硫酸乙酰肝素酶在NGF诱导的PC12神经突生成中的作用及硫酸乙酰肝素类似物的结构和构象及其活性研究
发布时间：2018-07-25 19:24
【摘要】： 硫酸乙酰肝素酶(heparanase)是哺乳动物细胞中β-D-糖苷内切酶,能降解硫酸乙酰肝素蛋白聚糖糖链(heparan sulfate proteoglycans,HSPGs),参与细胞外基质和基底膜重建。此外,其酶活性和肿瘤侵袭和炎症等生理病理过程。同时,该酶也表达于粒细胞,星形胶质细胞,神经元和大鼠脊髓白质神经胶质等正常细胞。在发育过程中,硫酸乙酰肝素酶出现在小鼠早期胚胎及鸡血管和神经系统,但其功能少有报道。 大鼠嗜铬瘤PC12细胞在神经生长因子(NGF)作用下能分化为交感神经样细胞。与该过程表型变化相关的是神经递质的合成,电可兴奋性的获得,及通过神经突生成的过程(neuritogenesis)产生被称为神经突的神经元样伸展。神经突生成是一复杂的过程,牵涉到生长的神经突(neurite)与细胞外基质之间复杂多样的作用。由于硫酸乙酰肝素酶广泛表达于神经系统,故我们以NGF诱导PC12细胞神经突生成为模型,研究硫酸乙酰肝素酶在神经突生成中的作用。 研究发现,50ng/ml NGF作用于PC12细胞16小时,硫酸乙酰肝素酶基因(HSPE)的mRNA水平显著升高;硫酸乙酰肝素酶化学抑制剂苏拉明(suramin,50uM)能显著抑制NGF诱导的PC12神经突生成。利用短发夹RNA(short hairpinRNA,shRNA)干扰技术沉默HSPE,筛选稳定细胞株。经RT-PCR,western blot方法检测出了稳定低表达硫酸乙酰肝素酶的细胞株sh-7。转染人HSPE全长cDNA至PC12细胞,挑选稳定高表达人源硫酸乙酰肝素酶的细胞株,经过RT-PCR和western blot检测,获得了稳定高表达人硫酸乙酰肝素酶的细胞株9406。以未转染的PC12细胞及转染相应空质粒的细胞为对照,利用NGF(25ng/ml)分别诱导sh-7,9406细胞神经突生成。结果发现,NGF作用72小时,9406细胞神经突生成增强,而sh-7的神经突生成受到了抑制。进一步机制研究发现,9406中p38 MAPK磷酸化水平增强,而sh-7中p38 MAPK磷酸化水平降低。 Heparanase能在特定位点催化HSPG的HS链降解,从而释放出HS片段。HS由N-乙酰葡萄糖和葡萄糖醛酸二糖重复单元组成,经不同的酶修饰后,在其不同位点会有硫酸基团取代形成高负电性分子,能与数百种功能性蛋白结合,从而影响细胞增殖、分化和形态发生。受此启发,我们尝试制备了HS结构有类似的多糖类硫酸化衍生物,并对其生物活性进行了研究。 从中药葛根(Pueraria lobata(Willd.)Ohwi)中提取了粗多糖PLB。依次利用DEAE-纤维素柱层析和G-150凝胶柱层析分离纯化,获得均一多糖组分PLB-2C。利用糖组成分析、甲基化分析等方法结合核磁共振技术(~1H NMR、~(13)C NMR、HSQC、HMBC)研究了PLB-2C的化学结构,表明PLB-2C为一线型(1,6)-α-D-葡聚糖。尺寸排除色谱和激光光散射联用仪(SEC-LLS)揭示PLB-2C的构象为无规线团。利用氯磺酸-吡啶法制备了HS类似物——PLB-2C的硫酸化衍生物PLB-2CS。~(13)C NMR证实PLB-2CS为2,3,4位发生硫酸化取代的(1,6)-α-D-葡聚糖。体外活性试验表明PLB-2C和PLB-2CS都不能诱导PC12细胞神经突生成。然而,用MTT方法对PLB-2CS的生物活性进行研究时,发现PLB-2CS具有减低H_2O_2对PC12的损伤作用。 综上所述,硫酸乙酰肝素酶很可能通过增强p38 MAPK磷酸化信号通路促进NGF诱导的PC12神经突生成;制备的硫酸乙酰肝素类似物不能诱导PC12细胞神经突生成,这也暗示或许是HS结合的生长因子才起增强神经突生成的作用。PLB-2CS具有抗氧化作用,提示HS类似物也可作为抗氧化剂。
[Abstract]:Heparanase sulfate (heparanase) is a beta -D- glycoside endonuclease in mammalian cells, which degrades heparan sulfate proteoglycans (HSPGs), and is involved in the reconstruction of extracellular matrix and basement membrane. In addition, its enzyme activity, tumor invasion and inflammation and other physiological and pathological processes. Normal cells such as astrocytes, astrocytes, neurons and rat spinal cord white matter neuroglia. During development, heparanase sulfate appears in early mouse embryos and chicken blood vessels and nervous system, but its function is less reported.
The chromaffin PC12 cells of rats can differentiate into sympathetic nerve like cells under the action of nerve growth factor (NGF), which are related to the synthesis of neurotransmitters, the acquisition of electrical excitability, and the production of neurite like neurites called neurites through the process of neurite formation (neuritogenesis). The process involves the complex and diverse role of the growth of the neurite and the extracellular matrix. As the heparanase is widely expressed in the nervous system, we induce the neurite production of PC12 cells by NGF as a model to study the role of heparanase in the formation of neurite process.
It was found that 50ng/ml NGF acted on PC12 cells for 16 hours, and the mRNA level of heparanase sulfate gene (HSPE) was significantly higher; the chemical inhibitor of heparanase, Sura Ming (suramin, 50uM), could significantly inhibit the formation of PC12 neurite induced by NGF, and the stability and refinement were screened by short hairpin RNA (short hairpinRNA) interference technique. RT-PCR, Western blot method was used to detect the stable low expression of heparanase acid heparanase, sh-7. transfected into HSPE full length cDNA to PC12 cells, and selected stable high expression human source of heparanase sulfate. After RT-PCR and Western blot, a cell line of stable high apparent human heparanase sulfate 9406. was obtained. Compared with the untransfected PC12 cells and the cells transfected with the corresponding empty plasmids, NGF (25ng/ml) was used to induce the formation of neurite process in sh-79406 cells. The results showed that the action of NGF was enhanced in 9406 cells for 72 hours, and the formation of sh-7 was inhibited. Further mechanism study found that the phosphorylation level of p38 MAPK was enhanced in 9406. In sh-7, the phosphorylation level of p38 MAPK decreased.
Heparanase can catalyze the degradation of the HS chain of HSPG at the special location, thus releasing the HS fragment.HS composed of N- acetyl glucose and glucuronic acid two sugar repeating unit. After modified by different enzymes, there will be a sulphuric acid group replacing the high negative power molecule at its different point, which can bind to hundreds of functional proteins and affect cell proliferation. Inspired by this, we have tried to prepare sulfated derivatives of HS with similar structure and studied their biological activities.
The crude polysaccharide PLB. was extracted from Pueraria lobata (Pueraria Lobata (Willd.) Ohwi) and purified by DEAE- cellulose column chromatography and G-150 gel column chromatography in order to obtain the composition analysis of the homogeneous polysaccharide component, and the methylation analysis combined with the nuclear magnetic resonance technique (~1H NMR, ~ (13) C NMR). The structure of PLB-2C is a linear (1,6) - alpha -D- glucan. The conformation of PLB-2C is revealed to be a random line by size exclusion chromatography and laser light scattering (SEC-LLS). HS analogs are prepared by the chlorsulfonic acid pyridine method, the sulfate derivative of PLB-2C, PLB-2CS.~ (13) C NMR, is substituted for the sulfation of the 2,3,4 position. In vitro activity test showed that both PLB-2C and PLB-2CS did not induce the generation of neurite in PC12 cells. However, when the biological activity of PLB-2CS was studied by MTT method, it was found that PLB-2CS could reduce the damage effect of H_2O_2 on PC12.
In summary, heparanase sulfate is likely to promote the formation of PC12 neurites induced by NGF by enhancing the p38 MAPK phosphorylation signal pathway; the prepared heparan sulfate analogues can not induce the production of PC12 cells, which may also suggest that the growth factor of HS binding can enhance the production of neuroprotrusion, and.PLB-2CS has antioxidant activity. The effect suggests that HS analogues can also be used as antioxidants.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R341;R285
本文编号：2144826
[Abstract]:Heparanase sulfate (heparanase) is a beta -D- glycoside endonuclease in mammalian cells, which degrades heparan sulfate proteoglycans (HSPGs), and is involved in the reconstruction of extracellular matrix and basement membrane. In addition, its enzyme activity, tumor invasion and inflammation and other physiological and pathological processes. Normal cells such as astrocytes, astrocytes, neurons and rat spinal cord white matter neuroglia. During development, heparanase sulfate appears in early mouse embryos and chicken blood vessels and nervous system, but its function is less reported.
The chromaffin PC12 cells of rats can differentiate into sympathetic nerve like cells under the action of nerve growth factor (NGF), which are related to the synthesis of neurotransmitters, the acquisition of electrical excitability, and the production of neurite like neurites called neurites through the process of neurite formation (neuritogenesis). The process involves the complex and diverse role of the growth of the neurite and the extracellular matrix. As the heparanase is widely expressed in the nervous system, we induce the neurite production of PC12 cells by NGF as a model to study the role of heparanase in the formation of neurite process.
It was found that 50ng/ml NGF acted on PC12 cells for 16 hours, and the mRNA level of heparanase sulfate gene (HSPE) was significantly higher; the chemical inhibitor of heparanase, Sura Ming (suramin, 50uM), could significantly inhibit the formation of PC12 neurite induced by NGF, and the stability and refinement were screened by short hairpin RNA (short hairpinRNA) interference technique. RT-PCR, Western blot method was used to detect the stable low expression of heparanase acid heparanase, sh-7. transfected into HSPE full length cDNA to PC12 cells, and selected stable high expression human source of heparanase sulfate. After RT-PCR and Western blot, a cell line of stable high apparent human heparanase sulfate 9406. was obtained. Compared with the untransfected PC12 cells and the cells transfected with the corresponding empty plasmids, NGF (25ng/ml) was used to induce the formation of neurite process in sh-79406 cells. The results showed that the action of NGF was enhanced in 9406 cells for 72 hours, and the formation of sh-7 was inhibited. Further mechanism study found that the phosphorylation level of p38 MAPK was enhanced in 9406. In sh-7, the phosphorylation level of p38 MAPK decreased.
Heparanase can catalyze the degradation of the HS chain of HSPG at the special location, thus releasing the HS fragment.HS composed of N- acetyl glucose and glucuronic acid two sugar repeating unit. After modified by different enzymes, there will be a sulphuric acid group replacing the high negative power molecule at its different point, which can bind to hundreds of functional proteins and affect cell proliferation. Inspired by this, we have tried to prepare sulfated derivatives of HS with similar structure and studied their biological activities.
The crude polysaccharide PLB. was extracted from Pueraria lobata (Pueraria Lobata (Willd.) Ohwi) and purified by DEAE- cellulose column chromatography and G-150 gel column chromatography in order to obtain the composition analysis of the homogeneous polysaccharide component, and the methylation analysis combined with the nuclear magnetic resonance technique (~1H NMR, ~ (13) C NMR). The structure of PLB-2C is a linear (1,6) - alpha -D- glucan. The conformation of PLB-2C is revealed to be a random line by size exclusion chromatography and laser light scattering (SEC-LLS). HS analogs are prepared by the chlorsulfonic acid pyridine method, the sulfate derivative of PLB-2C, PLB-2CS.~ (13) C NMR, is substituted for the sulfation of the 2,3,4 position. In vitro activity test showed that both PLB-2C and PLB-2CS did not induce the generation of neurite in PC12 cells. However, when the biological activity of PLB-2CS was studied by MTT method, it was found that PLB-2CS could reduce the damage effect of H_2O_2 on PC12.
In summary, heparanase sulfate is likely to promote the formation of PC12 neurites induced by NGF by enhancing the p38 MAPK phosphorylation signal pathway; the prepared heparan sulfate analogues can not induce the production of PC12 cells, which may also suggest that the growth factor of HS binding can enhance the production of neuroprotrusion, and.PLB-2CS has antioxidant activity. The effect suggests that HS analogues can also be used as antioxidants.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R341;R285
【参考文献】
相关期刊论文 前1条
1 瞿小婷;赵华军;张中华;侯永泰;奚涛;;乙酰肝素酶稳定表达细胞株的建立[J];中国药科大学学报;2006年01期
，本文编号：2144826
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