健脾祛湿和络方调控肾小球足细胞及系膜细胞的分子机制研究

发布时间：2018-08-02 19:31

【摘要】：第一章:文献综述健脾祛湿和络方常用于缓解肾病的蛋白尿及血尿,具有减轻肾损害,保护肾功能的作用。目前关于肾脏的生理病理研究表明,足细胞作为高度分化的终末期细胞,较易受到致病刺激的影响,受损后影响了肾小球滤过屏障的完整,进而导致蛋白尿的产生;此外,GMCs受到致病刺激后,会过度增殖并表达大量ECM,导致肾小球内系膜区膨胀,挤压毛细血管袢影响肾小球正常结构,进而引发血尿和蛋白尿。近年相关实验研究表明,多种中草药或中药复方对减轻足细胞、系膜细胞损伤,缓解肾脏疾病具有显著作用。故本研究旨在通过探索健脾祛湿和络方对肾小球内足细胞及GMCs的作用,构建该方临床疗效的实验基础。第二章:健脾祛湿和络方对损伤足细胞作用机制研究研究目的:通过氨基核苷嘌呤霉素(PAN)诱导体外足细胞损伤。探索健脾祛湿和络方含药血清对损伤模型的相关标志蛋白及mTOR相关自噬因子表达的影响。探索健脾祛湿和络方对损伤足细胞的作用机制。研究方法:(1)健脾祛湿和络方及替米沙坦、RAP、激素大鼠灌胃提取含药血清。(2)用10%FBS+DMEM配成的培养基于细胞箱内培养足细胞。(3)使用氨基核苷嘌呤霉素(PAN)诱导足细胞损伤模型。(4)检测各组含药血清对正常足细胞的毒性后,将含药血清作用于足细胞损伤模型,采用CCK-8法,酶标仪检测各组OD值,检测损伤模型在各组血清作用后的活性变化。(5)western blot法检测健脾祛湿和络方作用于足细胞模型后,细胞标志蛋白nephrin、podocalyxin及细胞内mTOR相关自噬蛋白表达的水平,以判断健脾祛湿和络方对损伤模型的作用。研究结果:(1)健脾祛湿和络方及对照组的血清对足细胞无毒副作用;(2)嘌呤氨基核苷(PAN)对足细胞具有损伤作用,100μg/mlPAN作用下,足细胞损伤为正常组50%左右,为较为理想的造模药物浓度;(3)健脾祛湿和络方含药血清作用于损伤足细胞后,低、中含药血清组与正常组间差别无统计学意义(P0.05),高剂量健脾祛湿和络方含药血清较模型组相比,可减轻足细胞的损伤(P0.05),高含药血清组与西药对照组间差别无统计学意义(P0.05);(4)健脾祛湿和络方组成药物防己的提取物——汉防己甲素,其药量在6.25μg/ml以下对足细胞无毒性作用,6.25μg/ml以上对足细胞损伤作用明显(P0.05);(5)健脾祛湿和络方可改善损伤足细胞模型的细胞活性,促进足细胞标志蛋白nephrin、podocalyxin的表达,且安全无毒性;(6)PAN诱导的损伤足细胞模型内mTOR自噬相关因子明显激活,其下游蛋白P-P70S6K、P-4EBP1等合成显著增多,同时,损伤模型内与自噬水平呈正相关的LC3-Ⅱ含量明显减少。表明PAN诱导的损伤模型内mTOR活性增强且正常的自噬程度降低。健脾祛湿和络方高剂量组含药血清可明显上调细胞模型内LC3-Ⅱ表达,减少mTOR及其相关因子的合成,改善细胞内降低的自噬水平。且健脾祛湿和络方高剂量组与西药对照组间的差别不具有统计学意义。(p0.05)。研究结论:健脾祛湿和络方对PAN诱导的损伤足细胞有修复作用。第三章:健脾祛湿和络方对增殖GMCs作用研究研究目的:研究健脾祛湿和络方对脂多糖(LPS)介导的体外GMCs增殖模型分泌层粘连蛋白、纤维连接蛋白及生长因子TGF-β1的影响。探讨健脾祛湿和络方临床可治疗慢性肾炎的细胞作用机制。研究方法:健脾祛湿和络方及对照组药物给实验大鼠灌胃后制备各组含药血清。用10%FBS+DMEM配制培养基于体外培养脂多糖(LPS)介导的增殖GMCs模型。采用酶联免疫法吸附试验(ELISA)测定含药血清作用于增殖的GMCs后,GMCs上清液中细胞外基质主要组成物质(LN、FN、TGF-β1)含量。研究结果:(1)LPS可诱导增殖GMCs大量分泌ECM组成成分,且作用时间越长,分泌越多;(2)健脾祛湿和络方中高剂量含药血清在24h、48h、72h对LPS诱导的增殖GMCs细胞上清液中LN、FN、TGF-β1表达均有抑制作用,差值具有统计学意义(P0.05)。且随健脾祛湿和络方药物浓度的升高及作用时间的增加,GMCs分泌量越少。健脾祛湿和络方低药量组与正常组间差值不具有统计学意义(P0.05)。研究结论:健脾祛湿和络方能显著抑制病理状态下LPS诱导的增殖GMCs过度分泌层粘连蛋白(LN)、纤维连接蛋白(FN)、转化生长因子(TGF-β1)。这可能是健脾祛湿和络方治疗慢性肾炎血尿和蛋白尿,预防肾脏纤维化的作用机制。第四章:问题与展望
[Abstract]:Chapter 1: literature review of Jianpi dispelling dampness and collaterals is often used to relieve proteinuria and hematuria in nephrosis. It has the effect of reducing renal damage and protecting renal function. At present, the physiological and pathological study of kidney shows that podocyte as highly differentiated end-stage cells is more susceptible to the effect of pathogenic stimulation, and it affects the glomerular filtration barrier after damage. In addition, GMCs is caused by the formation of proteinuria; in addition, after being stimulated by the pathogenic stimulation, it will proliferate and express a large number of ECM, which causes the expansion of the mesangial region, and the capillary loop affects the normal structure of the glomeruli, and then causes hematuria and proteinuria. The purpose of this study is to explore the effect of invigorating spleen and dispelling dampness and collaterals on the glomerular podocytes and GMCs, and to construct the experimental basis for the clinical effect of this prescription. The second chapter: the aim of the study on the mechanism of the effect of the spleen and eliminating dampness and collaterals on the injured foot cells: through the amidoside purinine mould In order to explore the effect of strengthening spleen and dispelling dampness and blood serum on the expression of related autophagy factors related to the damage model and the expression of mTOR related autophagy factors. Explore the mechanism of the action of Jianpi dispelling dampness and collaterals on the injured foot cells. Study methods: (1) extract the spleen and dispel dampness and collaterals and telmisartan, RAP, and hormone rats to extract the contents of the stomach. (2) the cultivation of podocytes in cell box with 10%FBS+DMEM. (3) using amidoside purinamycin (PAN) to induce podocyte injury model. (4) after testing the toxicity of serum to normal podocytes in each group, the serum containing drug was acted on the model of foot cell injury, and CCK-8 method and enzyme labeling instrument were used to detect all groups of OD values, and the detection loss was detected. The changes in the activity of the injury model after the action of the serum in each group. (5) Western blot method was used to detect the effect of invigorating spleen and dampness and collaterals on the expression level of nephrin, podocalyxin and mTOR related autophagic protein in the cells in order to judge the effect of invigorating spleen and dampness and collaterals on the damage model. (1) invigorating spleen and eliminating dampness and collaterals No side effects of serum on podocytes, and (2) purine amino nucleoside (PAN) had damage to podocytes. Under the action of 100 mu g/mlPAN, the injury of podocyte was about 50% of the normal group, which was the ideal concentration of drug making drugs. (3) the effect of spleen removing dampness and collaterals containing drug serum on the injury of podocytes. There was no statistically significant difference between the two groups (P0.05). Compared with the model group, the high dose of Jianpi dispelling dampness and collateral prescription could reduce the injury of podocyte (P0.05), and there was no significant difference between the high drug serum group and the western medicine control group (P0.05); (4) the extract of tetrandrine, an extract of Jianpi dispelling dampness and collaterals, was 6.2 of tetrandrine. No toxic effect on podocytes under 5 g/ml, and more than 6.25 mu g/ml in foot cell damage (P0.05); (5) invigorating the spleen and removing dampness and collaterals can improve the cell activity of the injured foot cell model, promote the expression of the podocyte protein nephrin, podocalyxin, and be safe and non-toxic; (6) the autophagy related causes of mTOR in the PAN induced foot cell model. The content of the downstream protein P-P70S6K, P-4EBP1 and so on increased significantly. At the same time, the content of LC3- II, which was positively correlated with the autophagy level in the damage model, was obviously reduced. It showed that the mTOR activity in the PAN induced injury model and the normal degree of autophagy decreased. The expression of internal LC3- II reduces the synthesis of mTOR and its related factors and improves the level of autophagy in the cell. The difference between the high dose group and the western medicine control group is not statistically significant. (P0.05). Conclusion: strengthening spleen and dampness and collaterals has the repair effect on PAN induced injury of foot cells. The third chapter: Invigorating Spleen and eliminating dampness and collaterals Objective: To study the effect of Jianpi dispelling dampness and collaterals on the secretion of laminin, fibronectin and growth factor TGF- beta 1 mediated by lipopolysaccharide (LPS) GMCs proliferation model in vitro. The mechanism of the effect of strengthening spleen and eliminating dampness and collaterals on the treatment of chronic nephritis was studied. The drug serum was prepared from the experimental rats after gavage. The proliferation of GMCs model based on the culture of lipopolysaccharide (LPS) in vitro was prepared by 10%FBS+DMEM. The main components of the extracellular matrix (LN, FN, TGF- beta 1) in the GMCs supernatant were determined by enzyme linked immunosorbent assay (ELISA). The results were as follows: (1) LPS could induce the proliferation of GMCs to secrete a large number of ECM components, and the longer the action time, the more secreted; (2) the high dose serum of the spleen and dispelling dampness and collaterals in 24h, 48h, and 72h had inhibitory effect on LN, FN, TGF- beta 1 in the LPS induced proliferation of GMCs cell supernatant, and the difference was statistically significant (P0.05). The increase of drug concentration and the increase of action time, the less GMCs secreted, the difference between the low dose group and the normal group was not statistically significant (P0.05). Conclusion: strengthening the spleen and eliminating dampness and collaterals can significantly inhibit the LPS induced GMCs oversecreted laminin (LN) and fiber connection in the pathological state. Protein (FN), transforming growth factor (TGF- beta 1). This may be the mechanism of strengthening spleen and eliminating dampness and collaterals in the treatment of chronic nephritis and proteinuria and preventing renal fibrosis. The fourth chapter: Problems and Prospects
【学位授予单位】：中国中医科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R277.5

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