刺参岩藻多糖对神经干/前体细胞的增殖作用及其机制研究
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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。
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 structure
The 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 vitro
Firstly, 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 pathway
In 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 NSPCs
NF-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 NSPCs
HS 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.
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。
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 structure
The 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 vitro
Firstly, 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 pathway
In 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 NSPCs
NF-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 NSPCs
HS 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.
引文
[1]周世文,徐传福.多糖的免疫药理作用.中国生化药物杂志,1994,15(2)：143-147.
[2]黄芳,蒙义文.活性多糖的研究进展.天然产物开发与研究1996,11(5)：90-97.
[3]魏经建,邵树军,王天元.硫酸酯化多糖化学及临床应用研究进展.中国生化药物杂志1999,20(5)：260-262.
[4]郭振环,胡元亮,马霞,赵晓娜,王德云,赵炳凯,郭利伟,刘萍,郝利华.硫酸化香菇多糖对新城疫疫苗免疫效果的影响.南京农业大学学报,2010,33(1)：76-80.
[5]高贵珍,陈群,李绪亮.硫酸化茯苓多糖药理性质研究.安徽大学学报(自然科学版),2009,33(6)：72-77.
[6]杨铁虹,贾敏,姚秀娟,孟嘉,孟静茹,梅其炳.当归多糖硫酸酯的合成及其抗小鼠L6565逆转录病毒的作用.中国新药杂志2006,15(10)：783-786.
[7]孙汉文,许城燕,赵燕燕,梁淑轩.枸杞多糖硫酸酯化修饰及其对Hela细胞的体外抑制作用.河北大学学报(自然科学版),2009,29(6)：591-595.
[8]史宝军,聂小华,许泓渝,陶文沂.灰树花多糖硫酸酯的制备及其抗肿瘤活性.中国医药工业杂志,2003,34(8)：383-385.
[9]Kenned YJF, White CA. Bioactive carbohydrates M. Chichester:Ellis Horwood, Ltd,1993.
[10]吴东儒.糖类的生物化学[M]北京：高等教育出版社,1987
[11]张惟杰.糖复合物生化研究技术[M].杭州：浙江大学出版社,1999
[12]来鲁华,杨显婷.寡糖的构象分析.生物化学与生物物理进展,1995,22(4)：290-294.
[13]Peters T, Meyer B, Stuike-Prill R, Somorjai R, Brisson JR. A Monte Carlo method for conformational analysis of saccharides.Carbohydr Res.1993,238:49-73.
[14]Imberty A, Perez S, Hricovini M, Shah RN, Carver JP. Flexibility in a tetrasaccharide fragment from the high mannose type of N-linked oligosaccharides. Int J Biol Macromol 1993,15:17-23.
[15]Silvestri LJ, Hurst RE, Simpson L,Settine JM. Analysis of sulfate in complex carbohydrates. Anal Biochea.1982,123:303-309.
[16]Dodgson KS, Price RG. A note oil the determination of ester sulfate content of sulfated polysaccharides. J Biochem,1962,84:106-110.
[17]张惠芬,李宝才,范家恒.盐酸水解一硫酸钡重量法测定硫酸酯化多糖硫酸基含量方法考察.食品科学,2002,23(5)：107-111.
[18]张丽萍,汉丽萍,王月秋等.硫酸化高山红景天多糖的制备及鉴定.分子科学学报,1999,15(4)：205-209.
[19]黄小燕,孔祥峰,王德云,胡元亮.多糖硫酸化修饰和多糖硫酸酯的研究.天然产物研究与开发,2007,19：328-332,355
[20]Ghosh P, Adhikari U, Prodyot KG. In vitro anti-herpetic activity of sulfated polysaccharide fraction from caulerpa racemosa. Phytochemistry,2004,65: 3151-3157.
[21]刘玉红,茶藨子木层孔菌多糖及其硫酸化衍生物的制备、结构分析与生物活性研究[D].山东大学,2008.
[22]Katsyara K, Nakashima H, Yamamoto N, Uryu T. Synthesis of sulfated oligosaccharide glycosides having high anti-HIV activity and the relationship between activity and chemical structure.Carbohydr.Res,1999,315:234-242.
[23]魏经建,邵树军,王天元.硫酸酯多糖化学及临床应用研究进展.中国生化药物杂志.1999,20(5)：260-262.
[24]He ZJ, Chen JW, L i X. Advances in studies of sulfated polysaccharide. J Chin Tradit Herb Drugs,2003,21:2087-2088.
[25]Doctor VM, Lewis D, Coleman M. Anticoagulant properties of semisynthetic polysaccharide sulfates. Thromb Res,1991,64:413-425.
[26]Yang TH, Shang P, Mei QB, et al. Effects of angelica polysaccharide and its sulfates on coagulation and platelet aggregation. Chin Tradit Herb Drugs,2002,33: 1010-1013.
[27]Hoshino T, Hayashi T, Hayashi K, et al. An antivirally active sulfated polysaccharide from argassum homeri (TURNER) C. AGARDH. Biol Pharm Bull, 1998,21:730-734.
[28]Beress A, Wassermann O, Tahhan S, et al. A new procedure for the isolation of anti-HIV compound (polysaccharides and polyphenols) from the marine alga fucus vesiculosus. J Nat Prod,1993,56:478-483.
[29]Hayashi K, Hayashi T, Kojima I. A nature sulfated polysaccharide, calcium sp irulan, isolated from spirulina p latensis:in vitro and ex vivo evaluation of anti-herpes simp lex virus and anti-human immunodeficiency virus activities. Aids Res Hum Retroviruses,1996,12:1463-1468.
[30]Xing XL, Ding H, Geng MY, et al. Studies of the anti-AIDS effects of marine polysaccharids drug 911 and its related mechanisms of action. Chin J Marine Drugs, 2000, (6):428-435.
[31]Ono L, Wollinger W, Rocco IM, Coimbra TL, Gorin PA, Sierakowski MR. In vitro and in vivo antiviral properties of sulfated galactomannans against yellow fever virus (BeH111 strain) and dengue virus (Hawaii strain), Antiviral Res,2003,60: 201-208.
[32]Talarico LB, Pujol CA, Zibetti RG, Faria PC, Noseda MD, Duarte ME, Damonte EB. The antiviral activity of sulfated polysaccharides against dengue virus is dependent on virus serotype and host cell, Antiviral Res,2005,66:103-110.
[33]Hidari KI, Takahashi N, Arihara M, Nagaoka M, Morita K, Suzuki T. Structure and anti-dengue virus activity of sulfated polysaccharide from a marine alga. Biochem Biophys Res Commun,2008,376:91-95.
[34]Zhu GJ, Zhao ZF, Fang HY. The relationship between structure and function of polysaccharides. Journal of Wiuxi University of Light Indus,2002,21:209-212.
[35]Tian GY, Li ST, Song ML, et al. Synthesis of achyranthes bidentata polysaccharide sulfate and its antivirus activity. Acta Pharm Sinica,1995,30: 107-111.
[36]An WL, Pu Q, Meng YW. Preparation of sulfated of two polygonatum polysaccharides & their antiviral activities. Nat Prod Res Dev,2000,3:60-65.
[37]Zhang LP, Zhang YS, Sun F, et al. The Influence of sulfation on the conformation and biological activity of polysaccharides from pleurotus citrinop ileatus. Acta Biochimica & Biophysica Sinica,1994,26:417-421.
[38]Chen CY, Hunag XH, Zhou JY, et al.Sulfation of polsaccharides isolated from indocalamus tesselatus and their anticytopathic effect on human immunodeficencv virus type I. Acta Pharm Sinica,1998,33:264-269.
[39]Chen CY, J iang Y. Effects of ruoye polysacchrides and their derivatives on mice infected with LP2BM5MuLV, amurine AIDSmodel. Chine Pharm Bull,1999,8,15: 336-342.
[40]Yang TH, Jia M, Shang P, et al. Synthesis of angelica sinensis polysaccharide sulfate and their effects on splenocyte proliferation in vitro. J Fourth Mil Med Univ 2001,22:432-435.
[41]Wei WQ, Cong JB, Xian H, et al. The effect of polysaccharides from sea weed (SPS) on regulating the immune function in mice. Chin JNew Drugs,2001,10: 671-675.
[42]Wei WQ, Cong JB, Xian H,et al. Inhibitory effects and its mechanism of sulfated polysaccharides from sea weed (SPS) on the lymphocyte apoptosis induced by oxidative stress. Chin Pharm J,2002,37:664-667.
[43]薛静波,刘希英,张鸿芬.海带多糖对小鼠腹腔巨噬细胞的激活作用.中国海洋药物,1999,3：23-25.
[44]Okai, Yasuji. Detection of immunomodulating activities in an extract of Japanese edible seaweed,Laminariaja ponica. J Sci Food Agric,1996,72 (4):455-459.
[45]Zvyagintseva TN, Shevchenko NM, Nazarova IV, Scobun AS, Luk'yanov PA, Elyakova LA, Inhibition of complement activation by water-soluble polysaccharides of some fareastenr brown seaweeds. Comp Biochem Physiol, PartC:Toxicol Pharmacol,2000,126:209-215.
[46]苗本春,耿美玉,李静等.海洋硫酸多糖911免疫增强作用的探讨.中国海洋
药物,2002,5：1-4.
[47]黄益丽,郑忠辉,苏文金.二色桌片参的化学成分研究；Ⅲ二色桌片参多糖一Ⅰ一岩藻聚糖的免疫调节.海洋通报,2001,20(1)：88-91.
[48]陈文星,吴皓,金亦涛.珠蚌多糖的免疫调节作用研究.中药药理与临床,2001,17(5)：16-18.
[49]朱爱民,扬晓峰,李刚.螺旋多糖对小鼠免疫系统的作用.山东医药工业,2001,30(6)：44-45.
[50]陈绍缓,莫卫民,潘远江.海洋药物研究(Ⅲ)一羊栖菜多糖.兰州大学学报(自然科学版),1998,34(4)：110-113.
[51]Bobek V, Kovarik J. Antitumor and antimetastatic effect of warfarin and heparins. Biomed Pharmacother.2004,58:213-219.
[52]朱国臣,肖大江,张永胜,袁渊,吴四海,沈莉莉,郑晓彬.乙酰肝素酶与鼻咽癌侵袭、转移和血管生成的相关性.中国耳鼻咽喉头颈外科,2009,(12)：669-672.
[53]马秀梅,王栓柱,于宏,怀娜.沉默乙酰肝素酶对人胃癌细胞侵袭迁移能力的影响.肿瘤,2009,(10)：924-928.
[54]鄢俊安,宋波,陈志文,宋彩萍.乙酰肝素酶在膀胱移行细胞癌中的表达及与临床特征的关系.第三军医大学学报,2007,(23)：2280-2282.
[55]Iozzo RV. Matrix proteoglycans:from molecular design to cellular function. Annu Rev Biochem.1998,67:609-652.
[56]Wegrowski Y, Maquart FX. Involvement of stromal proteoglycans in tumour progression. Crit Rev Oncol Hematol.2004,49:259-268.
[57]Theocharis AD. Human colon adenocarcinoma is associated with specific post-translational modifications of versican and decorin. Biochim Biophys Acta,2002, 1588:165-172.
[58]Yamada S, Sugahara K. Potential therapeutic application of chondroitin sulfate/dermatan sulfate. Curr Drug Discov Technol.2008,5:289-301.
[59]曲显俊,崔淑香,解砚英.螺旋藻多糖抗癌作用的实验研究.中国海洋药物,2000,4：10-14.
[60]许东晖,许实波,王兵等.皱纹盘鲍多糖抗肿瘤药理作用研究.热带海洋,1999,18(4)：86-90.
[61]刘秋英,孟庆勇,刘志辉.海藻多糖抗肿瘤作用的研究进展.中国海洋药物,2003,4：45-48.
[62]师然新,徐祖洪,李智恩.降解的角叉菜多糖的抗肿瘤活性.海洋与湖泊,2000,31(6)：653-656.
[63]Kumar GP, Sudheesh S, Vijayalakshmi NR. Hypoglycaemic effect of Coccinia indica:mechanism of action. Planta Med.1993,59(4):330-332.
[64]侯建明,蓝进.海洋生物多糖(AC-1)防治高脂血症的实验报告.海军医学杂志2001,22(3)：197-199.
[65]王远红,徐家敏,耿美玉.海洋硫酸多糖916对实验性高脂血症大鼠血浆中含硫氨基酸的影响.中国海洋药物,2000,4：23-24.
[66]王雪松,郑芸,方积年.降血糖多糖及寡糖的研究进展.药学学报,2004,39(12)：1028-1033.
[67]徐中平,李福川,王海仁.昆布多糖硫酸酯的抑制血管生成和抗肿瘤作用.中草药1999,30(7)：551-553.
[68]Liu CH, Li XD, Li YH, Feng Y, Zhou S, Wang FS. Structural characterisation and antimutagenic activity of a novel polysaccharide isolated from Sepiella maindroni ink; Food Chemistry,2008,110:807-813.
[69]Wang J, Wang F, Zhang Q, Zhang Z, Shi X, Li P. Synthesized different derivatives of low molecular fucoidan extracted from Laminaria japonica and their potential antioxidant activity in vitro. Int J Biol Macromol.2009,44:379-384.
[70]Wang J, Zhang Q, Zhang Z, Zhang J, Li P. Synthesized phosphorylated and aminated derivatives of fucoidan and their potential antioxidant activity in vitro. Int J Biol Macromol.2009,44:170-174.
[71]Wang J, Zhang Q, Zhang Z, Li Z. Antioxidant activity of sulfated polysaccharide fractions extracted from Laminaria japonica. Int J Biol Macromol.2008,42:127-132.
[72]Mori H, Kanemura Y, Onaya,J, Hara M, Miyake J, Yamasaki M, Kariya Y. Effects of heparin and its 6-O-and 2-O-desulfated derivatives with low anticoagulant activity on proliferation of human neural stem/progenitor cells. J Biosci Bioeng,2005, 100,54-61.
[73]李绪亮,焦庆才,陈群,刘茜.硫酸化茯苓多糖对大鼠慢性肾功能衰竭的防治作用.中国药学杂志2005,40(12)：908-911.
[74]Mourao PA, Giumaraes B, Mulloy B, Thomas S, Gray E. Antithrombotic activity of a fucosylated chondroitin sulphate from echinoderm:sulphated fucose branches on the polysaccharide account for its antithrombotic action. Br J Haematol.1998,101: 647-652.
[75]Tapon-Bretaudiere J, Chabut D, Zierer M, Matou S, Helley D, Bros A, Mourao, PA, Fischer,AM. A Fucosylated chondroitin sulfate from Echinoderm modulates in Vitro fibroblast growth factor 2-dependent angiogenesis. Mol Cancer Res,2002, 1:96-102.
[76]Nikitovic D, Assouti M, Sifaki M, Katonis P, Krasagakis K, Karamanos NK, Tzanakakis GN. Chondroitin sulfate and heparan sulfate-containing proteoglycans are both partners and targets of basic fibroblast growth factor-mediated proliferation in human metastatic melanoma cell lines. Int JBiochem Cell Biol.2008,40:72-83.
[77]Taylor KR, Rudisill JA, Gallo RL. Structural and sequence motifs in dermatan sulfate for promoting fibroblast growth factor-2 (FGF-2) and FGF-7 activity. J Biol Chem.2005,280:5300-5306.
[78]Wang CY, Guang HS. Advances in studies of antiviral activity of polysaccharide Ⅱ. antiviral activity of sulfated polysaccharide. Advances of Biolagical Project.2000, (2):328-330.
[79]Ashikari-Hada S, Habuchi H, Kariya Y, Kimata K. Heparin regulates vascular endothelial growth factor 165-dependent mitogenic activity, tube formation, and its receptor phosphorylation of human endothelial cells. J Biol Chem.2005,280: 31508-31515.
[80]Li F, Shetty AK, Sugahara K. Neuritogenic activity of chondroitin/dermatan sulfate hybrid chains of embryonic pig brain and their mimicry from shark liver. Involvement of the pleiotrophin and hepatocyte growth factor signaling pathways. J Biol Chem.2007,282:2956-2966.
[81]Alban S, Franz G. Characterization of the anticoagulant actions of a semisynthetic curdlan sulfate. Thromb Res,2000,99:377-388.
[82]GaoY, Fukuda A, Katsuraya K, et al. Synthesis of regioselective substituted curdlan sulfates with medium molecular weights and their specific anti-HIV-1 activities. Macromolecules,1997,30:3224-3228.
[83]Calazans GMT, Lima RC, de Franca FP, Lopes CE. Molecular weight and antitumour activity of Zymomonas mobilis levans. Int J Biol Macromol.2000,12; 27(4):245-247.
[84]石磊,几种多糖的分离纯化、结构解析和生物活性研究[D],山东大学,2007.
[85]顾文莉,硫酸软骨素蛋白多糖在神经前体细胞增殖及其分化细胞中的表达和调控[D],上海交通大学,2007.
[86]Bjorklund A, Lindvall O. Cell replacement therapies for central nervous system disorders. Nat Neurosci,2000,3:537-544.
[87]McBride JL, Behrstock SP, Chen EY et al. Human neural stem cell transplants improve motor function in a rat model of Huntington's disease. J Comp Neurol.2004, 475:211-219.
[88]Sugaya K, Alvarez A, Marutle A, Kwak YD, Choumkina E. Stem cell strategies for Alzheimer's disease therapy. Panminerva Med.2006,48(2):87-96.
[89]Yu D, Silva GA. Stem cell sources and therapeutic approaches for central nervous system and neural retinal disorders. Neurosurg Focus.2008,24(3-4):Ell.
[90]Lee JP, Jeyakumar M, Gonzalez R. Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic disease. Nat Med,2007,13:439-447.
[91]Lim DA, Huang YC, Alvarez-Buylla A. The adult neural stem cell niche:lessons for future neural cell replacement strategies. Neurosurg Clin NAm,2007,18:81-92.
[92]varez-Buylla A, Lim DA. For the long run:maintaining germinal niches in the adult brain. Neuron.2004,41:683-686.
[93]Mercier F, Kitasako JT, Hatton GI. Anatomy of the brain neurogenic zones revisited:fractones and the fibroblast/macrophage network. J Comp Neurol.2002, 451:170-188.
[94]Pitman M, Emery B, Binder M, Wang S, Butzkueven H, Kilpatrick TJ. LIF receptor signaling modulates neural stem cell renewal, Mol Cell Neurosci.2004,27: 255-266.
[95]Kokuzawa J, Yoshimura S, Kitajima H, Shinoda J, Kaku Y, Iwama T, Morishita R, Shimazaki T, Okano H, Kunisada T, Sakai N. Hepatocyte growth factor promotes proliferation and neuronal differentiation of neural stem cells from mouse embryos, Mol Cell Neurosci.2003,24:190-197.
[96]Kosaka M, Kodama H, Sasaki Y, Yamamoto F, Takeshita Y, Takahama H, Sakamoto T, Kato M, Terada T, Ochiya. FGF-4 regulates neural progenitor cell proliferation and neuronal differentiation, FASEB J.2006,20:1484-1485.
[97]Lum M, Croze E, Wagner C, McLenachan S, Mitrovic B, Turnley AM, Inhibition of neurosphere proliferation by IFN-gamma but not IFN-beta is coupled to neuronal differentiation, JNeuroimmunol.2009,206:32-38.
[98]Galderisi U, Cipollaro M, Giordano A. Stem cells and brain cancer. Cell Death Differ.2006,13(1):5-11.
[99]Ida M, Shuo T, Hirano K, Tokita Y, Nakanishi K, Matsui F, Aono S, Fujita H, Fujiwara Y, Kaji T, Oohira A. Identification and functions of chondroitin sulfate in the milieu of neural stem cells. JBiol Chem,2006,281:5982-5991.
[100]Sirko S, von Holst A, Wizenmann A. et al. Chondroitin sulfate glycosaminoglycans control proliferation, radial glia cell differentiation and neurogenesis in neural stem/progenitor cells. Development.2007,134:2727-2738.
[101]Robert K. Yu, Makoto Yanagisawa. Glycobiology of neural stem cells. CNS & Neurological Disorders-Drug Targets,2006,5:415-423.
[102]Akita K, von Holst A, Furukawa Y, Mikami T, Sugahara K, Faissner A. Expression of multiple chondroitin/dermatan sulfotransferases in the neurogenic regions of the embryonic and adult CNS imply that complex chondroitin sulfates have a role in neural stem cell maintenance. Stem Cells,2008,26:798-809
[103]Sugahara K, Mikami T. Chondroitin/dermatan sulfate in the central nervous system. Curr Opin Struct Biol,2007,17:536-545.
[104]Kabos P, Matundan H, Zandian M, Bertolotto C, Robinson ML, Davy BE, Yu JS, Krueger RC Jr. Neural precursors express multiple chondroitin sulfate proteoglycans, including the lectican family.Biochem Biophys Res Commun.2004; 318(4):955-963.
[105]Zou P, Muramatsu H, Miyata T, Muramatsu T. Midkine, a heparin-binding growth factor, is expressed in neural precursor cells and promotes their growth. J. Neurochem.2006,9:1470-1479.
[106]Brickman YG, Ford MD, Small DH, Bartlett PF, Nurcombe V. Heparan sulfates mediate the binding of basic fibroblast growth factor to a specific receptor on neural precursor cells. JBiol Chem,1995,270,24941-24948.
[107]Nurcombe V, Ford MD, Wildschut JA. Bartlett PF. Developmental regulation of neural response to FGF-1 and FGF-2 by heparan sulfate proteoglycan. Science,1993, 260:103-106.
[108]Hagihara K, Watanabe K, Chun J. Yamaguchi Y. Glypican-4 is an FGF2-binding heparan sulfate proteoglycan expressed in neural precursor cells. Dev Dyn,2000,219: 353-367.
[109]Caldwell MA, Svendsen CN. Heparin, but not other proteoglycans potentiates the mitogenic effects of FGF-2 on mesencephalic precursor cells. Exp Neurol. 1998;152(1):1-10.
[110]Landolt RM, Vaughan L, Winterhalter KH, Zimmermann DR. Versican is selectively expressed in embryonic tissues that act as barriers to neural crest cell migration and axon outgrowth. Development,1995,121,2303-2312.
[111]Gu WL, Fu SL, Wang YX, Li Y, Lu HZ, Xu XM, Lu PH. Chondroitin sulfate proteoglycans regulate the growth, differentiation and migration of multipotent neural precursor cells through the integrin signaling pathway. BMC Neurosci,2009,10: 128(1-15).
[112]Kearns SM, Laywell ED, Kukekov VK, Steindler DA. Extracellular matrix effects on neurosphere cell motility. Exp Neurol.2003,182:240-244.
[113]Nagayasu T, Miyata S, Hayashi N, Takano R, Kariya Y, Kamei K. Heparin structures in FGF-2-dependent morphological transformation of astrocytes. J Biomed Mater Res A.2005,74(3):374-380.
[114]Yamada T, Sawada R, Tsuchiya T. The effect of sulfated hyaluronan on the morphological transformation and activity of cultured human astrocytes. Biomaterials. 2008,29 (26):3503-3513.
[115]Ahmed S, Tsuchiya T, Nagahata-Ishiguro M, Sawada R, Banu N, Nagira T. Enhancing action by sulfated hyaluronan on connexin-26,-32, and-43 gene expressions during the culture of normal human astrocytes. J Biomed Mater Res A, 2009,90 (3):713-719.
[116]Inatani M, Irie F, Plump AS, Tessier-Lavigne M. Yamaguchi Y. Mammalian brain morphogenesis and midline axon guidance require heparan sulfate. Science, 2003,302:1044-1046.
[117]Ai X, Kitazawa T, Do AT, Kusche-Gullberg M, Labosky PA, Emerson CP Jr. SULF1 and SULF2 regulate heparan sulfate-mediated GDNF signaling for esophageal innervation. Development,2007,134:3327-3338.
[118]Casu B. Structure and biological activity of heparin. Adv Carbohydr Chem Biochem,1985; 43:51-134.
[119]Comper D. Heparin and related polysaccharides. Vol.7, Gordan and Breach, 1981; 41:158-172.
[120]Rosenberg D, L Lam. Correlation between structure and function of heparin, Proc Natl Acad Sci USA,1979,76:1218-1222.
[121]Cardin AD, Weintraub HJR. Molecular modeling of protein-glycosaminoglycan interactions. Arteriosclerosis,1989,9:21-32.
[122]Rosenberg D, Schworak NW, Liu J.Schwartz J J, Zhang L, Molecular cloning and expression of mouse and human cDNAs encoding heparan sulfate d-Glucosaminyl 3-O-Sulfotransferase. JClin Invest,1997,99,2062-2070.
[123]Walker A, Turnbull JE, Gallagher JT. Specific heparan sulphate saccharides mediate the activity of basic fibroblast growth factor, JBiol Chem,1994,269:931-935.
[124]Wang HM, ToidaT, KimYS, Capila I, Hileman RE, Bernfield M, Linhardt RJ. Glycosaminoglycans can influence fibroblast growth factor-2 mitogenicity without significant growth factor binding, Biochem Biophys Res Commun,1997; 235, 369-373.
[125]Coombe DR, Kett WC. Heparan sulfate-protein interactions:therapeutic potential through structurefunction insights. Cell Mol Life Sci,2005,62:410-424.
[126]Shikari S, Habuchi H, Kimata K. Characterization of heparan sulfate oligosaccharides that bind to hepatocyte growth factor. J Biol Chem,1995,270, 29586-29593.
[127]Gordon MY, Riley G P, Watt SM, Greaves M F. Compartmentalization of a haematopoietic growth factor (GM-CSF) by glycosaminoglycans in the bone marrow microenvironment. Nature,1987,326,403-405.
[128]Roberts R, Gallagher J, Spooncer E, Allen T D, Bloomfield F, Dexter TM. Heparan sulphate bound growth factors:a mechanism for stromal cell mediated haemopoiesis. Nature,1988,332,376-378.
[129]Takada T, Katagiri T, Ifuku M, Morimura N, Kobayashi M, Hasegawa K, Ogamo A, Kamijo, R. Sulfated polysaccharides enhance the biological activities of bone morphogeneticproteins. J Biol Chem.2003.278,43229-43235.
[130]Lakshmi TS, Shanmugasundaram N, Shanmuganathan S, Karthikeyan K, Meenakshi J, Babu M. Controlled release of 2,3 desulfated heparin exerts its anti-inflammatory activity by effectively inhibiting E-selectin. J Biomed Mater Res A. 2010,95(1):118-128.
[131]Perez-Pinera P, Chang Y, Deuel TF. Pleiotrophin, a multifunctional tumor promoter through induction of tumor angiogenesis, remodeling of the tumor microenvironment, and activation of stromal fibroblasts. Cell Cycle.2007; 6 (23):2877-2883.
[132]Deakin JA, Lyon M. Differential regulation of hepatocyte growth factor/scatter factor by cell surface proteoglycans and free glycosaminoglycan chains. J Cell Sci, 1999; 112. (Ptl2):1999-2009.
[133]樊绘曾.海参：海中人参：关于海参及其成分保健医疗功能的研究与开发.中国海洋药物,2001,20(4)：37-44.
[134]姜健,杨宝灵,邰阳.海参资源及生物活性物质的研究.生物技术通讯2004,15(5)：537-540.
[135]樊绘曾,陈菊,吕培宏.玉足海参酸性多糖的研究.药学学报,1983,18：2031-2032.
[136]Bulgakov AA, Nazarenko EL, Petrova IY, Eliseikina MG, Vakhrusheva NM, Zubkov VA. Isolation and properties of a mannan-binding lectin from the coelomic fluid of the holothurian Cucumaria japonica. Biochemistry (Mosc).2000; 65 (8): 933-939.
[137]Ribeiro AC,Vieira RP, Mourao PA. Mulloy B. A sulfated alpha-L-fucan from sea cucumber. Carbohydr Res,1994,255:225-240.
[138]Kariya Y, Watabe S, Kyogashima M. Structure of fucose branches in the glycosam inoglycan from the body wall of the sea cucumber, Stichopus japonicu. Carbohydr Res,1997,297:273-278.
[139]盛文静,薛长湖,赵庆喜,徐杰,李兆杰,孙妍.不同海参多糖的化学组成分析比较.中国海洋药物杂志2007,26：44-49.
[140]Li JZ, Bao CX, Chen GZ. Antithrombin activity and platelet aggregation by acid mucopo lysaccharides isolated from stichopusj aponicus selenka.China. Acta Pharma cologica Sinica,1985,6:107-112.
[141]王学锋,李志广,储海燕.海参糖胺聚糖抗血栓形成机制的研究.中国新药与临床杂志2002,21(12)：718-721.
[142]杨晓光,陈关珍,罗晓玲.Sjamp对纤溶系统影响的初步观察.中国医学科学院学报1990,2：1871-1873
[143]Fonseca RJ, Mourao PA. Fucosylated chondroitin sulfate as a new oral antithrombotic agent. Thromb Haemost.2006,96:822-829.
[144]Mourao PA, Pereira MS, Pavao MS, Mulloy B, Tollefsen DM, Mowinckel MC, Abildgaard U. Structure and anticoagulant activity of a fucosylated chondroitin sulfate from echinoderm. Sulfated fucose branches on the polysaccharide account for its high anticoagulant action. JBiol Chem.1996,271 (39):23973-23984.
[145]陈玲,于壮,宋扬,张笑雪,李明君.刺参黏多糖对人宫颈癌细胞凋亡的影响.齐鲁医学杂志,2009,24：95-98.
[146]王静凤,王奕,赵林,逢龙,薛长湖.日本刺参的抗肿瘤及免疫调节作用研究.中国海洋大学学报,2007,37：93-96.
[147]王强基,李春艳.玉足海参酸性粘多糖对小鼠免疫功能的影响.海洋药物,1984,12-14.
[148]黄益利,郑忠辉,苏文金等.二色桌片参的化学成分研究-二色桌片参多糖的免疫调节作用.全国第三届海津生命活性物质与天热生化药物学术讨论会论文集,海南,2000：129.
[149]李艳菊,刘辉,郭月秋.海参免疫调节作用的实验研究.中国海洋药物杂志2006,25：49-50.
[150]纪静,王笑峰,马忠兵,罗兵.现刺参糖胺聚糖抗仙台病毒作用机制的探讨.现代生物医学进展2009,9：1060-1063.
[151]罗兵,马忠兵,王笑峰,王云.刺参糖胺聚糖对单纯疱疹病毒Ⅰ型抑制作用的实验研究.中国海洋药物杂志,2008,27：44-48.
[152]高森,王静风,王玉明,刘治东,李晓林,薛长湖.冰岛刺参调节血脂及其作用机制.武汉大学学报(理学版),2009,55：324-328.
[153]王静风,逢龙,黄平,赵芹,薛长湖.北极刺参和日本刺参对大鼠血脂水平调节和血管内皮保护作用的研究.中国海洋药物杂志2007,26：10-13.
[154]胡晓倩,王玉明,任兵兴,常耀光,王静凤,薛长湖.海参主要活性成分对大鼠脂质代谢影响的比较研究.食品科学,2009,30：393-396.
[155]王静凤,逢龙,薛勇,董平,盛文静,薛长湖.日本刺参酸性粘多糖、皂苷和胶原蛋白多肽对血管内皮细胞的保护作用.中国药理学通报2008,24：227-232.
[156]逄龙,王静凤,王玉明,盛文静,薛长湖.北极刺参多糖、皂苷和胶原蛋白多肽对血管内皮细胞的保护作用.中国药科大学学报2007,38：437-441.
[157]朱宗涛,蔡生业,姚成芳,王丽,王华亭,张维东.复方花刺参粘多糖对髂动脉内皮剥脱家兔内膜增生的影响及机制.中国动脉硬化杂志2004,12：43-46.
[158]王华亭,蔡生业,姚成芳,朱宗涛,王丽,张维东.复方花刺参粘多糖对家兔血管成形术后内皮功能及超微结构的影响.中国动脉硬化杂志,2004,5：497-501.
[159]王曼玲,徐建民.海参糖胺聚糖抑制血小板-内皮细胞粘附及调节血小板粘附分子表达.中国临床医学,2006,13：431-433.
[160]王利民,蔡生业,姚成芳,王丽,周宪宾,王华亭,王恒孝.花刺参粘多糖对大鼠血管平滑肌细胞粘附分子表达的影响.中国动脉硬化杂志,2006,14：565-568.
[161]Tapon-Bretaudiere J, Drouet B, Matou S, Mourao PA, Bros A, Letourneur D, Fischer AM. Modulation of vascular human endothelial and rat smooth muscle cell growth by a fucosylated chondroitin sulfate from echinoderm. Thromb Haemost.2000, 84:332-337.
[162]Kariya Y, Mulloy B, Imai K, Tominaga A, Kaneko T, Asari, Suzuki K, Masuda H, Kyogashima, Ishii T. Isolation and partial characterization of fucan sulfates from the body wall of sea cucumber Stichopus japonicus and their ability to inhibit osteoclastogenesis. Carbohydr Res,2004,339:1339-1346.
[163]迟玉森,庄桂东,黄福祥,安桂香,徐鹏.海参多糖对小白鼠伤口愈合的影响.食品科学,2005,26：211-214.
[164]邱鹏新,黎明涛,唐孝礼,苏兴文,林穗珍,颜光美.黑海参多糖对β-淀粉样蛋白诱导的皮质神经元凋亡的保护作用.中草药2000,31：271-274.
[165]Borsig L, Wang L, Cavalcante MC, Cardilo-Reis L, Ferreira PL, Mourao PA, Esko JD, Pavao MS. Selectin blocking activity of a fucosylated chondroitin sulfate glycosaminoglycan from sea cucumber. Effect on tumor metastasis and neutrophil recruitment. JBiol Chem.2007,282 (20):14984-14991.
[166]Landeira-Fernandez AM, Aiello KR, Aquino RS, Silva LC, Meis L, Mourao PA. A sulfated polysaccharide from the sarcoplasmic reticulum of sea cucumber smooth muscle is an endogenous inhibitor of the Ca(2+)-ATPase. Glycobiology.2000,10: 773-779.
[167]朱玉强.刺参多糖急性毒理研究及其安全性评价.四川食品与发酵,2005,41：48-51.
[168]段君.刺参酸性粘多糖的提取及其性质与结构分析[D],2003.
[169]方积年.天然药物-多糖的主要生物活性及分离纯化方法.中国天然药物,2007,5：338-347.
[170]唐孝礼,邱鹏新,黎明涛,苏兴文,颜光美.黑海参酸性粘多糖的分离纯化.中药材,1999,22：223-225.
[171]郑艾初,陈健,彭超英.糙海参酸性粘多糖的提取纯化工艺探讨.现代食品科技2007,23：65-67.
[172]Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem,1956,28:350-356.
[173]Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Chem,1976, 72:248-254.
[174]Filisetti-Cozzi, TMCC, Carpita, NC. Measurement of uronic acids without interference from neutral sugars. Analytical Biochemistry,1991,197:157-162.
[175]Saito H, Yamagata T, Suzuki S. Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates. J Biol Chem,1968,242:1536-1542.
[176]Takeda M, Nomoto S, Koizumi J. Structural analysis of the extracellular polysaccharide produced by Sphaerotilus natans. Biosci Biotechnol Biochem.2002, 66(7):1546-1551.
[177]Matsuhiro, B, Zuniga, E, Jashesl, M, Guacucanol M. Sulfated polysaccharides from Durvillaea antarctica. Hydrobiologia,1996,321:77-81.
[178]Caceres PJ, Faundez CA, Matsuhiro B, Vasquez JA J. Carrageenophyte identification by second-derivative Fourier transform infrared spectroscopy. Journal of Applied Phycology,1997,8:523-527.
[179]Barker SA, Bourne EJ, Stacey M, et al. Infra-red spectra of carbohydrates. Part Ⅰ. Some derivatives of d-glucopyranose. J Chem Soci,1954,6:34-39.
[180]陈菊娣,樊绘曾,刑蕊凝等.花刺参酸性粘多糖的分离研究.中国海洋药物,1994,1：24-26.
[181]Mourao PA, Bastos IG. Highly acidic glycans from sea cucumbers. Isolation and fractionation of fucose-rich sulfated polysaccharides from the body wall of Ludwigothurea grisea.Eur J Biochem,1987,166:639-645.
[182]Zhao J, Wu M, Kang H, Zeng W, Liang H, Li Z. Low molecular weight fractions and preparation of a fucosylated glycosaminoglycan from sea cucumber. CN patent,200910110114.0.
[183]王玲,陈健,姜建国,郑艾初.沙海参多糖的分离和特性研究.现代食品科技,2008,24：655-657.
[184]Lee JP, Jeyakumar M, Gonzalez R. Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic disease. Nat Med 2007,13 (4): 439-447.
[185]Lim DA, Huang YC, Alvarez-Buylla A. The adult neural stem cell niche: lessons for future neural cell replacement strategies. Neurosurg Clin NAm 2007,18 (1):81-92.
[186]Ahmed S. The culture of neural stem cells, J Cell Biochem.2009,106:1-6.
[187]Coles-Takabe BL, Brain I, Purpura KA, Karpowicz P, Zandstra PW, Morshead CM, van der Kooy D. Don't look:growing clonal versus nonclonal neural stem cell colonies. Stem Cells,2008,26 (11):2938-2944.
[188]蔡文琴.发育神经生物学[M],北京：科学出版社,2002：223.
[189]Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system, Science,1992,255:1707-1710.
[190]Malavaki C, Mizumoto S, Karamanos N, Sugahara K. Recent advances in the structural study of functional chondroitin sulfate and dermatan sulfate in health and disease. Connect Tissue Res,2008,49 (3):133-139.
[191]Jiang J, Chen X, Shen J, Wei Y, Wu T, Yang Y, Wang H, Zong H, Yang J, Zhang S, Xie J, Kong X, Liu W, Gu J. Beta1,4-galactosyltransferase V functions as a positive growth regulator in glioma. JBiol Chem,2006,281:9482-9489.
[192]杨雪松.岩藻糖基转移酶Ⅳ对A431细胞增殖与凋亡的影响[D].2007.
[193]王秋雁.α-1,3-岩藻糖转移酶-Ⅶ对人肝癌细胞信号转导、增殖和凋亡的影响[D].2004.
[194]Fawcett J. Repair of spinal cord injuries:where are we, where are we going? Spinal Cord.2002,40:615-623.
[195]McLenachan S, Lum MG, Waters MJ, Turnley AM. Growth hormone promotes proliferation of adult neurosphere cultures. Growth Horm IGF Res.2009,19 (3): 212-218.
[196]Fonseca RJ, Mourao PA. Fucosylated chondroitin sulfate as a new oral antithrombotic agent. Thromb Haemost.2006,96(6):822-829.
[197]Sweeney EA, Priestley GV, Nakamoto B, Collins RG, Beaudet AL, Papayannopoulou T. Mobilization of stem/progenitor cells by sulfated polysaccharides does not require selectin presence, Proc Natl Acad Sci USA,2000,97: 6544-6549.
[198]Matou S, Helley D, Chabut D, Bros A, Fischer AM. Effect of fucoidan on fibroblast growth factor-2-induced angiogeriesis in vitro. Thromb Res,2002,106: 213-221.
[199]Islam MO, Kanemura Y, Tajria J, Mori H, Kobayashi S, Shofuda T, Miyake J, Hara M, Yamasaki M, Okano H. Characterization of ABC transporter ABCB1 expressed in human neural stem/progenitor cells. FEBS Lett,2005,579:3473-3480.
[200]Wang TY, Sen A, Behie LA, Kallos MS, Dynamic behavior of cells within neurospheres in expanding populations of neural precursors, Brain Res.2006,1107: 82-96.
[201]郑志竑,胡建石,陈文列,林玲,钟秀容,林建银.体外培养的神经干细胞球的超微结构.解剖学报,2003,34(6)：615-619.
[202]Mori H, Ninomiya K, Kino-oka M, Shofuda T, Islam MO, Yamasaki M, Okano H, Taya M, Kanemura Y. Effect of neurosphere size on the growth rate of human neural stem/progenitor cells. JNeurosci Res,2006,84:1682-1691.
[203]Widera D, Mikenberg I, Kaus A, Kaltschmidt C, Kaltschmidt B, Nuclear Factor-kappaB controls the reaggregation of 3D neurosphere cultures in vitro, Eur Cell Mater.2006,11:76-84.
[204]Widera D, Mikenberg I, Elvers M, Kaltschmidt C, Kaltschmidt B. Tumor necrosis factor alpha triggers proliferation of adult neural stem cells via IKK/NF-kappaB signaling, BMC Neurosci,2006,7:64-82.
[205]Campos LS, Leone DP, Relvas JB, Brakebusch C, Fassler R, Suter U, ffrench-Constant C. Betal integrins activate a MAPK signalling pathway in neuralstem cells that contributes to their maintenance. Development,2004,131(14): 3433-3444.
[206]Kimura A, Ohmori T, Ohkawa R, Madoiwa S, Mimuro J, Murakami T, Kobayashi E, Hoshino Y, Yatomi Y, Sakata Y. Essential roles of sphingosine 1-phosphate/S1P1 receptor axis in the migration of neural stem cells toward a site of spinal cord injury. Stem Cells.2007,25:115-124.
[207]Mori H, Fujitani T, Kanemura Y, Kino-Oka M, Taya M. Observational examination of aggregation and migration during early phase of neurosphere culture of mouse neural stem cells. J Biosci Bioeng,2007,104(3):231-234.
[208]Karin M, Lin A. NF-κB at the crossroads of life and death, Nat Immunol,2002, 3:221-227.
[209]Goldschmidt B, Wider D, Goldschmidt C. Signaling via NF-κB in the nervous system, Biochim Biophys Acta,2005,1745:287-299.
[210]Raballo R, Rhee J, Lyn-Cook R, Leckman JF, Schwartz ML, Vaccarino FM. Basic fibroblast growth factor (Fgf2) is necessary for cell proliferation and neurogenesis in the developing cerebral cortex, JNeurosci,2000,20:5012-5023.
[211]Qian X, Davis AA, Goderie SK, Temple S. FGF2 concentration regulates the generation of neurons and glia from multipotent cortical stem cells, Neuron,1997,18: 81-93.
[212]Sun L, Lee J, Fine HA. Neuronally expressed stem cell factor induces neural stem cell migration to areas of brain injury, JClin Invest,2004,113:1364-1374.
[213]Bell HS, Whittle IR, Walker M, Leaver HA, Wharton SB. The development of necrosis and apoptosis in glioma:experimental findings using spheroid culture systems. Neuropathol Appl Neurobiol,2001,27:291-304.
[214]Carlsson J, Acker H. Relations between pH, oxygen partial pressure and growth in cultured cell spheroids. Int J Cancer,1988,42:715-720.
[215]Glicklis R, Merchuk JC, Cohen S. Modeling mass transfer in hepatocyte spheroids via cell viability, spheroid size, and hepatocellular functions. Biotechnol Bioeng,2004,86:672-680.
[2]黄芳,蒙义文.活性多糖的研究进展.天然产物开发与研究1996,11(5)：90-97.
[3]魏经建,邵树军,王天元.硫酸酯化多糖化学及临床应用研究进展.中国生化药物杂志1999,20(5)：260-262.
[4]郭振环,胡元亮,马霞,赵晓娜,王德云,赵炳凯,郭利伟,刘萍,郝利华.硫酸化香菇多糖对新城疫疫苗免疫效果的影响.南京农业大学学报,2010,33(1)：76-80.
[5]高贵珍,陈群,李绪亮.硫酸化茯苓多糖药理性质研究.安徽大学学报(自然科学版),2009,33(6)：72-77.
[6]杨铁虹,贾敏,姚秀娟,孟嘉,孟静茹,梅其炳.当归多糖硫酸酯的合成及其抗小鼠L6565逆转录病毒的作用.中国新药杂志2006,15(10)：783-786.
[7]孙汉文,许城燕,赵燕燕,梁淑轩.枸杞多糖硫酸酯化修饰及其对Hela细胞的体外抑制作用.河北大学学报(自然科学版),2009,29(6)：591-595.
[8]史宝军,聂小华,许泓渝,陶文沂.灰树花多糖硫酸酯的制备及其抗肿瘤活性.中国医药工业杂志,2003,34(8)：383-385.
[9]Kenned YJF, White CA. Bioactive carbohydrates M. Chichester:Ellis Horwood, Ltd,1993.
[10]吴东儒.糖类的生物化学[M]北京：高等教育出版社,1987
[11]张惟杰.糖复合物生化研究技术[M].杭州：浙江大学出版社,1999
[12]来鲁华,杨显婷.寡糖的构象分析.生物化学与生物物理进展,1995,22(4)：290-294.
[13]Peters T, Meyer B, Stuike-Prill R, Somorjai R, Brisson JR. A Monte Carlo method for conformational analysis of saccharides.Carbohydr Res.1993,238:49-73.
[14]Imberty A, Perez S, Hricovini M, Shah RN, Carver JP. Flexibility in a tetrasaccharide fragment from the high mannose type of N-linked oligosaccharides. Int J Biol Macromol 1993,15:17-23.
[15]Silvestri LJ, Hurst RE, Simpson L,Settine JM. Analysis of sulfate in complex carbohydrates. Anal Biochea.1982,123:303-309.
[16]Dodgson KS, Price RG. A note oil the determination of ester sulfate content of sulfated polysaccharides. J Biochem,1962,84:106-110.
[17]张惠芬,李宝才,范家恒.盐酸水解一硫酸钡重量法测定硫酸酯化多糖硫酸基含量方法考察.食品科学,2002,23(5)：107-111.
[18]张丽萍,汉丽萍,王月秋等.硫酸化高山红景天多糖的制备及鉴定.分子科学学报,1999,15(4)：205-209.
[19]黄小燕,孔祥峰,王德云,胡元亮.多糖硫酸化修饰和多糖硫酸酯的研究.天然产物研究与开发,2007,19：328-332,355
[20]Ghosh P, Adhikari U, Prodyot KG. In vitro anti-herpetic activity of sulfated polysaccharide fraction from caulerpa racemosa. Phytochemistry,2004,65: 3151-3157.
[21]刘玉红,茶藨子木层孔菌多糖及其硫酸化衍生物的制备、结构分析与生物活性研究[D].山东大学,2008.
[22]Katsyara K, Nakashima H, Yamamoto N, Uryu T. Synthesis of sulfated oligosaccharide glycosides having high anti-HIV activity and the relationship between activity and chemical structure.Carbohydr.Res,1999,315:234-242.
[23]魏经建,邵树军,王天元.硫酸酯多糖化学及临床应用研究进展.中国生化药物杂志.1999,20(5)：260-262.
[24]He ZJ, Chen JW, L i X. Advances in studies of sulfated polysaccharide. J Chin Tradit Herb Drugs,2003,21:2087-2088.
[25]Doctor VM, Lewis D, Coleman M. Anticoagulant properties of semisynthetic polysaccharide sulfates. Thromb Res,1991,64:413-425.
[26]Yang TH, Shang P, Mei QB, et al. Effects of angelica polysaccharide and its sulfates on coagulation and platelet aggregation. Chin Tradit Herb Drugs,2002,33: 1010-1013.
[27]Hoshino T, Hayashi T, Hayashi K, et al. An antivirally active sulfated polysaccharide from argassum homeri (TURNER) C. AGARDH. Biol Pharm Bull, 1998,21:730-734.
[28]Beress A, Wassermann O, Tahhan S, et al. A new procedure for the isolation of anti-HIV compound (polysaccharides and polyphenols) from the marine alga fucus vesiculosus. J Nat Prod,1993,56:478-483.
[29]Hayashi K, Hayashi T, Kojima I. A nature sulfated polysaccharide, calcium sp irulan, isolated from spirulina p latensis:in vitro and ex vivo evaluation of anti-herpes simp lex virus and anti-human immunodeficiency virus activities. Aids Res Hum Retroviruses,1996,12:1463-1468.
[30]Xing XL, Ding H, Geng MY, et al. Studies of the anti-AIDS effects of marine polysaccharids drug 911 and its related mechanisms of action. Chin J Marine Drugs, 2000, (6):428-435.
[31]Ono L, Wollinger W, Rocco IM, Coimbra TL, Gorin PA, Sierakowski MR. In vitro and in vivo antiviral properties of sulfated galactomannans against yellow fever virus (BeH111 strain) and dengue virus (Hawaii strain), Antiviral Res,2003,60: 201-208.
[32]Talarico LB, Pujol CA, Zibetti RG, Faria PC, Noseda MD, Duarte ME, Damonte EB. The antiviral activity of sulfated polysaccharides against dengue virus is dependent on virus serotype and host cell, Antiviral Res,2005,66:103-110.
[33]Hidari KI, Takahashi N, Arihara M, Nagaoka M, Morita K, Suzuki T. Structure and anti-dengue virus activity of sulfated polysaccharide from a marine alga. Biochem Biophys Res Commun,2008,376:91-95.
[34]Zhu GJ, Zhao ZF, Fang HY. The relationship between structure and function of polysaccharides. Journal of Wiuxi University of Light Indus,2002,21:209-212.
[35]Tian GY, Li ST, Song ML, et al. Synthesis of achyranthes bidentata polysaccharide sulfate and its antivirus activity. Acta Pharm Sinica,1995,30: 107-111.
[36]An WL, Pu Q, Meng YW. Preparation of sulfated of two polygonatum polysaccharides & their antiviral activities. Nat Prod Res Dev,2000,3:60-65.
[37]Zhang LP, Zhang YS, Sun F, et al. The Influence of sulfation on the conformation and biological activity of polysaccharides from pleurotus citrinop ileatus. Acta Biochimica & Biophysica Sinica,1994,26:417-421.
[38]Chen CY, Hunag XH, Zhou JY, et al.Sulfation of polsaccharides isolated from indocalamus tesselatus and their anticytopathic effect on human immunodeficencv virus type I. Acta Pharm Sinica,1998,33:264-269.
[39]Chen CY, J iang Y. Effects of ruoye polysacchrides and their derivatives on mice infected with LP2BM5MuLV, amurine AIDSmodel. Chine Pharm Bull,1999,8,15: 336-342.
[40]Yang TH, Jia M, Shang P, et al. Synthesis of angelica sinensis polysaccharide sulfate and their effects on splenocyte proliferation in vitro. J Fourth Mil Med Univ 2001,22:432-435.
[41]Wei WQ, Cong JB, Xian H, et al. The effect of polysaccharides from sea weed (SPS) on regulating the immune function in mice. Chin JNew Drugs,2001,10: 671-675.
[42]Wei WQ, Cong JB, Xian H,et al. Inhibitory effects and its mechanism of sulfated polysaccharides from sea weed (SPS) on the lymphocyte apoptosis induced by oxidative stress. Chin Pharm J,2002,37:664-667.
[43]薛静波,刘希英,张鸿芬.海带多糖对小鼠腹腔巨噬细胞的激活作用.中国海洋药物,1999,3：23-25.
[44]Okai, Yasuji. Detection of immunomodulating activities in an extract of Japanese edible seaweed,Laminariaja ponica. J Sci Food Agric,1996,72 (4):455-459.
[45]Zvyagintseva TN, Shevchenko NM, Nazarova IV, Scobun AS, Luk'yanov PA, Elyakova LA, Inhibition of complement activation by water-soluble polysaccharides of some fareastenr brown seaweeds. Comp Biochem Physiol, PartC:Toxicol Pharmacol,2000,126:209-215.
[46]苗本春,耿美玉,李静等.海洋硫酸多糖911免疫增强作用的探讨.中国海洋
药物,2002,5：1-4.
[47]黄益丽,郑忠辉,苏文金.二色桌片参的化学成分研究；Ⅲ二色桌片参多糖一Ⅰ一岩藻聚糖的免疫调节.海洋通报,2001,20(1)：88-91.
[48]陈文星,吴皓,金亦涛.珠蚌多糖的免疫调节作用研究.中药药理与临床,2001,17(5)：16-18.
[49]朱爱民,扬晓峰,李刚.螺旋多糖对小鼠免疫系统的作用.山东医药工业,2001,30(6)：44-45.
[50]陈绍缓,莫卫民,潘远江.海洋药物研究(Ⅲ)一羊栖菜多糖.兰州大学学报(自然科学版),1998,34(4)：110-113.
[51]Bobek V, Kovarik J. Antitumor and antimetastatic effect of warfarin and heparins. Biomed Pharmacother.2004,58:213-219.
[52]朱国臣,肖大江,张永胜,袁渊,吴四海,沈莉莉,郑晓彬.乙酰肝素酶与鼻咽癌侵袭、转移和血管生成的相关性.中国耳鼻咽喉头颈外科,2009,(12)：669-672.
[53]马秀梅,王栓柱,于宏,怀娜.沉默乙酰肝素酶对人胃癌细胞侵袭迁移能力的影响.肿瘤,2009,(10)：924-928.
[54]鄢俊安,宋波,陈志文,宋彩萍.乙酰肝素酶在膀胱移行细胞癌中的表达及与临床特征的关系.第三军医大学学报,2007,(23)：2280-2282.
[55]Iozzo RV. Matrix proteoglycans:from molecular design to cellular function. Annu Rev Biochem.1998,67:609-652.
[56]Wegrowski Y, Maquart FX. Involvement of stromal proteoglycans in tumour progression. Crit Rev Oncol Hematol.2004,49:259-268.
[57]Theocharis AD. Human colon adenocarcinoma is associated with specific post-translational modifications of versican and decorin. Biochim Biophys Acta,2002, 1588:165-172.
[58]Yamada S, Sugahara K. Potential therapeutic application of chondroitin sulfate/dermatan sulfate. Curr Drug Discov Technol.2008,5:289-301.
[59]曲显俊,崔淑香,解砚英.螺旋藻多糖抗癌作用的实验研究.中国海洋药物,2000,4：10-14.
[60]许东晖,许实波,王兵等.皱纹盘鲍多糖抗肿瘤药理作用研究.热带海洋,1999,18(4)：86-90.
[61]刘秋英,孟庆勇,刘志辉.海藻多糖抗肿瘤作用的研究进展.中国海洋药物,2003,4：45-48.
[62]师然新,徐祖洪,李智恩.降解的角叉菜多糖的抗肿瘤活性.海洋与湖泊,2000,31(6)：653-656.
[63]Kumar GP, Sudheesh S, Vijayalakshmi NR. Hypoglycaemic effect of Coccinia indica:mechanism of action. Planta Med.1993,59(4):330-332.
[64]侯建明,蓝进.海洋生物多糖(AC-1)防治高脂血症的实验报告.海军医学杂志2001,22(3)：197-199.
[65]王远红,徐家敏,耿美玉.海洋硫酸多糖916对实验性高脂血症大鼠血浆中含硫氨基酸的影响.中国海洋药物,2000,4：23-24.
[66]王雪松,郑芸,方积年.降血糖多糖及寡糖的研究进展.药学学报,2004,39(12)：1028-1033.
[67]徐中平,李福川,王海仁.昆布多糖硫酸酯的抑制血管生成和抗肿瘤作用.中草药1999,30(7)：551-553.
[68]Liu CH, Li XD, Li YH, Feng Y, Zhou S, Wang FS. Structural characterisation and antimutagenic activity of a novel polysaccharide isolated from Sepiella maindroni ink; Food Chemistry,2008,110:807-813.
[69]Wang J, Wang F, Zhang Q, Zhang Z, Shi X, Li P. Synthesized different derivatives of low molecular fucoidan extracted from Laminaria japonica and their potential antioxidant activity in vitro. Int J Biol Macromol.2009,44:379-384.
[70]Wang J, Zhang Q, Zhang Z, Zhang J, Li P. Synthesized phosphorylated and aminated derivatives of fucoidan and their potential antioxidant activity in vitro. Int J Biol Macromol.2009,44:170-174.
[71]Wang J, Zhang Q, Zhang Z, Li Z. Antioxidant activity of sulfated polysaccharide fractions extracted from Laminaria japonica. Int J Biol Macromol.2008,42:127-132.
[72]Mori H, Kanemura Y, Onaya,J, Hara M, Miyake J, Yamasaki M, Kariya Y. Effects of heparin and its 6-O-and 2-O-desulfated derivatives with low anticoagulant activity on proliferation of human neural stem/progenitor cells. J Biosci Bioeng,2005, 100,54-61.
[73]李绪亮,焦庆才,陈群,刘茜.硫酸化茯苓多糖对大鼠慢性肾功能衰竭的防治作用.中国药学杂志2005,40(12)：908-911.
[74]Mourao PA, Giumaraes B, Mulloy B, Thomas S, Gray E. Antithrombotic activity of a fucosylated chondroitin sulphate from echinoderm:sulphated fucose branches on the polysaccharide account for its antithrombotic action. Br J Haematol.1998,101: 647-652.
[75]Tapon-Bretaudiere J, Chabut D, Zierer M, Matou S, Helley D, Bros A, Mourao, PA, Fischer,AM. A Fucosylated chondroitin sulfate from Echinoderm modulates in Vitro fibroblast growth factor 2-dependent angiogenesis. Mol Cancer Res,2002, 1:96-102.
[76]Nikitovic D, Assouti M, Sifaki M, Katonis P, Krasagakis K, Karamanos NK, Tzanakakis GN. Chondroitin sulfate and heparan sulfate-containing proteoglycans are both partners and targets of basic fibroblast growth factor-mediated proliferation in human metastatic melanoma cell lines. Int JBiochem Cell Biol.2008,40:72-83.
[77]Taylor KR, Rudisill JA, Gallo RL. Structural and sequence motifs in dermatan sulfate for promoting fibroblast growth factor-2 (FGF-2) and FGF-7 activity. J Biol Chem.2005,280:5300-5306.
[78]Wang CY, Guang HS. Advances in studies of antiviral activity of polysaccharide Ⅱ. antiviral activity of sulfated polysaccharide. Advances of Biolagical Project.2000, (2):328-330.
[79]Ashikari-Hada S, Habuchi H, Kariya Y, Kimata K. Heparin regulates vascular endothelial growth factor 165-dependent mitogenic activity, tube formation, and its receptor phosphorylation of human endothelial cells. J Biol Chem.2005,280: 31508-31515.
[80]Li F, Shetty AK, Sugahara K. Neuritogenic activity of chondroitin/dermatan sulfate hybrid chains of embryonic pig brain and their mimicry from shark liver. Involvement of the pleiotrophin and hepatocyte growth factor signaling pathways. J Biol Chem.2007,282:2956-2966.
[81]Alban S, Franz G. Characterization of the anticoagulant actions of a semisynthetic curdlan sulfate. Thromb Res,2000,99:377-388.
[82]GaoY, Fukuda A, Katsuraya K, et al. Synthesis of regioselective substituted curdlan sulfates with medium molecular weights and their specific anti-HIV-1 activities. Macromolecules,1997,30:3224-3228.
[83]Calazans GMT, Lima RC, de Franca FP, Lopes CE. Molecular weight and antitumour activity of Zymomonas mobilis levans. Int J Biol Macromol.2000,12; 27(4):245-247.
[84]石磊,几种多糖的分离纯化、结构解析和生物活性研究[D],山东大学,2007.
[85]顾文莉,硫酸软骨素蛋白多糖在神经前体细胞增殖及其分化细胞中的表达和调控[D],上海交通大学,2007.
[86]Bjorklund A, Lindvall O. Cell replacement therapies for central nervous system disorders. Nat Neurosci,2000,3:537-544.
[87]McBride JL, Behrstock SP, Chen EY et al. Human neural stem cell transplants improve motor function in a rat model of Huntington's disease. J Comp Neurol.2004, 475:211-219.
[88]Sugaya K, Alvarez A, Marutle A, Kwak YD, Choumkina E. Stem cell strategies for Alzheimer's disease therapy. Panminerva Med.2006,48(2):87-96.
[89]Yu D, Silva GA. Stem cell sources and therapeutic approaches for central nervous system and neural retinal disorders. Neurosurg Focus.2008,24(3-4):Ell.
[90]Lee JP, Jeyakumar M, Gonzalez R. Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic disease. Nat Med,2007,13:439-447.
[91]Lim DA, Huang YC, Alvarez-Buylla A. The adult neural stem cell niche:lessons for future neural cell replacement strategies. Neurosurg Clin NAm,2007,18:81-92.
[92]varez-Buylla A, Lim DA. For the long run:maintaining germinal niches in the adult brain. Neuron.2004,41:683-686.
[93]Mercier F, Kitasako JT, Hatton GI. Anatomy of the brain neurogenic zones revisited:fractones and the fibroblast/macrophage network. J Comp Neurol.2002, 451:170-188.
[94]Pitman M, Emery B, Binder M, Wang S, Butzkueven H, Kilpatrick TJ. LIF receptor signaling modulates neural stem cell renewal, Mol Cell Neurosci.2004,27: 255-266.
[95]Kokuzawa J, Yoshimura S, Kitajima H, Shinoda J, Kaku Y, Iwama T, Morishita R, Shimazaki T, Okano H, Kunisada T, Sakai N. Hepatocyte growth factor promotes proliferation and neuronal differentiation of neural stem cells from mouse embryos, Mol Cell Neurosci.2003,24:190-197.
[96]Kosaka M, Kodama H, Sasaki Y, Yamamoto F, Takeshita Y, Takahama H, Sakamoto T, Kato M, Terada T, Ochiya. FGF-4 regulates neural progenitor cell proliferation and neuronal differentiation, FASEB J.2006,20:1484-1485.
[97]Lum M, Croze E, Wagner C, McLenachan S, Mitrovic B, Turnley AM, Inhibition of neurosphere proliferation by IFN-gamma but not IFN-beta is coupled to neuronal differentiation, JNeuroimmunol.2009,206:32-38.
[98]Galderisi U, Cipollaro M, Giordano A. Stem cells and brain cancer. Cell Death Differ.2006,13(1):5-11.
[99]Ida M, Shuo T, Hirano K, Tokita Y, Nakanishi K, Matsui F, Aono S, Fujita H, Fujiwara Y, Kaji T, Oohira A. Identification and functions of chondroitin sulfate in the milieu of neural stem cells. JBiol Chem,2006,281:5982-5991.
[100]Sirko S, von Holst A, Wizenmann A. et al. Chondroitin sulfate glycosaminoglycans control proliferation, radial glia cell differentiation and neurogenesis in neural stem/progenitor cells. Development.2007,134:2727-2738.
[101]Robert K. Yu, Makoto Yanagisawa. Glycobiology of neural stem cells. CNS & Neurological Disorders-Drug Targets,2006,5:415-423.
[102]Akita K, von Holst A, Furukawa Y, Mikami T, Sugahara K, Faissner A. Expression of multiple chondroitin/dermatan sulfotransferases in the neurogenic regions of the embryonic and adult CNS imply that complex chondroitin sulfates have a role in neural stem cell maintenance. Stem Cells,2008,26:798-809
[103]Sugahara K, Mikami T. Chondroitin/dermatan sulfate in the central nervous system. Curr Opin Struct Biol,2007,17:536-545.
[104]Kabos P, Matundan H, Zandian M, Bertolotto C, Robinson ML, Davy BE, Yu JS, Krueger RC Jr. Neural precursors express multiple chondroitin sulfate proteoglycans, including the lectican family.Biochem Biophys Res Commun.2004; 318(4):955-963.
[105]Zou P, Muramatsu H, Miyata T, Muramatsu T. Midkine, a heparin-binding growth factor, is expressed in neural precursor cells and promotes their growth. J. Neurochem.2006,9:1470-1479.
[106]Brickman YG, Ford MD, Small DH, Bartlett PF, Nurcombe V. Heparan sulfates mediate the binding of basic fibroblast growth factor to a specific receptor on neural precursor cells. JBiol Chem,1995,270,24941-24948.
[107]Nurcombe V, Ford MD, Wildschut JA. Bartlett PF. Developmental regulation of neural response to FGF-1 and FGF-2 by heparan sulfate proteoglycan. Science,1993, 260:103-106.
[108]Hagihara K, Watanabe K, Chun J. Yamaguchi Y. Glypican-4 is an FGF2-binding heparan sulfate proteoglycan expressed in neural precursor cells. Dev Dyn,2000,219: 353-367.
[109]Caldwell MA, Svendsen CN. Heparin, but not other proteoglycans potentiates the mitogenic effects of FGF-2 on mesencephalic precursor cells. Exp Neurol. 1998;152(1):1-10.
[110]Landolt RM, Vaughan L, Winterhalter KH, Zimmermann DR. Versican is selectively expressed in embryonic tissues that act as barriers to neural crest cell migration and axon outgrowth. Development,1995,121,2303-2312.
[111]Gu WL, Fu SL, Wang YX, Li Y, Lu HZ, Xu XM, Lu PH. Chondroitin sulfate proteoglycans regulate the growth, differentiation and migration of multipotent neural precursor cells through the integrin signaling pathway. BMC Neurosci,2009,10: 128(1-15).
[112]Kearns SM, Laywell ED, Kukekov VK, Steindler DA. Extracellular matrix effects on neurosphere cell motility. Exp Neurol.2003,182:240-244.
[113]Nagayasu T, Miyata S, Hayashi N, Takano R, Kariya Y, Kamei K. Heparin structures in FGF-2-dependent morphological transformation of astrocytes. J Biomed Mater Res A.2005,74(3):374-380.
[114]Yamada T, Sawada R, Tsuchiya T. The effect of sulfated hyaluronan on the morphological transformation and activity of cultured human astrocytes. Biomaterials. 2008,29 (26):3503-3513.
[115]Ahmed S, Tsuchiya T, Nagahata-Ishiguro M, Sawada R, Banu N, Nagira T. Enhancing action by sulfated hyaluronan on connexin-26,-32, and-43 gene expressions during the culture of normal human astrocytes. J Biomed Mater Res A, 2009,90 (3):713-719.
[116]Inatani M, Irie F, Plump AS, Tessier-Lavigne M. Yamaguchi Y. Mammalian brain morphogenesis and midline axon guidance require heparan sulfate. Science, 2003,302:1044-1046.
[117]Ai X, Kitazawa T, Do AT, Kusche-Gullberg M, Labosky PA, Emerson CP Jr. SULF1 and SULF2 regulate heparan sulfate-mediated GDNF signaling for esophageal innervation. Development,2007,134:3327-3338.
[118]Casu B. Structure and biological activity of heparin. Adv Carbohydr Chem Biochem,1985; 43:51-134.
[119]Comper D. Heparin and related polysaccharides. Vol.7, Gordan and Breach, 1981; 41:158-172.
[120]Rosenberg D, L Lam. Correlation between structure and function of heparin, Proc Natl Acad Sci USA,1979,76:1218-1222.
[121]Cardin AD, Weintraub HJR. Molecular modeling of protein-glycosaminoglycan interactions. Arteriosclerosis,1989,9:21-32.
[122]Rosenberg D, Schworak NW, Liu J.Schwartz J J, Zhang L, Molecular cloning and expression of mouse and human cDNAs encoding heparan sulfate d-Glucosaminyl 3-O-Sulfotransferase. JClin Invest,1997,99,2062-2070.
[123]Walker A, Turnbull JE, Gallagher JT. Specific heparan sulphate saccharides mediate the activity of basic fibroblast growth factor, JBiol Chem,1994,269:931-935.
[124]Wang HM, ToidaT, KimYS, Capila I, Hileman RE, Bernfield M, Linhardt RJ. Glycosaminoglycans can influence fibroblast growth factor-2 mitogenicity without significant growth factor binding, Biochem Biophys Res Commun,1997; 235, 369-373.
[125]Coombe DR, Kett WC. Heparan sulfate-protein interactions:therapeutic potential through structurefunction insights. Cell Mol Life Sci,2005,62:410-424.
[126]Shikari S, Habuchi H, Kimata K. Characterization of heparan sulfate oligosaccharides that bind to hepatocyte growth factor. J Biol Chem,1995,270, 29586-29593.
[127]Gordon MY, Riley G P, Watt SM, Greaves M F. Compartmentalization of a haematopoietic growth factor (GM-CSF) by glycosaminoglycans in the bone marrow microenvironment. Nature,1987,326,403-405.
[128]Roberts R, Gallagher J, Spooncer E, Allen T D, Bloomfield F, Dexter TM. Heparan sulphate bound growth factors:a mechanism for stromal cell mediated haemopoiesis. Nature,1988,332,376-378.
[129]Takada T, Katagiri T, Ifuku M, Morimura N, Kobayashi M, Hasegawa K, Ogamo A, Kamijo, R. Sulfated polysaccharides enhance the biological activities of bone morphogeneticproteins. J Biol Chem.2003.278,43229-43235.
[130]Lakshmi TS, Shanmugasundaram N, Shanmuganathan S, Karthikeyan K, Meenakshi J, Babu M. Controlled release of 2,3 desulfated heparin exerts its anti-inflammatory activity by effectively inhibiting E-selectin. J Biomed Mater Res A. 2010,95(1):118-128.
[131]Perez-Pinera P, Chang Y, Deuel TF. Pleiotrophin, a multifunctional tumor promoter through induction of tumor angiogenesis, remodeling of the tumor microenvironment, and activation of stromal fibroblasts. Cell Cycle.2007; 6 (23):2877-2883.
[132]Deakin JA, Lyon M. Differential regulation of hepatocyte growth factor/scatter factor by cell surface proteoglycans and free glycosaminoglycan chains. J Cell Sci, 1999; 112. (Ptl2):1999-2009.
[133]樊绘曾.海参：海中人参：关于海参及其成分保健医疗功能的研究与开发.中国海洋药物,2001,20(4)：37-44.
[134]姜健,杨宝灵,邰阳.海参资源及生物活性物质的研究.生物技术通讯2004,15(5)：537-540.
[135]樊绘曾,陈菊,吕培宏.玉足海参酸性多糖的研究.药学学报,1983,18：2031-2032.
[136]Bulgakov AA, Nazarenko EL, Petrova IY, Eliseikina MG, Vakhrusheva NM, Zubkov VA. Isolation and properties of a mannan-binding lectin from the coelomic fluid of the holothurian Cucumaria japonica. Biochemistry (Mosc).2000; 65 (8): 933-939.
[137]Ribeiro AC,Vieira RP, Mourao PA. Mulloy B. A sulfated alpha-L-fucan from sea cucumber. Carbohydr Res,1994,255:225-240.
[138]Kariya Y, Watabe S, Kyogashima M. Structure of fucose branches in the glycosam inoglycan from the body wall of the sea cucumber, Stichopus japonicu. Carbohydr Res,1997,297:273-278.
[139]盛文静,薛长湖,赵庆喜,徐杰,李兆杰,孙妍.不同海参多糖的化学组成分析比较.中国海洋药物杂志2007,26：44-49.
[140]Li JZ, Bao CX, Chen GZ. Antithrombin activity and platelet aggregation by acid mucopo lysaccharides isolated from stichopusj aponicus selenka.China. Acta Pharma cologica Sinica,1985,6:107-112.
[141]王学锋,李志广,储海燕.海参糖胺聚糖抗血栓形成机制的研究.中国新药与临床杂志2002,21(12)：718-721.
[142]杨晓光,陈关珍,罗晓玲.Sjamp对纤溶系统影响的初步观察.中国医学科学院学报1990,2：1871-1873
[143]Fonseca RJ, Mourao PA. Fucosylated chondroitin sulfate as a new oral antithrombotic agent. Thromb Haemost.2006,96:822-829.
[144]Mourao PA, Pereira MS, Pavao MS, Mulloy B, Tollefsen DM, Mowinckel MC, Abildgaard U. Structure and anticoagulant activity of a fucosylated chondroitin sulfate from echinoderm. Sulfated fucose branches on the polysaccharide account for its high anticoagulant action. JBiol Chem.1996,271 (39):23973-23984.
[145]陈玲,于壮,宋扬,张笑雪,李明君.刺参黏多糖对人宫颈癌细胞凋亡的影响.齐鲁医学杂志,2009,24：95-98.
[146]王静凤,王奕,赵林,逢龙,薛长湖.日本刺参的抗肿瘤及免疫调节作用研究.中国海洋大学学报,2007,37：93-96.
[147]王强基,李春艳.玉足海参酸性粘多糖对小鼠免疫功能的影响.海洋药物,1984,12-14.
[148]黄益利,郑忠辉,苏文金等.二色桌片参的化学成分研究-二色桌片参多糖的免疫调节作用.全国第三届海津生命活性物质与天热生化药物学术讨论会论文集,海南,2000：129.
[149]李艳菊,刘辉,郭月秋.海参免疫调节作用的实验研究.中国海洋药物杂志2006,25：49-50.
[150]纪静,王笑峰,马忠兵,罗兵.现刺参糖胺聚糖抗仙台病毒作用机制的探讨.现代生物医学进展2009,9：1060-1063.
[151]罗兵,马忠兵,王笑峰,王云.刺参糖胺聚糖对单纯疱疹病毒Ⅰ型抑制作用的实验研究.中国海洋药物杂志,2008,27：44-48.
[152]高森,王静风,王玉明,刘治东,李晓林,薛长湖.冰岛刺参调节血脂及其作用机制.武汉大学学报(理学版),2009,55：324-328.
[153]王静风,逢龙,黄平,赵芹,薛长湖.北极刺参和日本刺参对大鼠血脂水平调节和血管内皮保护作用的研究.中国海洋药物杂志2007,26：10-13.
[154]胡晓倩,王玉明,任兵兴,常耀光,王静凤,薛长湖.海参主要活性成分对大鼠脂质代谢影响的比较研究.食品科学,2009,30：393-396.
[155]王静凤,逢龙,薛勇,董平,盛文静,薛长湖.日本刺参酸性粘多糖、皂苷和胶原蛋白多肽对血管内皮细胞的保护作用.中国药理学通报2008,24：227-232.
[156]逄龙,王静凤,王玉明,盛文静,薛长湖.北极刺参多糖、皂苷和胶原蛋白多肽对血管内皮细胞的保护作用.中国药科大学学报2007,38：437-441.
[157]朱宗涛,蔡生业,姚成芳,王丽,王华亭,张维东.复方花刺参粘多糖对髂动脉内皮剥脱家兔内膜增生的影响及机制.中国动脉硬化杂志2004,12：43-46.
[158]王华亭,蔡生业,姚成芳,朱宗涛,王丽,张维东.复方花刺参粘多糖对家兔血管成形术后内皮功能及超微结构的影响.中国动脉硬化杂志,2004,5：497-501.
[159]王曼玲,徐建民.海参糖胺聚糖抑制血小板-内皮细胞粘附及调节血小板粘附分子表达.中国临床医学,2006,13：431-433.
[160]王利民,蔡生业,姚成芳,王丽,周宪宾,王华亭,王恒孝.花刺参粘多糖对大鼠血管平滑肌细胞粘附分子表达的影响.中国动脉硬化杂志,2006,14：565-568.
[161]Tapon-Bretaudiere J, Drouet B, Matou S, Mourao PA, Bros A, Letourneur D, Fischer AM. Modulation of vascular human endothelial and rat smooth muscle cell growth by a fucosylated chondroitin sulfate from echinoderm. Thromb Haemost.2000, 84:332-337.
[162]Kariya Y, Mulloy B, Imai K, Tominaga A, Kaneko T, Asari, Suzuki K, Masuda H, Kyogashima, Ishii T. Isolation and partial characterization of fucan sulfates from the body wall of sea cucumber Stichopus japonicus and their ability to inhibit osteoclastogenesis. Carbohydr Res,2004,339:1339-1346.
[163]迟玉森,庄桂东,黄福祥,安桂香,徐鹏.海参多糖对小白鼠伤口愈合的影响.食品科学,2005,26：211-214.
[164]邱鹏新,黎明涛,唐孝礼,苏兴文,林穗珍,颜光美.黑海参多糖对β-淀粉样蛋白诱导的皮质神经元凋亡的保护作用.中草药2000,31：271-274.
[165]Borsig L, Wang L, Cavalcante MC, Cardilo-Reis L, Ferreira PL, Mourao PA, Esko JD, Pavao MS. Selectin blocking activity of a fucosylated chondroitin sulfate glycosaminoglycan from sea cucumber. Effect on tumor metastasis and neutrophil recruitment. JBiol Chem.2007,282 (20):14984-14991.
[166]Landeira-Fernandez AM, Aiello KR, Aquino RS, Silva LC, Meis L, Mourao PA. A sulfated polysaccharide from the sarcoplasmic reticulum of sea cucumber smooth muscle is an endogenous inhibitor of the Ca(2+)-ATPase. Glycobiology.2000,10: 773-779.
[167]朱玉强.刺参多糖急性毒理研究及其安全性评价.四川食品与发酵,2005,41：48-51.
[168]段君.刺参酸性粘多糖的提取及其性质与结构分析[D],2003.
[169]方积年.天然药物-多糖的主要生物活性及分离纯化方法.中国天然药物,2007,5：338-347.
[170]唐孝礼,邱鹏新,黎明涛,苏兴文,颜光美.黑海参酸性粘多糖的分离纯化.中药材,1999,22：223-225.
[171]郑艾初,陈健,彭超英.糙海参酸性粘多糖的提取纯化工艺探讨.现代食品科技2007,23：65-67.
[172]Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem,1956,28:350-356.
[173]Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Chem,1976, 72:248-254.
[174]Filisetti-Cozzi, TMCC, Carpita, NC. Measurement of uronic acids without interference from neutral sugars. Analytical Biochemistry,1991,197:157-162.
[175]Saito H, Yamagata T, Suzuki S. Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates. J Biol Chem,1968,242:1536-1542.
[176]Takeda M, Nomoto S, Koizumi J. Structural analysis of the extracellular polysaccharide produced by Sphaerotilus natans. Biosci Biotechnol Biochem.2002, 66(7):1546-1551.
[177]Matsuhiro, B, Zuniga, E, Jashesl, M, Guacucanol M. Sulfated polysaccharides from Durvillaea antarctica. Hydrobiologia,1996,321:77-81.
[178]Caceres PJ, Faundez CA, Matsuhiro B, Vasquez JA J. Carrageenophyte identification by second-derivative Fourier transform infrared spectroscopy. Journal of Applied Phycology,1997,8:523-527.
[179]Barker SA, Bourne EJ, Stacey M, et al. Infra-red spectra of carbohydrates. Part Ⅰ. Some derivatives of d-glucopyranose. J Chem Soci,1954,6:34-39.
[180]陈菊娣,樊绘曾,刑蕊凝等.花刺参酸性粘多糖的分离研究.中国海洋药物,1994,1：24-26.
[181]Mourao PA, Bastos IG. Highly acidic glycans from sea cucumbers. Isolation and fractionation of fucose-rich sulfated polysaccharides from the body wall of Ludwigothurea grisea.Eur J Biochem,1987,166:639-645.
[182]Zhao J, Wu M, Kang H, Zeng W, Liang H, Li Z. Low molecular weight fractions and preparation of a fucosylated glycosaminoglycan from sea cucumber. CN patent,200910110114.0.
[183]王玲,陈健,姜建国,郑艾初.沙海参多糖的分离和特性研究.现代食品科技,2008,24：655-657.
[184]Lee JP, Jeyakumar M, Gonzalez R. Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic disease. Nat Med 2007,13 (4): 439-447.
[185]Lim DA, Huang YC, Alvarez-Buylla A. The adult neural stem cell niche: lessons for future neural cell replacement strategies. Neurosurg Clin NAm 2007,18 (1):81-92.
[186]Ahmed S. The culture of neural stem cells, J Cell Biochem.2009,106:1-6.
[187]Coles-Takabe BL, Brain I, Purpura KA, Karpowicz P, Zandstra PW, Morshead CM, van der Kooy D. Don't look:growing clonal versus nonclonal neural stem cell colonies. Stem Cells,2008,26 (11):2938-2944.
[188]蔡文琴.发育神经生物学[M],北京：科学出版社,2002：223.
[189]Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system, Science,1992,255:1707-1710.
[190]Malavaki C, Mizumoto S, Karamanos N, Sugahara K. Recent advances in the structural study of functional chondroitin sulfate and dermatan sulfate in health and disease. Connect Tissue Res,2008,49 (3):133-139.
[191]Jiang J, Chen X, Shen J, Wei Y, Wu T, Yang Y, Wang H, Zong H, Yang J, Zhang S, Xie J, Kong X, Liu W, Gu J. Beta1,4-galactosyltransferase V functions as a positive growth regulator in glioma. JBiol Chem,2006,281:9482-9489.
[192]杨雪松.岩藻糖基转移酶Ⅳ对A431细胞增殖与凋亡的影响[D].2007.
[193]王秋雁.α-1,3-岩藻糖转移酶-Ⅶ对人肝癌细胞信号转导、增殖和凋亡的影响[D].2004.
[194]Fawcett J. Repair of spinal cord injuries:where are we, where are we going? Spinal Cord.2002,40:615-623.
[195]McLenachan S, Lum MG, Waters MJ, Turnley AM. Growth hormone promotes proliferation of adult neurosphere cultures. Growth Horm IGF Res.2009,19 (3): 212-218.
[196]Fonseca RJ, Mourao PA. Fucosylated chondroitin sulfate as a new oral antithrombotic agent. Thromb Haemost.2006,96(6):822-829.
[197]Sweeney EA, Priestley GV, Nakamoto B, Collins RG, Beaudet AL, Papayannopoulou T. Mobilization of stem/progenitor cells by sulfated polysaccharides does not require selectin presence, Proc Natl Acad Sci USA,2000,97: 6544-6549.
[198]Matou S, Helley D, Chabut D, Bros A, Fischer AM. Effect of fucoidan on fibroblast growth factor-2-induced angiogeriesis in vitro. Thromb Res,2002,106: 213-221.
[199]Islam MO, Kanemura Y, Tajria J, Mori H, Kobayashi S, Shofuda T, Miyake J, Hara M, Yamasaki M, Okano H. Characterization of ABC transporter ABCB1 expressed in human neural stem/progenitor cells. FEBS Lett,2005,579:3473-3480.
[200]Wang TY, Sen A, Behie LA, Kallos MS, Dynamic behavior of cells within neurospheres in expanding populations of neural precursors, Brain Res.2006,1107: 82-96.
[201]郑志竑,胡建石,陈文列,林玲,钟秀容,林建银.体外培养的神经干细胞球的超微结构.解剖学报,2003,34(6)：615-619.
[202]Mori H, Ninomiya K, Kino-oka M, Shofuda T, Islam MO, Yamasaki M, Okano H, Taya M, Kanemura Y. Effect of neurosphere size on the growth rate of human neural stem/progenitor cells. JNeurosci Res,2006,84:1682-1691.
[203]Widera D, Mikenberg I, Kaus A, Kaltschmidt C, Kaltschmidt B, Nuclear Factor-kappaB controls the reaggregation of 3D neurosphere cultures in vitro, Eur Cell Mater.2006,11:76-84.
[204]Widera D, Mikenberg I, Elvers M, Kaltschmidt C, Kaltschmidt B. Tumor necrosis factor alpha triggers proliferation of adult neural stem cells via IKK/NF-kappaB signaling, BMC Neurosci,2006,7:64-82.
[205]Campos LS, Leone DP, Relvas JB, Brakebusch C, Fassler R, Suter U, ffrench-Constant C. Betal integrins activate a MAPK signalling pathway in neuralstem cells that contributes to their maintenance. Development,2004,131(14): 3433-3444.
[206]Kimura A, Ohmori T, Ohkawa R, Madoiwa S, Mimuro J, Murakami T, Kobayashi E, Hoshino Y, Yatomi Y, Sakata Y. Essential roles of sphingosine 1-phosphate/S1P1 receptor axis in the migration of neural stem cells toward a site of spinal cord injury. Stem Cells.2007,25:115-124.
[207]Mori H, Fujitani T, Kanemura Y, Kino-Oka M, Taya M. Observational examination of aggregation and migration during early phase of neurosphere culture of mouse neural stem cells. J Biosci Bioeng,2007,104(3):231-234.
[208]Karin M, Lin A. NF-κB at the crossroads of life and death, Nat Immunol,2002, 3:221-227.
[209]Goldschmidt B, Wider D, Goldschmidt C. Signaling via NF-κB in the nervous system, Biochim Biophys Acta,2005,1745:287-299.
[210]Raballo R, Rhee J, Lyn-Cook R, Leckman JF, Schwartz ML, Vaccarino FM. Basic fibroblast growth factor (Fgf2) is necessary for cell proliferation and neurogenesis in the developing cerebral cortex, JNeurosci,2000,20:5012-5023.
[211]Qian X, Davis AA, Goderie SK, Temple S. FGF2 concentration regulates the generation of neurons and glia from multipotent cortical stem cells, Neuron,1997,18: 81-93.
[212]Sun L, Lee J, Fine HA. Neuronally expressed stem cell factor induces neural stem cell migration to areas of brain injury, JClin Invest,2004,113:1364-1374.
[213]Bell HS, Whittle IR, Walker M, Leaver HA, Wharton SB. The development of necrosis and apoptosis in glioma:experimental findings using spheroid culture systems. Neuropathol Appl Neurobiol,2001,27:291-304.
[214]Carlsson J, Acker H. Relations between pH, oxygen partial pressure and growth in cultured cell spheroids. Int J Cancer,1988,42:715-720.
[215]Glicklis R, Merchuk JC, Cohen S. Modeling mass transfer in hepatocyte spheroids via cell viability, spheroid size, and hepatocellular functions. Biotechnol Bioeng,2004,86:672-680.