健脾祛濕和絡(luò)方調(diào)控腎小球足細(xì)胞及系膜細(xì)胞的分子機(jī)制研究
發(fā)布時(shí)間：2018-08-02 19:31
【摘要】：第一章:文獻(xiàn)綜述健脾祛濕和絡(luò)方常用于緩解腎病的蛋白尿及血尿,具有減輕腎損害,保護(hù)腎功能的作用。目前關(guān)于腎臟的生理病理研究表明,足細(xì)胞作為高度分化的終末期細(xì)胞,較易受到致病刺激的影響,受損后影響了腎小球?yàn)V過(guò)屏障的完整,進(jìn)而導(dǎo)致蛋白尿的產(chǎn)生;此外,GMCs受到致病刺激后,會(huì)過(guò)度增殖并表達(dá)大量ECM,導(dǎo)致腎小球內(nèi)系膜區(qū)膨脹,擠壓毛細(xì)血管袢影響腎小球正常結(jié)構(gòu),進(jìn)而引發(fā)血尿和蛋白尿。近年相關(guān)實(shí)驗(yàn)研究表明,多種中草藥或中藥復(fù)方對(duì)減輕足細(xì)胞、系膜細(xì)胞損傷,緩解腎臟疾病具有顯著作用。故本研究旨在通過(guò)探索健脾祛濕和絡(luò)方對(duì)腎小球內(nèi)足細(xì)胞及GMCs的作用,構(gòu)建該方臨床療效的實(shí)驗(yàn)基礎(chǔ)。第二章:健脾祛濕和絡(luò)方對(duì)損傷足細(xì)胞作用機(jī)制研究研究目的:通過(guò)氨基核苷嘌呤霉素(PAN)誘導(dǎo)體外足細(xì)胞損傷。探索健脾祛濕和絡(luò)方含藥血清對(duì)損傷模型的相關(guān)標(biāo)志蛋白及mTOR相關(guān)自噬因子表達(dá)的影響。探索健脾祛濕和絡(luò)方對(duì)損傷足細(xì)胞的作用機(jī)制。研究方法:(1)健脾祛濕和絡(luò)方及替米沙坦、RAP、激素大鼠灌胃提取含藥血清。(2)用10%FBS+DMEM配成的培養(yǎng)基于細(xì)胞箱內(nèi)培養(yǎng)足細(xì)胞。(3)使用氨基核苷嘌呤霉素(PAN)誘導(dǎo)足細(xì)胞損傷模型。(4)檢測(cè)各組含藥血清對(duì)正常足細(xì)胞的毒性后,將含藥血清作用于足細(xì)胞損傷模型,采用CCK-8法,酶標(biāo)儀檢測(cè)各組OD值,檢測(cè)損傷模型在各組血清作用后的活性變化。(5)western blot法檢測(cè)健脾祛濕和絡(luò)方作用于足細(xì)胞模型后,細(xì)胞標(biāo)志蛋白nephrin、podocalyxin及細(xì)胞內(nèi)mTOR相關(guān)自噬蛋白表達(dá)的水平,以判斷健脾祛濕和絡(luò)方對(duì)損傷模型的作用。研究結(jié)果:(1)健脾祛濕和絡(luò)方及對(duì)照組的血清對(duì)足細(xì)胞無(wú)毒副作用;(2)嘌呤氨基核苷(PAN)對(duì)足細(xì)胞具有損傷作用,100μg/mlPAN作用下,足細(xì)胞損傷為正常組50%左右,為較為理想的造模藥物濃度;(3)健脾祛濕和絡(luò)方含藥血清作用于損傷足細(xì)胞后,低、中含藥血清組與正常組間差別無(wú)統(tǒng)計(jì)學(xué)意義(P0.05),高劑量健脾祛濕和絡(luò)方含藥血清較模型組相比,可減輕足細(xì)胞的損傷(P0.05),高含藥血清組與西藥對(duì)照組間差別無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);(4)健脾祛濕和絡(luò)方組成藥物防己的提取物——漢防己甲素,其藥量在6.25μg/ml以下對(duì)足細(xì)胞無(wú)毒性作用,6.25μg/ml以上對(duì)足細(xì)胞損傷作用明顯(P0.05);(5)健脾祛濕和絡(luò)方可改善損傷足細(xì)胞模型的細(xì)胞活性,促進(jìn)足細(xì)胞標(biāo)志蛋白nephrin、podocalyxin的表達(dá),且安全無(wú)毒性;(6)PAN誘導(dǎo)的損傷足細(xì)胞模型內(nèi)mTOR自噬相關(guān)因子明顯激活,其下游蛋白P-P70S6K、P-4EBP1等合成顯著增多,同時(shí),損傷模型內(nèi)與自噬水平呈正相關(guān)的LC3-Ⅱ含量明顯減少。表明PAN誘導(dǎo)的損傷模型內(nèi)mTOR活性增強(qiáng)且正常的自噬程度降低。健脾祛濕和絡(luò)方高劑量組含藥血清可明顯上調(diào)細(xì)胞模型內(nèi)LC3-Ⅱ表達(dá),減少mTOR及其相關(guān)因子的合成,改善細(xì)胞內(nèi)降低的自噬水平。且健脾祛濕和絡(luò)方高劑量組與西藥對(duì)照組間的差別不具有統(tǒng)計(jì)學(xué)意義。(p0.05)。研究結(jié)論:健脾祛濕和絡(luò)方對(duì)PAN誘導(dǎo)的損傷足細(xì)胞有修復(fù)作用。第三章:健脾祛濕和絡(luò)方對(duì)增殖GMCs作用研究研究目的:研究健脾祛濕和絡(luò)方對(duì)脂多糖(LPS)介導(dǎo)的體外GMCs增殖模型分泌層粘連蛋白、纖維連接蛋白及生長(zhǎng)因子TGF-β1的影響。探討健脾祛濕和絡(luò)方臨床可治療慢性腎炎的細(xì)胞作用機(jī)制。研究方法:健脾祛濕和絡(luò)方及對(duì)照組藥物給實(shí)驗(yàn)大鼠灌胃后制備各組含藥血清。用10%FBS+DMEM配制培養(yǎng)基于體外培養(yǎng)脂多糖(LPS)介導(dǎo)的增殖GMCs模型。采用酶聯(lián)免疫法吸附試驗(yàn)(ELISA)測(cè)定含藥血清作用于增殖的GMCs后,GMCs上清液中細(xì)胞外基質(zhì)主要組成物質(zhì)(LN、FN、TGF-β1)含量。研究結(jié)果:(1)LPS可誘導(dǎo)增殖GMCs大量分泌ECM組成成分,且作用時(shí)間越長(zhǎng),分泌越多;(2)健脾祛濕和絡(luò)方中高劑量含藥血清在24h、48h、72h對(duì)LPS誘導(dǎo)的增殖GMCs細(xì)胞上清液中LN、FN、TGF-β1表達(dá)均有抑制作用,差值具有統(tǒng)計(jì)學(xué)意義(P0.05)。且隨健脾祛濕和絡(luò)方藥物濃度的升高及作用時(shí)間的增加,GMCs分泌量越少。健脾祛濕和絡(luò)方低藥量組與正常組間差值不具有統(tǒng)計(jì)學(xué)意義(P0.05)。研究結(jié)論:健脾祛濕和絡(luò)方能顯著抑制病理狀態(tài)下LPS誘導(dǎo)的增殖GMCs過(guò)度分泌層粘連蛋白(LN)、纖維連接蛋白(FN)、轉(zhuǎn)化生長(zhǎng)因子(TGF-β1)。這可能是健脾祛濕和絡(luò)方治療慢性腎炎血尿和蛋白尿,預(yù)防腎臟纖維化的作用機(jī)制。第四章:問(wèn)題與展望
[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
【學(xué)位授予單位】：中國(guó)中醫(yī)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R277.5
[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
【學(xué)位授予單位】：中國(guó)中醫(yī)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R277.5
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