本文選題：天麻多糖　切入點：硫酸酯化天麻多糖　出處：《武漢大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:本實驗采用CORT誘導損傷PC12細胞模型,以及C6神經膠質瘤細胞模型,通過CCK-8法檢測細胞存活率、檢測LDH釋放率、檢測細胞內ROS水平、觀察細胞形態(tài)變化、熒光染色、劃痕實驗、WB等試驗方法,研究天麻多糖及其硫酸酯化物的神經保護作用和機制,并探索了硫酸酯化天麻多糖的抗腫瘤活性。為將天麻多糖開發(fā)成一種新型的、安全有效的神經保護的天然藥物提供了相應的理論基礎;同時,也為尋找硫酸酯化天麻多糖新的生物活性指明了方向。方法:對于天麻多糖及其酯化物的神經保護研究,采用CORT誘導損傷PC12細胞模型,實驗分為正常對照組、CORT損傷組(200μMCORT)、低濃度組(200μM CORT+250μg/ml GEP/GEPs)、中濃度組(200μM CORT+500μg/ml GEP/GEPs)和高濃度組(200μMCORT+1000μg/mlGEP/GEPs)。實驗給藥時,正常對照組換上空白培養(yǎng)基,CORT損傷組換上含有200μMCORT的空白培養(yǎng)基,其他分組先用相應濃度的天麻多糖和硫酸酯化天麻多糖孵育30 min。采用CCK-8法檢測細胞存活率,檢測細胞LDH釋放量,檢測細胞內ROS水平,Hoschst33258/PI雙染法檢測細胞凋亡,ER熒光染色和WB檢測細胞中GRP78、XBP-1、GADD153、caspase9和caspase12的表達量,以探究天麻多糖的神經保護活性機制。采用CCK-8法檢測細胞存活率,檢測細胞LDH釋放量,觀察細胞形態(tài)學變化,DAPI熒光染色和WB檢測細胞中BAX和Bcl-2的表達量,以探究硫酸酯化天麻多糖的神經保護作用。對于硫酸酯化天麻多糖抑制神經膠質瘤細胞生長作用的活性研究,將實驗分為正常對照組、0.1 mg/ml GEPs組、0.25 mg/ml GEPs組、0.5 mg/ml GEPs組、1 mg/ml GEPs組、1.5 mg/ml GEPs組。采用CCK-8法檢測細胞存活率,觀察細胞形態(tài)變化、DAPI熒光染色和劃痕實驗。結果:天麻多糖的預處理使PC12細胞存活率上升,LDH釋放率下降,細胞內的ROS水平降低,細胞凋亡數下降,ER形態(tài)染色的熒光強度減弱,且WB檢測結果顯示 PC12 細胞中的 GRP78、XBP-1、GADD153、caspase9 和 caspase12 的表達水平經CORT誘導損傷后均顯著上升(p0.01),但天麻多糖的預處理有效降低了這些蛋白的表達量。硫酸酯化天麻多糖的預處理,同樣使PC12細胞存活率上升,LDH釋放量下降,同時WB檢測結果顯示,PC12細胞中的Bcl-1/BAX因CORT作用而升高,而硫酸酯化天麻多糖降低了這一蛋白比例。最后,硫酸酯化天麻多糖降低了 C6細胞的存活率,使C6細胞凋亡,能有效抑制C6細胞的遷移,并且呈現出濃度依賴關系。結論:天麻多糖對CORT誘導損傷PC12細胞的保護作用是通過抑制內質網應激通路實現的。硫酸酯化天麻多糖對CORT誘導損傷PC12細胞也具有一定的保護作用,但與天麻多糖相比,此保護作用沒有顯著提高。最后,發(fā)現硫酸酯化天麻多糖具有抑制神經膠質瘤細胞生長的活性,并可有效抑制神經膠質瘤細胞的遷移。
[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.

【學位授予單位】：武漢大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R285.5

【參考文獻】

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本文編號：1559333