刺参糖复合物的分离纯化及其对神经前体细胞作用的初步研究
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摘要
刺参(Stichopus japonicus)是我国北方唯一的食用海参，主要分布在渤海湾、南至江苏连云港等周边海域。目前对于刺参多糖及其复合物药理活性的研究主要集中于抗凝血、增强免疫力、抗肿瘤等方面，关于刺参多糖及其复合物对原代培养的神经前体细胞活性方面，尤其是诱导其去分化方面的报道还较少见。另外多数研究是以干刺参为原材料，从中提取粗多糖或者进行初步的纯化，而本实验则采用鲜活刺参为原材料，从中提取并纯化了糖复合物，这在国内外文献报道中也是较少出现的。
本文以刺参粗提物为材料，研究了它对原代培养的胎鼠神经前体细胞的作用，根据结果推测刺参粗提物在神经前体细胞贴壁后可以诱导其发生去分化。为了确定是何种活性物质在起作用，以便进一步深入地研究这种活性物质及其活性，我们对刺参粗提物进行了分离纯化和各组分活性跟踪，得到了HS-1和HS-2两个糖复合物活性组分，并对其结构和活性两方面进行了初步研究。本文的主要研究内容如下：
1．活性组分的提取和分离纯化。采用双酶解然后醇沉的方法得到刺参粗提物，用大孔吸附树脂柱脱色，然后用DEAE-Sephrose柱进行第一次分离纯化，用氯化钠溶液进行线性洗脱，紫外210nm、280nm结合苯酚硫酸法监测流出的各组分，得到A、B、C、D四个糖复合物组分，然后将其中有活性的组分(组分C、D)分别用Superdex 200柱进行第二次分离纯化，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，冷冻干燥，再分别利用Sephadex G-25柱脱盐，冷冻干燥后进行活性检测，得到两个糖复合物活性组分：HS-1和HS-2。
2．刺参糖复合物HS-1和HS-2的初步结构分析。对HS-1、HS-2进行紫外光谱全波长扫描和Superdex 200柱层析分析，结果表明二者均为均一组分；利用硫酸钡沉淀的原理对HS-1和HS-2进行了硫酸基的定性鉴别，结果表明二者均含有硫酸基；通过苯酚硫酸法测得HS-1、HS-2的糖含量分别为63.22％和58.90％，硫酸咔唑法测得HS-1、HS-2的己糖醛酸含量分别为38.55％和32.29％，考马斯亮蓝法测得HS-1、HS-2中的蛋白含量分别为1.19％和1.69％；采用凝胶色谱法测得HS-1、HS-2的分子量分别为631 000Dal和707 900Dal；采用簿层层析和高效液相色谱法测定了HS-1的单糖组成及摩尔比，结果表明酸解后的HS-1中含有大量岩藻糖和少量半乳糖等其他单糖，岩藻糖和半乳糖的摩尔比为14.29：1；还对HS-1进行了红外光谱和核磁共振分析，根据结果推知HS-1的糖苷键可能主要为β-型，且HS-1为硫酸化的糖复合物。
3．刺参糖复合物HS-1和HS-2对原代培养胎鼠神经前体细胞的作用的初步研究。在原代培养的神经前体细胞贴壁后向其中加入HS-1、HS-2，若干小时后细胞团便呈现出聚团现象，且细胞团之间有较粗的连接，随着培养时间的延长，细胞团变得大而致密。浓度梯度试验结果表明这种聚团现象具有剂量依赖性，随着刺参糖复合物浓度的增高，聚团所需的时间逐渐缩短。免疫荧光染色结果显示贴壁后的神经前体细胞已经逐渐分化为神经元，但HS-1、HS-2作用10d后的细胞早Nestin阳性和NSE弱阳性，即表现出神经干细胞的特征，而此时正常对照组和NGF阳性对照组则表现为Nestin阴性和NSE阳性，即仍然为神经元。而且台盼蓝染色结果显示实验组细胞团绝大部分细胞仍为活细胞，因此我们初步推测，在神经前体细胞贴壁后，刺参糖复合物HS-1和HS-2具有诱导其去分化的作用。另外，利用MTT法检测发现HS-1、HS-2在较低浓度时具有与NGF相似的提高神经前体细胞存活率的作用。
As the only edible sea cucumber in north of China, Stichopus japonicus (selenka) mainly distributed over Bohai Sea estuary and sea area of Lianyungang City. There are many reports about the pharmacological effects of Stichopus japonicus polysaccharide and glycoconjugate, such as anticoagulant, immunity enhance, anticancer activity, etc. But there is few report about the effect of Stichopus japonicus glycoconjugate on neural precursor cells, especially about the dedifferentiation. Besides, raw polysaccharide or preliminary depurated polysaccharide extracted from dried Stichopus japonicus rather than homogeneous glycoconjugate extracted from fresh Stichopus japonicus was used in previously research.
In this paper, we obtained two homogeneous Stichopus japonicus glycoconjugates. We studied the structure and their pharmacological effects on neural precursor cells preliminarily. The main contents of the papers are as follows:
1. The extraction, separation and purification of raw Stichopus japonicus extract. Hydrolyzed by double-enzyme, the raw extract was precipitated by ethanol. After decolored by macroporous adsorptive resins, the polysaccharides was first separated by DEAE-Sepharose column. Monitored by 210nm, 280nm ultraviolet light and phenol-sulphuric acid colorimetric method, four main components named A, B, C, D were obtained. Only C and D have the dedifferentiation effects on neural precursor cells. So C and D were then separated by Superdex 200 column separately. We collected every main components, and they were desalted by Sephadex G-25 column. After screening according to their dedifferentiation effects on neural precursor cells, we got two fine purification Stichopus japonicus glycoconjugates named HS-1 and HS-2.
2. The study of the structure of HS-1 and HS-2. UV spectrum and Superdex 200 column chromatography showed that the purified Stichopus japonicus glycoconjugates (HS-1 and HS-2) were homogeneous; the form of Barium Sulfate showed that HS-1 and HS-2 both were sulfated; saccharides contents of HS-1 and HS-2 determined by phenol-sulphuric acid colorimetric method were 63.22% and 58.90% separately; uronic acides contents of HS-1 and HS-2 determined by sulphutic acid carbazole reaction were 38.55% and 32.29% separately; protein contents of HS-1 and HS-2 determined by Bradford method were 1.19% and 1.69% separately: the molecular weight of HS-1 and HS-2 determined by Gel Permeation Chromatography were 631 000Dal and 707 900Dal separately; the results of TLC and HPLC showed that the polysaccharide part of HS-1 was mainly composed of fucose and some other monosaccharide, and the mol ratio of fucose and galactose was 14.29:1; the results of IR and NMR showed that the glucosidicbond configuration of HS-1 was mainlyβ-configuration and HS-1 was a sulfated glycoconjugate.
3. The study of pharmacological effects of the purified Stichopus japonicus glycoconjugate. We found that HS-1 and HS-2 can aggregate adherenced neural precursor cells. And the higher the Stichopus japonicus glycoconjugate concentrations are, the less the time needed for aggregating. The results of immunofluorescence staining showed that cells aggregated together were Nestin positive and NSE lepto-positive while normal cells were Nestin negative and NSE positive. The result of Trypan blue method showed that most aggregated cells were still alive. According to aforesaid results, we presumed that HS-1 and HS-2 can induce the dedifferentiation of adherenced neural precursor cells. Besides, MTT method results showed that cells in low concentration Stichopus japonicus glycoconjugate experimental group had similar survival rate with that of cells in low concentration NGF experimental group.
本文以刺参粗提物为材料，研究了它对原代培养的胎鼠神经前体细胞的作用，根据结果推测刺参粗提物在神经前体细胞贴壁后可以诱导其发生去分化。为了确定是何种活性物质在起作用，以便进一步深入地研究这种活性物质及其活性，我们对刺参粗提物进行了分离纯化和各组分活性跟踪，得到了HS-1和HS-2两个糖复合物活性组分，并对其结构和活性两方面进行了初步研究。本文的主要研究内容如下：
1．活性组分的提取和分离纯化。采用双酶解然后醇沉的方法得到刺参粗提物，用大孔吸附树脂柱脱色，然后用DEAE-Sephrose柱进行第一次分离纯化，用氯化钠溶液进行线性洗脱，紫外210nm、280nm结合苯酚硫酸法监测流出的各组分，得到A、B、C、D四个糖复合物组分，然后将其中有活性的组分(组分C、D)分别用Superdex 200柱进行第二次分离纯化，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，冷冻干燥，再分别利用Sephadex G-25柱脱盐，冷冻干燥后进行活性检测，得到两个糖复合物活性组分：HS-1和HS-2。
2．刺参糖复合物HS-1和HS-2的初步结构分析。对HS-1、HS-2进行紫外光谱全波长扫描和Superdex 200柱层析分析，结果表明二者均为均一组分；利用硫酸钡沉淀的原理对HS-1和HS-2进行了硫酸基的定性鉴别，结果表明二者均含有硫酸基；通过苯酚硫酸法测得HS-1、HS-2的糖含量分别为63.22％和58.90％，硫酸咔唑法测得HS-1、HS-2的己糖醛酸含量分别为38.55％和32.29％，考马斯亮蓝法测得HS-1、HS-2中的蛋白含量分别为1.19％和1.69％；采用凝胶色谱法测得HS-1、HS-2的分子量分别为631 000Dal和707 900Dal；采用簿层层析和高效液相色谱法测定了HS-1的单糖组成及摩尔比，结果表明酸解后的HS-1中含有大量岩藻糖和少量半乳糖等其他单糖，岩藻糖和半乳糖的摩尔比为14.29：1；还对HS-1进行了红外光谱和核磁共振分析，根据结果推知HS-1的糖苷键可能主要为β-型，且HS-1为硫酸化的糖复合物。
3．刺参糖复合物HS-1和HS-2对原代培养胎鼠神经前体细胞的作用的初步研究。在原代培养的神经前体细胞贴壁后向其中加入HS-1、HS-2，若干小时后细胞团便呈现出聚团现象，且细胞团之间有较粗的连接，随着培养时间的延长，细胞团变得大而致密。浓度梯度试验结果表明这种聚团现象具有剂量依赖性，随着刺参糖复合物浓度的增高，聚团所需的时间逐渐缩短。免疫荧光染色结果显示贴壁后的神经前体细胞已经逐渐分化为神经元，但HS-1、HS-2作用10d后的细胞早Nestin阳性和NSE弱阳性，即表现出神经干细胞的特征，而此时正常对照组和NGF阳性对照组则表现为Nestin阴性和NSE阳性，即仍然为神经元。而且台盼蓝染色结果显示实验组细胞团绝大部分细胞仍为活细胞，因此我们初步推测，在神经前体细胞贴壁后，刺参糖复合物HS-1和HS-2具有诱导其去分化的作用。另外，利用MTT法检测发现HS-1、HS-2在较低浓度时具有与NGF相似的提高神经前体细胞存活率的作用。
As the only edible sea cucumber in north of China, Stichopus japonicus (selenka) mainly distributed over Bohai Sea estuary and sea area of Lianyungang City. There are many reports about the pharmacological effects of Stichopus japonicus polysaccharide and glycoconjugate, such as anticoagulant, immunity enhance, anticancer activity, etc. But there is few report about the effect of Stichopus japonicus glycoconjugate on neural precursor cells, especially about the dedifferentiation. Besides, raw polysaccharide or preliminary depurated polysaccharide extracted from dried Stichopus japonicus rather than homogeneous glycoconjugate extracted from fresh Stichopus japonicus was used in previously research.
In this paper, we obtained two homogeneous Stichopus japonicus glycoconjugates. We studied the structure and their pharmacological effects on neural precursor cells preliminarily. The main contents of the papers are as follows:
1. The extraction, separation and purification of raw Stichopus japonicus extract. Hydrolyzed by double-enzyme, the raw extract was precipitated by ethanol. After decolored by macroporous adsorptive resins, the polysaccharides was first separated by DEAE-Sepharose column. Monitored by 210nm, 280nm ultraviolet light and phenol-sulphuric acid colorimetric method, four main components named A, B, C, D were obtained. Only C and D have the dedifferentiation effects on neural precursor cells. So C and D were then separated by Superdex 200 column separately. We collected every main components, and they were desalted by Sephadex G-25 column. After screening according to their dedifferentiation effects on neural precursor cells, we got two fine purification Stichopus japonicus glycoconjugates named HS-1 and HS-2.
2. The study of the structure of HS-1 and HS-2. UV spectrum and Superdex 200 column chromatography showed that the purified Stichopus japonicus glycoconjugates (HS-1 and HS-2) were homogeneous; the form of Barium Sulfate showed that HS-1 and HS-2 both were sulfated; saccharides contents of HS-1 and HS-2 determined by phenol-sulphuric acid colorimetric method were 63.22% and 58.90% separately; uronic acides contents of HS-1 and HS-2 determined by sulphutic acid carbazole reaction were 38.55% and 32.29% separately; protein contents of HS-1 and HS-2 determined by Bradford method were 1.19% and 1.69% separately: the molecular weight of HS-1 and HS-2 determined by Gel Permeation Chromatography were 631 000Dal and 707 900Dal separately; the results of TLC and HPLC showed that the polysaccharide part of HS-1 was mainly composed of fucose and some other monosaccharide, and the mol ratio of fucose and galactose was 14.29:1; the results of IR and NMR showed that the glucosidicbond configuration of HS-1 was mainlyβ-configuration and HS-1 was a sulfated glycoconjugate.
3. The study of pharmacological effects of the purified Stichopus japonicus glycoconjugate. We found that HS-1 and HS-2 can aggregate adherenced neural precursor cells. And the higher the Stichopus japonicus glycoconjugate concentrations are, the less the time needed for aggregating. The results of immunofluorescence staining showed that cells aggregated together were Nestin positive and NSE lepto-positive while normal cells were Nestin negative and NSE positive. The result of Trypan blue method showed that most aggregated cells were still alive. According to aforesaid results, we presumed that HS-1 and HS-2 can induce the dedifferentiation of adherenced neural precursor cells. Besides, MTT method results showed that cells in low concentration Stichopus japonicus glycoconjugate experimental group had similar survival rate with that of cells in low concentration NGF experimental group.
引文
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