硫酸乙酰肝素酶在NGF誘導(dǎo)的PC12神經(jīng)突生成中的作用及硫酸乙酰肝素類似物的結(jié)構(gòu)和構(gòu)象及其活性研究
發(fā)布時間：2018-07-25 19:24
【摘要】： 硫酸乙酰肝素酶(heparanase)是哺乳動物細(xì)胞中β-D-糖苷內(nèi)切酶,能降解硫酸乙酰肝素蛋白聚糖糖鏈(heparan sulfate proteoglycans,HSPGs),參與細(xì)胞外基質(zhì)和基底膜重建。此外,其酶活性和腫瘤侵襲和炎癥等生理病理過程。同時,該酶也表達(dá)于粒細(xì)胞,星形膠質(zhì)細(xì)胞,神經(jīng)元和大鼠脊髓白質(zhì)神經(jīng)膠質(zhì)等正常細(xì)胞。在發(fā)育過程中,硫酸乙酰肝素酶出現(xiàn)在小鼠早期胚胎及雞血管和神經(jīng)系統(tǒng),但其功能少有報(bào)道。 大鼠嗜鉻瘤PC12細(xì)胞在神經(jīng)生長因子(NGF)作用下能分化為交感神經(jīng)樣細(xì)胞。與該過程表型變化相關(guān)的是神經(jīng)遞質(zhì)的合成,電可興奮性的獲得,及通過神經(jīng)突生成的過程(neuritogenesis)產(chǎn)生被稱為神經(jīng)突的神經(jīng)元樣伸展。神經(jīng)突生成是一復(fù)雜的過程,牽涉到生長的神經(jīng)突(neurite)與細(xì)胞外基質(zhì)之間復(fù)雜多樣的作用。由于硫酸乙酰肝素酶廣泛表達(dá)于神經(jīng)系統(tǒng),故我們以NGF誘導(dǎo)PC12細(xì)胞神經(jīng)突生成為模型,研究硫酸乙酰肝素酶在神經(jīng)突生成中的作用。 研究發(fā)現(xiàn),50ng/ml NGF作用于PC12細(xì)胞16小時,硫酸乙酰肝素酶基因(HSPE)的mRNA水平顯著升高;硫酸乙酰肝素酶化學(xué)抑制劑蘇拉明(suramin,50uM)能顯著抑制NGF誘導(dǎo)的PC12神經(jīng)突生成。利用短發(fā)夾RNA(short hairpinRNA,shRNA)干擾技術(shù)沉默HSPE,篩選穩(wěn)定細(xì)胞株。經(jīng)RT-PCR,western blot方法檢測出了穩(wěn)定低表達(dá)硫酸乙酰肝素酶的細(xì)胞株sh-7。轉(zhuǎn)染人HSPE全長cDNA至PC12細(xì)胞,挑選穩(wěn)定高表達(dá)人源硫酸乙酰肝素酶的細(xì)胞株,經(jīng)過RT-PCR和western blot檢測,獲得了穩(wěn)定高表達(dá)人硫酸乙酰肝素酶的細(xì)胞株9406。以未轉(zhuǎn)染的PC12細(xì)胞及轉(zhuǎn)染相應(yīng)空質(zhì)粒的細(xì)胞為對照,利用NGF(25ng/ml)分別誘導(dǎo)sh-7,9406細(xì)胞神經(jīng)突生成。結(jié)果發(fā)現(xiàn),NGF作用72小時,9406細(xì)胞神經(jīng)突生成增強(qiáng),而sh-7的神經(jīng)突生成受到了抑制。進(jìn)一步機(jī)制研究發(fā)現(xiàn),9406中p38 MAPK磷酸化水平增強(qiáng),而sh-7中p38 MAPK磷酸化水平降低。 Heparanase能在特定位點(diǎn)催化HSPG的HS鏈降解,從而釋放出HS片段。HS由N-乙酰葡萄糖和葡萄糖醛酸二糖重復(fù)單元組成,經(jīng)不同的酶修飾后,在其不同位點(diǎn)會有硫酸基團(tuán)取代形成高負(fù)電性分子,能與數(shù)百種功能性蛋白結(jié)合,從而影響細(xì)胞增殖、分化和形態(tài)發(fā)生。受此啟發(fā),我們嘗試制備了HS結(jié)構(gòu)有類似的多糖類硫酸化衍生物,并對其生物活性進(jìn)行了研究。 從中藥葛根(Pueraria lobata(Willd.)Ohwi)中提取了粗多糖PLB。依次利用DEAE-纖維素柱層析和G-150凝膠柱層析分離純化,獲得均一多糖組分PLB-2C。利用糖組成分析、甲基化分析等方法結(jié)合核磁共振技術(shù)(~1H NMR、~(13)C NMR、HSQC、HMBC)研究了PLB-2C的化學(xué)結(jié)構(gòu),表明PLB-2C為一線型(1,6)-α-D-葡聚糖。尺寸排除色譜和激光光散射聯(lián)用儀(SEC-LLS)揭示PLB-2C的構(gòu)象為無規(guī)線團(tuán)。利用氯磺酸-吡啶法制備了HS類似物——PLB-2C的硫酸化衍生物PLB-2CS。~(13)C NMR證實(shí)PLB-2CS為2,3,4位發(fā)生硫酸化取代的(1,6)-α-D-葡聚糖。體外活性試驗(yàn)表明PLB-2C和PLB-2CS都不能誘導(dǎo)PC12細(xì)胞神經(jīng)突生成。然而,用MTT方法對PLB-2CS的生物活性進(jìn)行研究時,發(fā)現(xiàn)PLB-2CS具有減低H_2O_2對PC12的損傷作用。 綜上所述,硫酸乙酰肝素酶很可能通過增強(qiáng)p38 MAPK磷酸化信號通路促進(jìn)NGF誘導(dǎo)的PC12神經(jīng)突生成;制備的硫酸乙酰肝素類似物不能誘導(dǎo)PC12細(xì)胞神經(jīng)突生成,這也暗示或許是HS結(jié)合的生長因子才起增強(qiáng)神經(jīng)突生成的作用。PLB-2CS具有抗氧化作用,提示HS類似物也可作為抗氧化劑。
[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.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R341;R285
本文編號：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.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R341;R285
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 瞿小婷;趙華軍;張中華;侯永泰;奚濤;;乙酰肝素酶穩(wěn)定表達(dá)細(xì)胞株的建立[J];中國藥科大學(xué)學(xué)報(bào);2006年01期
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