类代谢以及组织发生等。近年来,硫酸化多糖对神经干/前体细胞(NSPCs)发育的影响成为神经生物学及糖生物学研究的新热点。硫酸化多糖作为中枢神经系统(CNS)细胞外基质的重要成分,其在神经细胞增殖、分化和迁移等过程中发挥极其重要的调节作用。然而,单纯的硫酸化多糖,特别是海洋生物来源的酸性硫酸化多糖对NSPCs发育的研究较少。我们从鲜刺参体壁中分离纯化得到单一组分的岩藻多糖(HS),对其理化性质和结构进行分析；研究HS对NSPCs存活、增殖、聚集和凋亡的影响,结果发现HS明显地促进神经球的形成；并进一步探讨其促进NSPCs形成神经球的原因以及可能的作用机制。期望为深入认识硫酸化多糖在CNS中的生物学功能提供新的线索,同时为NSPCs移植用于神经系统退行性疾病及CNS损伤的临床治疗提供一些新的思路。本论文的研究内容和结果主要有以下几个方面。1刺参岩藻多糖的提取、纯化以及活性多糖组分的筛选首先采用胃蛋白酶/胰蛋白酶双酶解,60%乙醇沉淀获得刺参粗多糖。经大孔吸附树脂柱脱色后,采用DEAE-Sepharose柱进行第一次分离纯化。用2M氯化钠溶液进行线性洗脱,紫外210nnm、280nm结合苯酚硫酸法监测流出的各组分,得到A、B、C、D四个糖组分,然后将有活性的组分(组分D)用Superdex 200柱进行二次分离纯化。用0.15mol/L氯化钠溶液洗脱,紫外210nm和280nm同时监测,收集各组分,再利用Sephadex G-25柱脱盐,最后冷冻干燥得到活性组分HS。粗多糖(CHS)得率为0.23%,精制多糖(FHS)得率为0.053%。2刺参岩藻多糖(HS)理化性质和结构的分析凝胶过滤法和紫外扫描初步确定HS为均一物质,分子量为4.23×105Da。HS为白色絮状物,无色、无味,易吸湿；不含蛋白质和核酸。HS中岩藻糖含量为38.12%,糖醛酸含量为16.52%,硫酸基含量为32.64%。单糖组成分析结果表明,HS含有岩藻糖,且相对含量较高；微量的半乳糖,岩藻糖与半乳糖的摩尔比为14.29。红外光谱(IR)显示HS具有硫酸化多糖的特征吸收峰,即1250.96cm-1的S=O的伸缩振动峰和850.78 cm-1的C-O-S的伸缩振动峰,且提示HS由p-D-吡喃糖组成。1HNMR及13C NMR波谱数据进一步提示HS中糖残基可能为β-构型。3刺参岩藻多糖(HS)对体外培养NSPCs的增殖作用首先,建立NSPCs悬浮培养体系。采用酶解与机械吹打相结合的方法从孕14天大鼠的大脑皮质分离NSPCs,以无血清悬浮培养方式体外培养NSPCs并形成神经球,确定适合的种板密度(2-3×105cells/mL)、培养时间(72-96 h)以及HS的剂量范围；采用免疫细胞化学方法对其干细胞特性和多向分化潜能进行鉴定。然后,通过MTT法、BrdU掺入法以及神经球形成实验测定HS对NSPCs的增殖作用。结果表明HS能够以剂量依赖性的方式增加NSPCs的活力；在与生长因子同时作用时,HS可以增加FGF-2对细胞增殖的促进作用,而对于EGF的增殖作用没有影响。BrdU掺入法证实了HS能够促进NSPCs的增殖,并且与FGF-2具有协同性。在较高浓度范围(1-8μg/mL)内,单独的HS能够以剂量依赖性的方式促进NSPCs形成神经球。同样,HS可以增加FGF-2对于神经球形成的促进作用。HS与FGF-2协同促进神经球形成的有效剂量在4-8μg/mL之间。有血清培养条件下,HS同样可以促进神经球的形成,但神经元突起数量减少,神经球之间联系更加直接。最后,采用Hoechst33342/PI双染法检测HS对NSPCs凋亡的作用。结果显示,在HS和/或FGF-2各个处理组中,没有凋亡的细胞出现。4刺参岩藻多糖(HS)促进神经球形成的内在机制实验中我们观察到HS处理组细胞形成神经球的时间要早于对照组；同时,在有血清培养条件下,HS也可以促进已经分化的细胞聚集形成细胞团。从以上两方面综合分析,我们认为HS促进NSPCs形成神经球并不仅仅是由增殖作用引起的。因此,进一步从细胞聚集方面分析HS促进神经球快速形成的原因。结果表明,在NSPCs培养初期,HS能够促进单个细胞聚集,促使3-5个细胞的细胞团生成,这种微环境的形成有利于NSPCs进一步的增殖,从而加速了神经球的形成。但HS诱导的NSPCs聚集及运动性的影响不能用趋化性迁移来解释。进一步的细胞周期实验结果显示HS能够促使更多的细胞进入S期,HS处理组的S-期细胞数是对照组的2.8倍,加速细胞增殖。也就是说,HS的促增殖以及促聚集作用共同促进了神经球的快速生成,因此,HS有可能成为促进NSPCs增殖以及神经球形成的良好辅助分子。5刺参岩藻多糖(HS)对NSPCs的作用与NF-κB转录因子的激活有关NF-κB广泛地存在于神经系统,在神经发生、神经保护以及突触可塑性等方面具有重要作用。文献报道NF-κB信号途径可以调节细胞对于有丝分裂原的反应,而且TNF-α诱导的NSPCs聚集及增殖是通过NF-κB信号途径的激活实现的。因此,我们假定HS对于NSPCs增殖和聚集的作用是通过NF-κB的激活来实现的。采用NF-κBP65ELISA检测细胞核中P65蛋白的含量,以此表示转录因子的激活程度。结果显示,在5-50μg/mL剂量范围内,细胞核内的P65蛋白含量以剂量依赖性方式增加；在50μg/mL浓度时,细胞核内P65蛋白的含量比对照组增加了近50%,初步结果表明HS对NSPCs的作用与NF-κB转录因子的激活有关。6刺参岩藻多糖(HS)对NSPCs的作用并不影响神经球的干细胞特性以及多向分化潜能HS来源于海洋无脊椎动物,没有潜在的病毒感染；更为重要的是HS抗凝活性较低,不易引起内出血等不良反应,有可能成为促进NSPCs增殖以及神经球形成的良好辅助分子。因此,本文在研究发现HS促进NSPCs增殖和神经球形成的基础上,对其诱导生成的神经球干细胞特性及其多向分化潜能进行鉴定。结果显示,HS并不改变神经球的特征性蛋白Nestin的表达；经HS(4μg/mL)诱导形成的神经球仍具有多向分化潜能,可以分化成04+的少突胶质细胞、GFAP+的星型胶质细胞以及MAP2+的神经元。这些结果表明刺参岩藻多糖(HS)并不影响神经球的干细胞特性及其多向分化潜能。本研究取得的成果和结论主要有：(1)从刺参体壁中分离得到均一的硫酸化多糖组分HS,分子量为4.23×105Da,岩藻糖含量为38.12%,糖醛酸含量为16.52%,硫酸基含量为32.64%。(2)首次确定HS能够促进NSPCs增殖,并且与成纤维细胞生长因子(FGF-2)有协同作用。HS不会引起细胞凋亡。(3)首次确定HS能够促进NSPCs的聚集,在细胞培养初期能够促进神经球的快速形成；同时促进NSPCs分裂,使更多的细胞进入S期,这两种作用共同促进神经球的形成。(4)初步确定HS对于NSPCs的促增殖和促聚集作用与NF-κB信号途径的激活有关。更多还原

【Abstract】 Sea cucumber, a kind of marine invertebrate, belongs to Holothurioide genus, Stichopodidae family. As a precious food supplement, it has been used as Chinese folk medicine from time immemorial. The sulfated polysaccharides isolated from sea cucumber displayed various biological activities including anticoagulative effects, inhibition of osteoclastogenesis, modulation of angiogenesis, inhibition of tumor metastasis, inflammatory reactions and so on. In recent years, sulfated polysaccharides (SPS) have obtained much attention in nervous system development and in area of glycobiology. Proteoglycans consisting of SPS are prominent components of the ECM in the CNS and are assumed to play an important role in controlling proliferation, differentiation and migration of NSPCs. However, the study of SPS, especially SPS isolated from marine mollusca, effect on neural stem/progenitor cells (NSPCs) is rather rare.We isolated the polysaccharide from body wall of the fresh sea cucumber Stichopus japonicus by enzymolysis extraction, anion-exchange and gel-permeation chromatography. The homogeneous fraction, a kind of fucoidan named as HS, was obtained and a series of physicochemical properties of HS and its structure were analyzed. The effects on survival, proliferation, aggregation and apoptosis of NSPCs were studied for the first time, indicating HS promotes the proliferation of NSPCs and neurosphere formation, acting synergistically with FGF-2 but not EGF. Moreover, we investigated the possible mechanism leading to neurosphere formation and the related signaling pathway. All the results of HS were summarized as follows.1 Extraction and purification of Stichopus japonicus polysaccharide (HS)Hydrolyzed by double-enzyme, the crude extract (CHS) was precipitated by ethanol. After decolored by macroporous adsorptive resins, the polysaccharide (FHS) was first separated by DEAE-Sepharose column with monitoring of 210nm,280nm ultraviolet light assisted phenol-sulphuric acid colorimetric method. The results have shown that four main fractions named A, B, C and D were obtained, but only D fraction has proliferative effects on NSPCs based on the NSPC proliferative assay. Therefore, the D fraction eluted from DEAE column was further fractionated on a SuperdexTM 200 column which was eluted with 0.15 M sodium chloride at a flow rate of 30 mL/h. The main fraction (D1) was collected, desalted by Sephadex G25 gel column and lyophilized to obtain a purified polysaccharide (D1 fraction, named as HS). The rate of CHS and FHS was 0.23% and 0.053% respectively.2 Analysis of physicochemical properties of HS and its structureThe homogeneity of HS was verified initially by UV spectrum scan and Superdex 200 column chromatography. HS had a weight-average molecular weight of about 4.23×105 Da in reference to standard T-series Dextran. HS appeared as a white powder. It produced a negative response in the Bradford test and no absorption was found at 280 or 260 nm in the ultraviolet spectrum, indicating the absence of proteins and nucleic acid in the fraction. The total sugar content of HS was determined to be 38.12% and uronic acid content was 16.52%. It is noteworthy that the sulfate content of HS was as high as 32.64%, much higher than that of heparin. The monosaccharide composition analysis from TLC showed that HS was mainly composed of fucose and a trace amount of galactose. These results were further substantiated by HPLC. The molar ratio of fucose to galactose in HS was equivalent to 14.29:1 based on the conversion of the peak area of the two monosaccharides. The FT-IR spectrum showed that HS had the characteristic absorption peak of SPS, and that the pyranose residues of HS were linked inβ-configuration. The 1H and 13C NMR chemical shifts of HS were confirmed that HS were linked inβ-configuration.3 Proliferative effects on NSPCs of HS in vitroFirstly, the culture systems for the in vitro expansion of NSPCs using neurosphere in suspension had been established. NSPCs were obtained from the cerebral cortex of 14-day-embryonic Wistar rats and incubated in the free-serum growth medium containing DMEM/F-12 nutrient and B27 supplement. The sternness and the multi-lineage potential of neurospheres formed were identified by immunochemistry assay. Secondly, the effects of HS on the viability and proliferation of NSPCs in vitro were examined by MTT assay, BrdU labelling and neurosphere formation assay respectively. Our results showed that HS alone increased NSPC viability in a dose-dependent manner. Moreover, HS acted synergistically with basic fibroblast growth factor (FGF-2) but not epidermal growth factor (EGF) to enhance the proliferation of NSPCs. At a higher concentration ranging from 2～8μg/mL, HS significantly promoted neurosphere formation in a dose-dependent manner,also acted synergistically with FGF-2. The most effective dose of HS to promote the formation of neurospheres was between 4 and 8μg/mL. Besides, HS significantly promoted neurosphere formation even though incubating in the the medium containing 1% fetal bovine serum, but neurite branch was less than the control, and showed more elaborate networks of neurites. Finally, HS did not induce apoptosis of NSPCs among the treatment of HS and/or FGF-2.4 Possible mechanism leading to neurosphere formation and the related signaling pathwayIn the present study, we observed a significantly faster formation of neurosphere units on the same day of plating. And a larger free space between cells and/or neurosphere units appeared in HS cultures, as compared to the control group. Meanwhile, HS significantly promoted neurosphere formation even though incubating in the the medium containing 1% fetal bovine serum. Therefore, we thought that proliferation of NSPCs induced by HS was not the only reasons for the faster formation of neurosphere units. Thus we attempted to explore the mechanisms of the faster formation of neurosphere units from these two aspects:aggregation and proliferation of NSPCs. The results showed that several NSPCs dispersed on the plate begin to aggregate induced by HS at the early culture stage, and then 3-5 cells will aggregate to form neurosphere units. The cell aggregates formed might provide a favorable environment for the proliferation of NSPCs. However, the aggregation was not caused by chembtactic migration of NSPCs, as evidenced by the transwell chamber assay. On the other hand, cell cycle analysis showed that HS increased the percentage of cells in S phase by 2.8-fold, as compared with the control. Thus, we demonstrated that HS was able to promote cell proliferation and aggregation of NSPCs which could lead to the formation of neurospheres, and suggested that HS can serve as an adjuvant for promoting proliferation of NSPCs and formation of neurospheres. 5 NF-κB activation and the effects of HS on NSPCsNF-κB is ubiquitously expressed throughout the nervous system, and the pathway is activated by cell surface receptors that signal to degrade its inhibitor IκB, leading to NF-κB nuclear translocation. The NF-κB signaling pathway plays a central role in neuronal integrity, synaptic plasticity, neuroprotection and neurogenesis. Since NF-κB is known to control the proliferation and aggregation of NSPCs stimulated by TNF-α, we hypothesized that there was a relationship between the effects of HS on NSPCs and the activation of nuclear factor NF-κB. We used an ELISA kit to measure the amount of p65 in the nucleus of NSPCs that indicated the extent of activation of NF-κB. The results showed that HS significantly activated the translocation of NF-κB and the effects were dose-dependent. HS (50μg/mL) increased NF-κB nuclear translocation nearly 1.5-fold greater than the controls. These findings suggested that HS stimulation was related to the activation of NF-κB signaling pathway.6 Stemness and multi-lineage potential of NSPCsHS originated from marine invertebrate which is less likely to contain infectious agents, such as viruses or prions. More importantly, the anticoagulant activity of fucoidan from sea cucumber is lower than heparin, so that the hemorrhage risk is lowered for clinical use of transplanting NSPCs into the CNS if contaminated with fucoidan.Due to its safety, we suggested that HS can serve as an adjuvant for promoting the proliferation of NSPCs. Therefore, we examined the sternness and multi-lineage potential of neurospheres formed by HS. The results showed that HS-stimulated formation of neurospheres did not alter the lineage of after differentiation. Neurospheres were remained positive for intermediate filament protein (Nestin), a NSC marker, and the neurosphere formed with HS had the ability of multi-lineage potential, the markers of three neural lineages, such as oligodendrocytes marker O4, astrocytes marker GFAP and neuronal marker MAP2 did not change after HS treatment. These findings suggest that HS stimulation might not influence sternness of neurospheres and their multi-lineage potential. Originality of the article are the following:(1) A homogeneous sulfated polysaccharide named as HS was obtained from Stichopus japonicus. HS had a weight-average molecular weight of about 4.23×105 Da. The total sugar content and uronic acid content of HS was 38.12% and 16.52% respectively. The sulfate content of HS was as high as 32.64%.(2) The effects on survival, proliferation, aggregation and apoptosis of NSPCs were studied for the first time. The results indicated HS promotes the proliferation of NSPCs and neurosphere formation, acting synergistically with FGF-2. HS did not induce apoptosis of NSPCs.(3) The aggregation effects on NSPCs of HS were studied for the first time. At the early culture stage, HS induced a significantly rapid aggregation of NSPCs, resulting in formation of 3-5 cells aggregates. Meanwhile, HS increased the percentage of cells in S phase and promoted proliferation of NSPCs. These two roles of HS leaded to a rapid formation of neurospheres.(4) The effects on NSPCs of HS were related to the activation of NF-κB signaling pathway.更多还原