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低分子量褐藻多糖硫酸酯的制备及治疗肾纤维化的研究
李新朋1,2
学位类型博士
导师张全斌
2017-05-23
学位授予单位中国科学院大学
学位授予地点北京
学位专业海洋生物学
关键词低分子量褐藻多糖硫酸酯 急性肾损伤 肾脏纤维化 乙酰肝素酶 生长因子
摘要肾脏纤维化是慢性肾脏病(CKD)发展至终末期肾病(ESRD)的一种病理损伤过程。在纤维化形成的过程中,肾脏固有细胞在致纤维化因子、生长因子作用下,细胞的表型发生转化,最终导致纤维化。此外细胞外基质(ECM)成分的产生及降解失调也会进一步促进肾脏纤维化。急性肾损伤(AKI),是指肾功能短期内突然下降。在全球范围内AKI发病率持续攀升,其死亡率高居不下,对于AKI预防或治疗并无特别有效的药物,对于严重的患者只能选择肾脏代替治疗或血液透析治疗的方法。课题组前期实验已发现褐藻多糖硫酸酯(FPS)及低分子量褐藻多糖硫酸酯(LMWF)对慢性肾衰及糖尿病具有良好的治疗作用,但对于褐藻多糖硫酸酯抑制慢性肾衰的过程及机制尚不明确。本论文制备了低分子量海带褐藻多糖硫酸酯及其分级组分,并对其抗急性肾衰和肾纤维化的作用进行了探索研究。
(1)        以提取率和舒张血管活性筛选结果为依据,优化了LWMF的制备工艺,并对优化后的工艺进行了三批工艺验证。采用阴离子交换柱层析对LWMF进行分级纯化制备了两个LWMF分级组分F0.5和F1.0。
(2)        采用50%甘油后肢注射诱导大鼠AKI模型,大鼠出现典型的AKI模型症状。LMWF的分级组分F0.5、F1.0腹腔注射,分级组分F1.0可以明显降低BUN,SCr水平,肾脏重量与体重维持在正常水平,血糖也与正常组的大鼠血糖保持在同一水平,表明F1.0组分可以有效治疗AKI。
(3)        采用TGF-β1或FGF-2诱导HK-2细胞转分化(EMT),出现明显的纤维化形态。LMWF,F0.5,F1.0在蛋白和基因水平都可以明显降低HK-2细胞EMT标志物纤维连结蛋白(Fn)和平滑肌蛋白(α-SMA)的表达,具有明显的抗肾纤维化作用。LMWF,F0.5,F1.0通过降低乙酰肝素酶(HPSE)表达,可以间接调控细胞内糖蛋白SDC-1表达,促进基质金属蛋白酶-9(MMP-9)表达降低,从而使得ECM积聚减少,减轻纤维化症状。
(4)        褐藻多糖硫酸酯(FPS)对高糖诱导的NRK-52E细胞PKC-α、PKC-β表达具有抑制作用,显著下调其蛋白表达,在FPS处理的细胞中,TGF-β1表达也受到抑制。FPS也可以调节P-选择素表达,促进其调节高糖诱导的DN。结果表明FPS可以通过PKC涉及的信号通路和TGF-β信号通路来调节高糖诱导糖尿病肾病。
其他摘要Renal fibrosis is a pathogenesis that chronic kidney disease (CKD) develops to end stage renal disease (ESRD). In the process of fibrosis, kidney cells in the cause of fibrosis factors or growth factors, the cell phenotype transformation, and ultimately lead to fibrosis. In addition, the production and degradation imbalance of extracellular matrix (ECM) components will further promote renal fibrosis. Acute renal injury (AKI), refers to the sudden decline in renal function in the short term. All over the global range, AKI morbidity continues to rise, the mortality rate is so high. As so far, there is no effective drugs for AKI prevention or treatment, for severe patients can only choose kidney replacement therapy or hemodialysis treatment methods. Our precious studies found that fucoidan (FPS) and low molecular weight fucoidan (LMWF) have a good therapeutic effect on chronic renal failure and diabetes mellitus, but the process and mechanism are still unclear. In this paper, low molecular weight fucoidan and its fractions were prepared and explored in the study of renal fibrosis.
(1)     Based on the extraction rate and the results of diastolic vasoactive screening, the preparation process of LWMF was optimized, and the optimized process was verified by preparing three batches. Two fractions F0.5 and F1.0 were prepared by fractional purification of LWMF with anion exchange column chromatography.
(2)     AKI model was induced by 50% glycerol hindlimb injection, and the typical AKI model was observed in rats. The fractions F0.5 and F1.0 was received through intraperitoneal injection. The F1.0 fraction can significantly reduce the BUN, SCr levels, kidney weight and body weight maintained at normal levels, blood glucose level is as same as normal group. The results indicated that F1.0 fraction can be effective in the treatment of AKI.
(3)     HK-2 cell transdifferentiation (EMT) were induced by TGF-β1 or FGF-2, the cells were observed significant fibrosis morphology. LMWF, F0.5 and F1.0 could significantly reduce the expression of fibronectin (Fn) and α-smooth muscle protein (α-SMA) in HK-2 cells at both protein and gene levels which are EMT markers, they showed significantly anti-renal fibrosis effect. The LMWF, F0.5 and F1.0 could indirectly regulate the expression of intracellular glycoprotein SDC-1 and decrease the expression of matrix metalloproteinase-9 (MMP-9) by decreasing the expression of heparanase (HPSE) to reduce fibrosis symptoms.
(4)     Fucoidan (FPS) could inhibit the expression of PKC-α and PKC-β1 in NRK-52E cells which induced by high glucose. The expression of TGF-β1 was also down-regulated in FPS-treated cells. FPS could also regulate P-selectin expression and promote its regulation for high glucose-induced DN. The results showed that FPS could regulate hyperglycemia-induced diabetic nephropathy by PKC-related signaling pathway and TGF-β signaling pathway.
学科领域生物学
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/136567
专题实验海洋生物学重点实验室
作者单位1.中国科学院大学
2.中国科学院海洋研究所
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李新朋. 低分子量褐藻多糖硫酸酯的制备及治疗肾纤维化的研究[D]. 北京. 中国科学院大学,2017.
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