馬兜鈴酸對(duì)腎臟管旁毛細(xì)血管內(nèi)皮細(xì)胞的毒性作用
發(fā)布時(shí)間：2018-08-13 18:35
【摘要】：背景和目的： 馬兜鈴酸(Aristolochic Acid，AA)隸屬于馬兜鈴屬，長(zhǎng)期服用AA或含AA的藥物可引起患者腎小管壞死，腎間質(zhì)明顯纖維化，進(jìn)而發(fā)展為慢性腎功能衰竭，由此，國(guó)內(nèi)外學(xué)者將這類(lèi)腎臟疾病稱(chēng)之為馬兜鈴酸腎病(Aristolochic Acid Nephropathy，AAN)[1]。與其他腎臟疾病或中毒性腎病不同，AAN患者表現(xiàn)為腎小管上皮細(xì)胞變性、壞死、脫落，缺乏明顯的上皮細(xì)胞再生，基底膜裸露，間質(zhì)彌漫性增寬、進(jìn)行性纖維化。目前，這些特殊損傷病理表現(xiàn)機(jī)制尚不十分清楚，臨床治療更無(wú)有效措施，AAN患者常病情遷延不愈，最終進(jìn)展為終末期腎衰竭[2]。因此，深入探討AAN腎小管損傷及修復(fù)機(jī)制是很有必要的。圍繞AA的主要及重要靶點(diǎn)——腎小管，國(guó)內(nèi)外學(xué)者進(jìn)行了大量研究，確立了腎小管上皮細(xì)胞凋亡[3]、轉(zhuǎn)分化[4]、胞內(nèi)AA-DNA加合物形成[5]、泌尿系統(tǒng)腫瘤形成[6]等學(xué)說(shuō)，這些學(xué)說(shuō)能解釋AAN腎小管毀損的部分病理特點(diǎn)，然腎小管修復(fù)不良的原因仍然需要進(jìn)一步闡明。近年來(lái)，腎小管管旁毛細(xì)血管(peritubular capillary，PTC)損傷及丟失或內(nèi)皮細(xì)胞損傷[7]在急慢性腎臟疾病，特別是慢性腎臟疾病進(jìn)展中的作用引起了廣大學(xué)者的注意[8]。因此，，有必要觀察AAN過(guò)程中，PTC損傷及丟失情況，將其與腎小管損傷及增殖修復(fù)不良的關(guān)系作一系統(tǒng)分析，并深入研究AA對(duì)PTC損傷的細(xì)胞生物學(xué)毒性機(jī)制，以期提出治療馬兜鈴酸腎病的新機(jī)制和保護(hù)治療新策略。 本課題擬通過(guò)建立體內(nèi)動(dòng)物模型觀察PTC損傷及丟失情況，分析該損傷丟失與腎小管損傷、增殖修復(fù)的關(guān)系，并通過(guò)透射電鏡進(jìn)一步觀察PTC內(nèi)皮細(xì)胞超微結(jié)構(gòu)改變；體外建立AA對(duì)人臍靜脈內(nèi)皮細(xì)胞(HUVEC)損傷模型，觀察AA毒性下內(nèi)皮細(xì)胞增殖、凋亡及遷移、成管等細(xì)胞生物學(xué)變化，深入研究AA對(duì)內(nèi)皮細(xì)胞的損傷機(jī)制，以期為臨床治療AAN提供新的損傷機(jī)制和治療策略。 方法： 本課題擬分為體內(nèi)、體外兩部分進(jìn)行探討。 第一部分：體內(nèi)觀察AA對(duì)PTC的損傷作用及其與腎小管損傷、修復(fù)的相關(guān)性分析。 1. AAN小鼠模型建立。C57/BL/6小鼠分為4組：對(duì)照2W組，AA2W組，對(duì)照4W組，AA4W組。AA組小鼠以5mg/kg劑量每?jī)商旄骨蛔⑸湟淮蜛A溶液，對(duì)照組注射等體積的緩沖液(PBS)。 2.分別于2周、4周時(shí)收集小鼠血、尿、腎臟組織，進(jìn)行血尿生化分析及腎臟病理學(xué)觀察。 3.分析PTC損傷與小鼠腎小管損傷、增殖和修復(fù)的相關(guān)關(guān)系。 第二部分：體外研究AA損傷血管內(nèi)皮細(xì)胞的機(jī)制。 1.采用文獻(xiàn)濃度分組[1]，以不同濃度AA(0μg/ml，5g/ml，10g/ml，20g/ml)和不同時(shí)間(24h，48h，72h)作用人臍靜脈內(nèi)皮細(xì)胞株(HUVEC)，MTT法檢測(cè)AA對(duì)細(xì)胞增殖力的影響，篩選出藥物最適作用時(shí)間。 2. LDH釋放：不同濃度AA(0μg/ml，5g/ml，10g/ml，20g/ml)處理HUVEC48h(最佳時(shí)間)，檢測(cè)細(xì)胞質(zhì)LDH釋放情況。 3.內(nèi)皮細(xì)胞成管：不同濃度AA(0μg/ml，5g/ml，10g/ml，20g/ml)處理HUVEC48h后，收集各組細(xì)胞，轉(zhuǎn)種于基質(zhì)膠包被的96孔板，每孔103~104個(gè)細(xì)胞，孵育6h后觀察細(xì)胞成管情況。 4.內(nèi)皮細(xì)胞遷移：不同濃度AA(0μg/ml，5g/ml，10g/ml，20g/ml)處理HUVEC48h后，收集各組細(xì)胞，按105/孔接種于Transwell小室，下室加入含低濃度血清(2.5%FBS)的DMEM高糖培養(yǎng)基刺激細(xì)胞遷移，18~24h后，取出小室，固定染色后倒置顯微鏡下計(jì)數(shù)小室下層細(xì)胞。 5.內(nèi)皮細(xì)胞凋亡：HUVEC經(jīng)不同濃度AA(0μg/ml，5g/ml，10g/ml，20g/ml)作用48h后，每組收集≥105個(gè)細(xì)胞，Annexin V-FITC及PI染色后，于流式細(xì)胞儀檢測(cè)細(xì)胞凋亡情況。 6.線粒體膜電位變化：HUVEC經(jīng)不同濃度AA(0μg/ml，5g/ml，10g/ml，20g/ml)作用48h后，JC-1染色，激光共聚焦檢測(cè)細(xì)胞線粒體膜電位變化情況。 7.線粒體介導(dǎo)的凋亡途徑： HUVEC經(jīng)不同濃度AA(0μg/ml，5g/ml，10g/ml，20g/ml)作用48h后，收集細(xì)胞進(jìn)行western blot實(shí)驗(yàn)，觀察Caspase-3、Caspase-9、Bax、Bcl-2、Apaf-1、Cytochrome C(細(xì)胞色素C)表達(dá)情況。 結(jié)果： 第一部分： 1.經(jīng)腹腔注射AA的小鼠血Scr、BUN、尿NAG酶較對(duì)照組明顯升高，其中AA4W較AA2W組更為明顯。 2. AA組小鼠腎小管損傷、間質(zhì)纖維化、PTC損傷及丟失情況較對(duì)照組明顯，其中AA4W較AA2W組更為明顯；與AA4W相比，AA2W的小鼠腎小管上皮細(xì)胞增殖修復(fù)情況較好。相關(guān)分析顯示PTC丟失量與腎小管損傷積分高度相關(guān)，相關(guān)關(guān)系為正相關(guān)(P＜0.05)；與PCNA陽(yáng)性表達(dá)量高度相關(guān)，相關(guān)關(guān)系為負(fù)相關(guān)(P＜0.05)。 3. AA組小鼠PTC內(nèi)皮細(xì)胞中可見(jiàn)線粒體腫脹，部分線粒體脊紊亂、消失。 第二部分： 1. AA可誘導(dǎo)HUVEC活力降低、LDH釋放增加，并呈濃度依賴(lài)性。 2. AA可導(dǎo)致HUVEC體外成管量減少，成管不完整，且內(nèi)皮遷移能力下降，兩者均呈濃度依賴(lài)性。 3. AA能誘導(dǎo)HUVEC凋亡，表現(xiàn)為凋亡標(biāo)記蛋白表達(dá)增高(caspase-3、 BAX)、內(nèi)皮細(xì)胞線粒體膜電位下降、線粒體凋亡途徑的相關(guān)蛋白(Cytochrome C、Apaf-1、Caspase-9)表達(dá)上調(diào)，而抗凋亡的蛋白Bcl-2表達(dá)下調(diào)，提示AA可通過(guò)線粒體凋亡途徑誘導(dǎo)HUVEC凋亡。 結(jié)論： 1. AA能導(dǎo)致小鼠腎PTC損傷及丟失，并隨AA作用時(shí)間延長(zhǎng)，損傷逐漸加重，該損傷與腎小管損傷加重及增殖修復(fù)不良高度相關(guān)； 2. AA能誘導(dǎo)HUVEC增殖抑制、成管抑制、遷移抑制等細(xì)胞生物學(xué)活動(dòng)障礙，阻抑內(nèi)皮細(xì)胞損傷時(shí)的血管再生，此毒性作用可能影響PTC的修復(fù)和功能恢復(fù)； 3. AA能誘導(dǎo)HUVEC凋亡，該作用可能與AA激活線粒體凋亡途徑有關(guān)。
[Abstract]:Background and purpose:
Aristolochic acid (AA) belongs to the genus Aristolochic acid. Long-term use of AA or AA-containing drugs can cause renal tubular necrosis, renal interstitial fibrosis, and then develop into chronic renal failure. Therefore, scholars at home and abroad call this kind of renal disease Aristolochic acid nephropathy (AAN) [1]. Renal diseases or toxic nephropathy are different. AAN patients show renal tubular epithelial cell degeneration, necrosis, exfoliation, lack of obvious epithelial cell regeneration, basement membrane exposure, diffuse interstitial enlargement, progressive fibrosis. At present, the pathological manifestations of these special injuries are not very clear, clinical treatment is even less effective measures, AAN patients often condition. Therefore, it is necessary to explore the mechanism of tubular damage and repair in AAN. A great deal of research has been done around the main and important target of AA, tubules, and established the apoptosis, transdifferentiation, intracellular AA-DNA adduct formation, urinary system. In recent years, peritubular capillary (PTC) injury and loss or endothelial cell injury in acute and chronic kidney diseases, especially in chronic kidneys. Therefore, it is necessary to observe the damage and loss of PTC in the process of AAN, to make a systematic analysis of the relationship between PTC and renal tubular damage and poor proliferation and repair, and to further study the cytotoxic mechanism of AA on PTC injury, in order to propose a new mechanism for the treatment of aristolochic acid nephropathy. New strategies for protection and treatment.
The purpose of this study is to establish an in vivo animal model to observe the damage and loss of PTC, analyze the relationship between the loss of PTC and renal tubular injury, proliferation and repair, and further observe the ultrastructural changes of PTC endothelial cells by transmission electron microscopy; establish an in vitro injury model of human umbilical vein endothelial cells (HUVEC) by AA, and observe the toxicity of AA to endothelial cells. Cell biological changes such as proliferation, apoptosis and migration, tubules and so on, in-depth study of the mechanism of AA damage to endothelial cells, in order to provide a new mechanism and treatment strategy for clinical treatment of AAN.
Method:
This subject is divided into two parts: in vivo and in vitro.
Part one: in vivo observation of AA damage to PTC and its correlation with renal tubular injury and repair.
1. Establishment of AAN mice model. C57/BL/6 mice were divided into four groups: control 2W group, AA2W group, control 4W group, AA4W group. AA mice were injected AA solution intraperitoneally every two days at a dose of 5mg/kg, and control mice were injected with the same volume of buffer solution (PBS).
2. Blood, urine and kidney tissues were collected at 2 and 4 weeks respectively for biochemical analysis and pathological observation.
3. to analyze the relationship between PTC damage and renal tubular injury, proliferation and repair in mice.
The second part: in vitro study of the mechanism of AA damaging vascular endothelial cells.
1. Human umbilical vein endothelial cell line (HUVEC) was treated with different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml) and different time (24 h, 48 h, 72 h). MTT assay was used to detect the effect of AA on the proliferation of human umbilical vein endothelial cell line (HUVEC).
2. LDH release: Different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml) were used to treat HUVEC for 48h (the best time) to detect the release of LDH in cytoplasm.
3. Endothelial cell tube formation: After treated with different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml) for 48 hours, HUVEC cells were collected and transferred to 96-well plate coated with matrix gel. 103-104 cells per hole were incubated for 6 hours.
4. Endothelial cell migration: After 48 hours of treatment with different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml), the cells in each group were collected and inoculated into the Transwell chamber according to 105/hole. The cells were stimulated by DMEM high glucose medium containing low concentration of serum (2.5% FBS) in the lower chamber. After 18-24 hours, the cells were removed from the chamber and stained and counted under the inverted microscope.
5. Endothelial cell apoptosis: After 48 hours of exposure to different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml), more than 105 cells were collected from each group. After staining with Annexin V-FITC and PI, the apoptosis was detected by flow cytometry.
6. Mitochondrial membrane potential changes: HUVEC treated with different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml) for 48 hours, JC-1 staining, laser confocal detection of cell mitochondrial membrane potential changes.
7. Mitochondrial-mediated apoptosis pathway: After 48 hours of exposure to different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml), HUVEC cells were collected for Western blot assay to observe the expression of Caspase-3, Caspase-9, Bax, Bcl-2, Apaf-1, Cytochrome C.
Result:
Part one:
1. The levels of serum Scr, BUN and urinary NAG enzymes in AA-injected mice were significantly higher than those in the control group, especially in AA4W group.
2. The renal tubular injury, interstitial fibrosis, PTC injury and loss in AA group were more obvious than those in control group, especially in AA4W group. Compared with AA4W group, the proliferation and repair of renal tubular epithelial cells in AA2W group were better. Correlation analysis showed that the loss of PTC was highly correlated with the renal tubular injury score, and the correlation was positive (P < 0.05). It was highly correlated with the positive expression level of PCNA, and the correlation was negatively correlated (P < 0.05).
3. mitochondria of PTC endothelial cells in group AA showed swelling of mitochondria and partial mitochondrial ridge disappearance.
The second part:
1. AA could induce the decrease of HUVEC activity and the increase of LDH release in a concentration dependent manner.
2. AA can decrease the volume of tube formation, incomplete tube formation and the ability of endothelial migration of HUVEC in vitro, both of which are concentration-dependent.
3. AA can induce apoptosis of HUVEC, which is manifested by increased expression of apoptosis marker protein (caspase-3, BAX), decreased mitochondrial membrane potential, up-regulated expression of mitochondrial apoptosis pathway-related proteins (Cytochrome C, Apaf-1, Caspase-9), and down-regulated expression of anti-apoptotic proteins Bcl-2, suggesting that AA can induce apoptosis of HUVEC through mitochondrial apoptosis pathway.
Conclusion:
1. AA can cause the damage and loss of PTC in mice kidney, and with the prolongation of AA action time, the damage is gradually aggravated, which is highly related to the aggravation of renal tubular injury and poor proliferation and repair.
2. AA can induce proliferation inhibition, tubular inhibition and migration inhibition of HUVEC, and inhibit vascular regeneration during endothelial cell injury, which may affect the repair and functional recovery of PTC.
3. AA can induce HUVEC apoptosis, which may be related to AA activation of mitochondrial apoptotic pathway.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R692
本文編號(hào)：2181833
[Abstract]:Background and purpose:
Aristolochic acid (AA) belongs to the genus Aristolochic acid. Long-term use of AA or AA-containing drugs can cause renal tubular necrosis, renal interstitial fibrosis, and then develop into chronic renal failure. Therefore, scholars at home and abroad call this kind of renal disease Aristolochic acid nephropathy (AAN) [1]. Renal diseases or toxic nephropathy are different. AAN patients show renal tubular epithelial cell degeneration, necrosis, exfoliation, lack of obvious epithelial cell regeneration, basement membrane exposure, diffuse interstitial enlargement, progressive fibrosis. At present, the pathological manifestations of these special injuries are not very clear, clinical treatment is even less effective measures, AAN patients often condition. Therefore, it is necessary to explore the mechanism of tubular damage and repair in AAN. A great deal of research has been done around the main and important target of AA, tubules, and established the apoptosis, transdifferentiation, intracellular AA-DNA adduct formation, urinary system. In recent years, peritubular capillary (PTC) injury and loss or endothelial cell injury in acute and chronic kidney diseases, especially in chronic kidneys. Therefore, it is necessary to observe the damage and loss of PTC in the process of AAN, to make a systematic analysis of the relationship between PTC and renal tubular damage and poor proliferation and repair, and to further study the cytotoxic mechanism of AA on PTC injury, in order to propose a new mechanism for the treatment of aristolochic acid nephropathy. New strategies for protection and treatment.
The purpose of this study is to establish an in vivo animal model to observe the damage and loss of PTC, analyze the relationship between the loss of PTC and renal tubular injury, proliferation and repair, and further observe the ultrastructural changes of PTC endothelial cells by transmission electron microscopy; establish an in vitro injury model of human umbilical vein endothelial cells (HUVEC) by AA, and observe the toxicity of AA to endothelial cells. Cell biological changes such as proliferation, apoptosis and migration, tubules and so on, in-depth study of the mechanism of AA damage to endothelial cells, in order to provide a new mechanism and treatment strategy for clinical treatment of AAN.
Method:
This subject is divided into two parts: in vivo and in vitro.
Part one: in vivo observation of AA damage to PTC and its correlation with renal tubular injury and repair.
1. Establishment of AAN mice model. C57/BL/6 mice were divided into four groups: control 2W group, AA2W group, control 4W group, AA4W group. AA mice were injected AA solution intraperitoneally every two days at a dose of 5mg/kg, and control mice were injected with the same volume of buffer solution (PBS).
2. Blood, urine and kidney tissues were collected at 2 and 4 weeks respectively for biochemical analysis and pathological observation.
3. to analyze the relationship between PTC damage and renal tubular injury, proliferation and repair in mice.
The second part: in vitro study of the mechanism of AA damaging vascular endothelial cells.
1. Human umbilical vein endothelial cell line (HUVEC) was treated with different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml) and different time (24 h, 48 h, 72 h). MTT assay was used to detect the effect of AA on the proliferation of human umbilical vein endothelial cell line (HUVEC).
2. LDH release: Different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml) were used to treat HUVEC for 48h (the best time) to detect the release of LDH in cytoplasm.
3. Endothelial cell tube formation: After treated with different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml) for 48 hours, HUVEC cells were collected and transferred to 96-well plate coated with matrix gel. 103-104 cells per hole were incubated for 6 hours.
4. Endothelial cell migration: After 48 hours of treatment with different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml), the cells in each group were collected and inoculated into the Transwell chamber according to 105/hole. The cells were stimulated by DMEM high glucose medium containing low concentration of serum (2.5% FBS) in the lower chamber. After 18-24 hours, the cells were removed from the chamber and stained and counted under the inverted microscope.
5. Endothelial cell apoptosis: After 48 hours of exposure to different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml), more than 105 cells were collected from each group. After staining with Annexin V-FITC and PI, the apoptosis was detected by flow cytometry.
6. Mitochondrial membrane potential changes: HUVEC treated with different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml) for 48 hours, JC-1 staining, laser confocal detection of cell mitochondrial membrane potential changes.
7. Mitochondrial-mediated apoptosis pathway: After 48 hours of exposure to different concentrations of AA (0 ug/ml, 5 g/ml, 10 g/ml, 20 g/ml), HUVEC cells were collected for Western blot assay to observe the expression of Caspase-3, Caspase-9, Bax, Bcl-2, Apaf-1, Cytochrome C.
Result:
Part one:
1. The levels of serum Scr, BUN and urinary NAG enzymes in AA-injected mice were significantly higher than those in the control group, especially in AA4W group.
2. The renal tubular injury, interstitial fibrosis, PTC injury and loss in AA group were more obvious than those in control group, especially in AA4W group. Compared with AA4W group, the proliferation and repair of renal tubular epithelial cells in AA2W group were better. Correlation analysis showed that the loss of PTC was highly correlated with the renal tubular injury score, and the correlation was positive (P < 0.05). It was highly correlated with the positive expression level of PCNA, and the correlation was negatively correlated (P < 0.05).
3. mitochondria of PTC endothelial cells in group AA showed swelling of mitochondria and partial mitochondrial ridge disappearance.
The second part:
1. AA could induce the decrease of HUVEC activity and the increase of LDH release in a concentration dependent manner.
2. AA can decrease the volume of tube formation, incomplete tube formation and the ability of endothelial migration of HUVEC in vitro, both of which are concentration-dependent.
3. AA can induce apoptosis of HUVEC, which is manifested by increased expression of apoptosis marker protein (caspase-3, BAX), decreased mitochondrial membrane potential, up-regulated expression of mitochondrial apoptosis pathway-related proteins (Cytochrome C, Apaf-1, Caspase-9), and down-regulated expression of anti-apoptotic proteins Bcl-2, suggesting that AA can induce apoptosis of HUVEC through mitochondrial apoptosis pathway.
Conclusion:
1. AA can cause the damage and loss of PTC in mice kidney, and with the prolongation of AA action time, the damage is gradually aggravated, which is highly related to the aggravation of renal tubular injury and poor proliferation and repair.
2. AA can induce proliferation inhibition, tubular inhibition and migration inhibition of HUVEC, and inhibit vascular regeneration during endothelial cell injury, which may affect the repair and functional recovery of PTC.
3. AA can induce HUVEC apoptosis, which may be related to AA activation of mitochondrial apoptotic pathway.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R692
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 劉圓圓;李藝;王帥;官濤;鄭科;黃云劍;;馬兜鈴酸致腎小管周?chē)?xì)血管丟失的體內(nèi)實(shí)驗(yàn)研究[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2014年06期
本文編號(hào)：2181833
本文鏈接：http://sikaile.net/yixuelunwen/mjlw/2181833.html
最近更新
教材專(zhuān)著
