天麻多糖及其硫酸酯化物的神经保护作用及机制研究

发布时间：2018-03-03 03:21

本文选题：天麻多糖　切入点：硫酸酯化天麻多糖　出处：《武汉大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:本实验采用CORT诱导损伤PC12细胞模型,以及C6神经胶质瘤细胞模型,通过CCK-8法检测细胞存活率、检测LDH释放率、检测细胞内ROS水平、观察细胞形态变化、荧光染色、划痕实验、WB等试验方法,研究天麻多糖及其硫酸酯化物的神经保护作用和机制,并探索了硫酸酯化天麻多糖的抗肿瘤活性。为将天麻多糖开发成一种新型的、安全有效的神经保护的天然药物提供了相应的理论基础;同时,也为寻找硫酸酯化天麻多糖新的生物活性指明了方向。方法:对于天麻多糖及其酯化物的神经保护研究,采用CORT诱导损伤PC12细胞模型,实验分为正常对照组、CORT损伤组(200μMCORT)、低浓度组(200μM CORT+250μg/ml GEP/GEPs)、中浓度组(200μM CORT+500μg/ml GEP/GEPs)和高浓度组(200μMCORT+1000μg/mlGEP/GEPs)。实验给药时,正常对照组换上空白培养基,CORT损伤组换上含有200μMCORT的空白培养基,其他分组先用相应浓度的天麻多糖和硫酸酯化天麻多糖孵育30 min。采用CCK-8法检测细胞存活率,检测细胞LDH释放量,检测细胞内ROS水平,Hoschst33258/PI双染法检测细胞凋亡,ER荧光染色和WB检测细胞中GRP78、XBP-1、GADD153、caspase9和caspase12的表达量,以探究天麻多糖的神经保护活性机制。采用CCK-8法检测细胞存活率,检测细胞LDH释放量,观察细胞形态学变化,DAPI荧光染色和WB检测细胞中BAX和Bcl-2的表达量,以探究硫酸酯化天麻多糖的神经保护作用。对于硫酸酯化天麻多糖抑制神经胶质瘤细胞生长作用的活性研究,将实验分为正常对照组、0.1 mg/ml GEPs组、0.25 mg/ml GEPs组、0.5 mg/ml GEPs组、1 mg/ml GEPs组、1.5 mg/ml GEPs组。采用CCK-8法检测细胞存活率,观察细胞形态变化、DAPI荧光染色和划痕实验。结果:天麻多糖的预处理使PC12细胞存活率上升,LDH释放率下降,细胞内的ROS水平降低,细胞凋亡数下降,ER形态染色的荧光强度减弱,且WB检测结果显示 PC12 细胞中的 GRP78、XBP-1、GADD153、caspase9 和 caspase12 的表达水平经CORT诱导损伤后均显著上升(p0.01),但天麻多糖的预处理有效降低了这些蛋白的表达量。硫酸酯化天麻多糖的预处理,同样使PC12细胞存活率上升,LDH释放量下降,同时WB检测结果显示,PC12细胞中的Bcl-1/BAX因CORT作用而升高,而硫酸酯化天麻多糖降低了这一蛋白比例。最后,硫酸酯化天麻多糖降低了 C6细胞的存活率,使C6细胞凋亡,能有效抑制C6细胞的迁移,并且呈现出浓度依赖关系。结论:天麻多糖对CORT诱导损伤PC12细胞的保护作用是通过抑制内质网应激通路实现的。硫酸酯化天麻多糖对CORT诱导损伤PC12细胞也具有一定的保护作用,但与天麻多糖相比,此保护作用没有显著提高。最后,发现硫酸酯化天麻多糖具有抑制神经胶质瘤细胞生长的活性,并可有效抑制神经胶质瘤细胞的迁移。
[Abstract]:Objective: this experiment adopts the CORT PC12 cell injury model induced by C6, and glioma cell model by CCK-8 method to detect cell viability, detect the release rate of LDH and detection of ROS levels in cells, to observe the change of cell morphology, fluorescence staining, scratch test, WB test method of Gastrodia elata polysaccharide and its sulfated the neuroprotective effect and mechanism, and explore the sulfated polysaccharide anti-tumor activity. The polysaccharide was developed into a model, to provide the theoretical basis of natural neuroprotective drugs safe and effective; at the same time, it also points out the direction for sulfated polysaccharide new biological activity methods: in the study of neural protection of Gastrodia elata polysaccharide and its esters, using CORT PC12 cell injury model induced by experiment, divided into normal control group, CORT group (200 MCORT), low concentration group (200 M CORT+250 g/ml G EP/GEPs), middle dose group (200 M CORT+500 g/ml GEP/GEPs) and high concentration group (200 MCORT+1000 g/mlGEP/GEPs). The experimental drug, the normal control group in blank medium, CORT injury group for blank containing 200 MCORT medium, the other group with corresponding concentrations of Gastrodia elata sugar and sulfated polysaccharide were incubated for 30 min. cell viability was determined by CCK-8 assay, detection of cell LDH release, intracellular ROS assay to detect cell apoptosis, Hoschst33258/PI, GRP78, ER fluorescent staining and WB were detected in XBP-1, GADD153, expression of caspase9 and caspase12, with neuroprotective activity to explore the mechanism of Gastrodia polysaccharides. Cell viability was determined by CCK-8 assay, detection of cell LDH release, cell morphological changes and expression of BAX and Bcl-2 DAPI fluorescence staining and WB cells, to explore the sulfur acid polysaccharide nerve Protective effect. For the inhibition of sulfated polysaccharide in glioma cell growth, the experiment was divided into normal control group, 0.1 mg/ml GEPs 0.25 mg/ml group, GEPs group, GEPs group, 0.5 mg/ml, 1 mg/ml GEPs 1.5 mg/ml group, GEPs group. Cell viability by CCK-8 assay, cells were observed the morphological changes of DAPI staining and scratch test. Results: Gastrodia elata polysaccharide pretreatment to the survival rate of PC12 cells increased, the release rate of LDH decreased, the intracellular levels of ROS decreased the number of apoptotic cells decreased, decreased the fluorescence intensity of ER staining and morphology, WB assay showed that PC12 cells in GRP78, XBP-1, GADD153, the expression level of caspase9 and caspase12 induced by CORT after injury were significantly increased (P0.01), but the pretreatment of Gastrodia polysaccharides effectively reduced the expression of these proteins. Pretreatment of sulfated polysaccharide of Gastrodia elata, also make PC12 cell The survival rate of rise, the release amount of LDH decreased, while WB showed that PC12 cells in Bcl-1/BAX with CORT increased, while the sulfated polysaccharide decreased the protein ratio. Finally, the sulfated polysaccharide decreased the survival rate of C6 cells, C6 cells apoptosis, migration can effectively inhibit C6 cells, and showed a concentration dependent manner. Conclusion: the protective effect of Gastrodia elata polysaccharide on CORT induced PC12 cells by inhibiting the endoplasmic reticulum stress pathway. The sulfated polysaccharide on CORT induced also has certain protective effect on PC12 cell injury, but compared with the polysaccharide, the protective effect of no significantly improved. Finally, the sulfated polysaccharide has found that inhibiting nerve glioma cell growth activity, and can effectively inhibit the migration of glioma cells.

【学位授予单位】：武汉大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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