外源性一氧化碳对移植肾小管上皮—间质转化影响及其机制的实验研究
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摘要
肾移植是肾功能衰竭患者的有效也是最终治疗手段。目前移植肾的1年存活率己经超过90%,但其长期生存率并没有明显改善,接近半数的移植患者在10年内会出现移植肾纤维化和功能衰竭,这主要归因于移植肾脏的慢性排斥反应。尽管具体的分子和细胞机制尚不清楚,但目前可以肯定非免疫因素和免疫因素在慢性移植物功能障碍的发病机理中都起重要作用。
肾脏慢性排斥反应的主要病理特征是以实质细胞进行性减少和细胞外基质(ECM)的过度聚集为表现的肾脏纤维化。ECM主要由成纤维细胞产生。过度表达的成纤维细胞来源于发生了表型转化的肾小管上皮细胞。肾小管上皮细胞在特定条件下发生形态和功能的改变,转化为具有活动能力的间质细胞,即所谓的上皮-间质转化(epithelial-mesenchymal transition, EMT)。EMT的分子学表现为进行性的上皮细胞标志物E-钙粘素(E-cadherin)的表达下降和持续性的肌成纤维细胞标志物α-SMA的表达升高。
研究指出TGF-β1是调控肾小管EMT的关键细胞因子,其作用包括抑制上皮细胞标志物E-钙粘素(E-cadherin)的表达、上调肌成纤维细胞标志物α-SMA的表达,以及增加ECM的聚集等。但生理表达量的TGF-β1是一种强效的免疫抑制和抗炎因子,能显著抑制急性排斥反应和诱导免疫耐受,阻断其表达并不明智。研究指出,过量表达的TGF-p1相当一部分来自于T细胞及单核-巨噬细胞浸润,通过抑制免疫细胞浸润这一过程必然限制肾小管EMT进展。
血红素加氧酶-1(HO-1)被发现具有明确的抑制免疫细胞浸润作用。其在体内催化血红素分解生成胆绿素、一氧化碳(carbon monoxide, CO)和游离铁,而CO在几乎所有的损伤或疾病模型中部分甚至完全替代了HO-1所提供的保护作用。因此,我们在此基础上推测CO在肾移植背景下对免疫细胞具有确切的抑制功能。进一步推测CO对移植肾小管EMT可能有显著的抑制或逆转作用。
目的
1.先期探索并优化手术操作技术,建立稳定的DA-WF大鼠移植肾动物模型。
2.在已建立的移植肾模型基础上,通过相关染色检验明确移植肾组织间质纤维化的出现,并证实移植肾脏中上皮-间质转化(EMT)的存在。
3.应用一氧化碳释放分子CORM-2对DA-WF移植肾间质纤维化模型进行干预,以获取课题试验用组织标本。
4.通过对组织标本的相关检验进一步研究CO是否通过抑制免疫细胞浸润这一机
制来抑制或逆转移植肾EMT,进而显著延缓移植肾纤维化进程。
方法
本课题分四部分进行：
1.利用SD大鼠探索并优化同种异体肾移植手术操作技术。水合氯醛麻醉大鼠,腹主动脉直接插管原位灌注的方式处理供肾,以供、受体肾脏同名血管端端吻合、输尿管端端吻合的方式移植肾脏,受体自体肾采用“肾血管体外延迟结扎”方式处理。
2. Dark Agouti (DA)(RTlavl)大鼠为供体,Wistar-Furth (WF)(RTlu)大鼠为受体建立同种异体肾移植(DA-WF)模型并标记为实验组,WF-WF大鼠肾移植做对照组。术前3d及术后2w内WF受体大鼠接受10mg/kg/d的环孢素皮下注射。于术后第2w、4w、8w、12w测量大鼠体重并测血肌酐和尿素氮,动态监测大鼠一般情况和肾功能变化。取第12w实验组及对照组移植肾脏组织分别行H&E染色. Masson染色、E-cadherin免疫组化. a-SMA免疫组化。采用HPIAS-1000彩色病理图像分析仪分析、SPSS15.0统计软件处理数据。
3.建立DA-WF肾移植模型,随机编号分为两组,一组行CORM-2干预,一组行iCORM-2干预,WF-WF做空白参照。每组内的大鼠选取固定数目在术后第2w、4w、8w、12w分别称重并处死。留取处死受体大鼠移植肾组织及腔静脉血。移植肾组织备行进一步实验,腔静脉血检测血肌酐、尿素氮指标进行分析。使用SPSS15.0统计软件处理数据。
4.将第2w、4w、8w、12w共四批次行CORM-2干预,iCORM-2干预及空白参照移的植肾组织标本分别行H&E染色、Masson染色、E-cadherin免疫组化、α-SMA免疫组化、CD3免疫组化、CD163免疫组化。采用HPIAS-1000彩色病理图像分析仪分析、SPSS15.0统计软件处理数据。
结论
1.通过先期探索及练习,基本掌握了建立大鼠肾移植模型的相关技术,能基本保证模型的成活率。为课题实验提供了技术支持及相关经验。
2.通过建立并对比DA-WF、WF-WF两组肾移植模型的术后一般状况、移植肾功能比较及第12w移植肾组织的HE染色、Masson染色和移植肾组织的E-cadherin和α-SMA的免疫组化染色证实DA-WF大鼠肾移植模型诱导了慢性排斥反应,出现了肾脏间质纤维化改变,并且证实了移植肾脏模型中EMT的存在。
3.在对CORM-2干预,iCORM-2干预、空白参照三组大鼠肾移植模型的移植肾功能的检测结果分析中可以证实一氧化碳释放分子CORM-2可以抑制移植肾脏EMT的进程,预测CORM-2在体内释放的CO应具有某种移植肾保护作用。
4.通过对CORM-2干预,iCORM-2干预及空白参照移植肾组织标本的HE染色、Masson染色及E-cadherin、α-SMA、CD3T细胞和CD-163巨噬细胞的免疫组化研究分析得出结论,CORM-2释放的CO对移植肾小管EMT有显著的抑制或逆转作用,这种作用的可能机制为CO在肾移植背景下对T淋巴细胞、巨噬细胞具有确切的抑制功能。
Background:
Kidney transplantation is an effective and final treatment of patients with renal failure. Now, The1-year graft survival rate is more than90percent, but its long-term survival has no significant improvement. In nearly half of transplant patients'kindney will be in fibrosis and failure within10years, which is mainly attributed to chronic kidney transplant rejection. Although the specific molecular and cellular mechanisms are unclear, but the non-immunological factors and immune factors plays an important role are certainly.
The main pathological features of chronic renal rejection are the excessive accumulation of parenchymal cells and the progressive reduction of extracellular matrix (ECM). ECM is mainly generated from the fibroblasts. The over-expression fibroblasts derived from the phenotypic transition of tubular epithelial cells. In certain conditions, the renal tubular epithelial cells change it's morphological and functional, and transformate into the activity of mesenchymal cells, named the epithelial-mesenchymal transition (epithelial-mesenchymal transition EMT). EMT molecular manifestations of decreased expression of epithelial cell marker E-cadherin (E-cadherin) and continuous myofibroblast cell marker a-SMA.
The TGF-β1is a key cytokine in the regulation of renal tubular EMT. Its role is inhibit the expression of epithelial cell marker E-cadherin, raising the expression of myofibroblast marker α-SMA, and increase the ECM accumulation. The physiological expression of TGF-β1is a potent immunosuppressive and anti-inflammatory factor, it can inhibit acute rejection and induce the immune tolerance significantly. So, it is not wise to block its expression. Studies have indicated that the overexpressed TGF-β1is considerablly part from the T cells and monocytes-macrophage cells. The EMT progression should be limited by inhibiting the infiltration of immune cells.
Heme oxygenase-1(HO-1) was found to have the action of suppress of immune cell infiltration. It can catalysis heme to generate biliverdin, carbon monoxide (CO) and free iron in body. CO can completely or partly replace the protective effect of HO-1in almost all of the injury and disease models. On this basis, we speculate that the CO should have the exact suppression of immune cells action in the context of renal transplantation. Further speculated that the CO have the significantly action of inhibit or reverse the renal tubular EMT.
Objective:
1. Exploration and optimization of the operative techniques to establish a stable DA-WF rats transplanted kidney animal model.
2. Identify the graft interstitial fibrosis appears by relevant stained test on the basis of established renal transplantation model, and confirm the presence of epithelial-mesenchymal transition (EMT) in transplanted kidney.
3. Interference the DA-WF model with carbon monoxide released molecule (CORM-2), and gain the tissue.
4. Study CO inhibit or reverse the transplanted kidney's EMT, significantly delay the process of renal allograft fibrosis is in the mechanism of suppress the immune cell infiltration by testing this tissues.
Method
This topic is divided into four parts:
1. Exploration and optimization of the operative techniques of kidney transplantation surgery with SD rats. Chloral hydrate anesthetize the rats, the donor kidney perfusion with abdominal aortic intubation in situ. The donor kidney are transplant to the receptor in the same name renal vascular and ureteral. The receptor's another kidney is deal with the manner of blood vessels in vitro delayed ligation.
2. To establishe the model of kidney transplantation with Dark Agouti (DA)(RTlavl) rats for the donor, Wistar-Furth (WF)(RTlu) rats for the receptor, and marked as the experimental group. The WF-WF rats kidney transplantation marked as the control group. The WF receptors receive10mg/kg/d of cyclosporine subcutaneous injection preoperative3d and postoperative2w. Measure the body weight of rats, blood serum creatinine and blood urea nitrogen at postoperative2w,4w,8w,12w, to dynamic monitor the general situation of rats and kidney function. Take the12w experimental group and control group transplanted kidney tissue to perform H&E staine, Masson staine, E-cadherin immunohistochemistry, α-SMA immunohistochemistry. Analysis with HPIAS-1000analyzer, and SPSS15.0statistical software processing data.
3. Establishe the DA-WF kidney transplant model, random numbers and divide into two groups, one group of CORM-2intervention, another of iCORM-2intervention. The WF-WF are blank reference. Select a fixed number of rats in each group to weigh and sacrifice at postoperative2w,4w,8w,12w. Remain the sacrificed receptors's kidney and cavity blood. The kidney allograft tissues are equipped for further experiments, the cavity blood is to detect blood serum creatinine and urea nitrogen. Analysis the data with SPSS15.0statistical software.
4. Take the kidney allograft tissues intervention CORM-2, iCORM-2and blank reference at postoperative2w,4w.8w,12w, to perform H&E staine, Masson staine, E-cadherin immunohistochemistry, a-SMA immunohistochemistry, CD3immunohistochemistry, CD163immunohistochemistry. Analysis with HPIAS-1000analyzer, and SPSS15.0statistical software processing data.
Conclusion
1. Through the early exploration and practice, grasp the technic of establish the rat kidney transplant model, to guarantee the survival rate of the model, and to provide technical support and relevant experience for the subject of experiments
2. To establish and compare the two groups of kidney transplantation of DA-WF, WF-WF postoperative's general condition, graft function, and the12w transplanted kidney tissue HE staine, Masson staine, E-cadherin immunohistochemical, α-SMA immunohistochemical to confirm that of DA-WF rat renal transplantation model induce chronic rejection, a renal interstitial fibrosis, and confirm the presence of the EMT in the kidney transplant model.
3. By the results of detection of the three groups'(CORM-2intervention, iCORM-2intervention, blank reference) renal function, we can confirm that the carbon monoxide release molecule (CORM-2) can inhibit the process EMT of kidney transplant, prediction that the CO released by CORM-2in vivo should have some sort of transplant renal protective effects.
4. By the analysis of CORM-2intervention, iCORM-2intervention and blank reference tissue's H&E staine, Masson staine, E-cadherin immunohistochemistry, α-SMA immunohistochemistry, CD3immunohistochemistry, CD163immunohistochemistry. We can confirm that the CO released by CORM-2should significantly inhibit or reverse the transplant renal tubular EMT. and the possible mechanism of this effect should the exact suppression of T lymphocytes, macrophages in the context of renal transplantation.
肾脏慢性排斥反应的主要病理特征是以实质细胞进行性减少和细胞外基质(ECM)的过度聚集为表现的肾脏纤维化。ECM主要由成纤维细胞产生。过度表达的成纤维细胞来源于发生了表型转化的肾小管上皮细胞。肾小管上皮细胞在特定条件下发生形态和功能的改变,转化为具有活动能力的间质细胞,即所谓的上皮-间质转化(epithelial-mesenchymal transition, EMT)。EMT的分子学表现为进行性的上皮细胞标志物E-钙粘素(E-cadherin)的表达下降和持续性的肌成纤维细胞标志物α-SMA的表达升高。
研究指出TGF-β1是调控肾小管EMT的关键细胞因子,其作用包括抑制上皮细胞标志物E-钙粘素(E-cadherin)的表达、上调肌成纤维细胞标志物α-SMA的表达,以及增加ECM的聚集等。但生理表达量的TGF-β1是一种强效的免疫抑制和抗炎因子,能显著抑制急性排斥反应和诱导免疫耐受,阻断其表达并不明智。研究指出,过量表达的TGF-p1相当一部分来自于T细胞及单核-巨噬细胞浸润,通过抑制免疫细胞浸润这一过程必然限制肾小管EMT进展。
血红素加氧酶-1(HO-1)被发现具有明确的抑制免疫细胞浸润作用。其在体内催化血红素分解生成胆绿素、一氧化碳(carbon monoxide, CO)和游离铁,而CO在几乎所有的损伤或疾病模型中部分甚至完全替代了HO-1所提供的保护作用。因此,我们在此基础上推测CO在肾移植背景下对免疫细胞具有确切的抑制功能。进一步推测CO对移植肾小管EMT可能有显著的抑制或逆转作用。
目的
1.先期探索并优化手术操作技术,建立稳定的DA-WF大鼠移植肾动物模型。
2.在已建立的移植肾模型基础上,通过相关染色检验明确移植肾组织间质纤维化的出现,并证实移植肾脏中上皮-间质转化(EMT)的存在。
3.应用一氧化碳释放分子CORM-2对DA-WF移植肾间质纤维化模型进行干预,以获取课题试验用组织标本。
4.通过对组织标本的相关检验进一步研究CO是否通过抑制免疫细胞浸润这一机
制来抑制或逆转移植肾EMT,进而显著延缓移植肾纤维化进程。
方法
本课题分四部分进行：
1.利用SD大鼠探索并优化同种异体肾移植手术操作技术。水合氯醛麻醉大鼠,腹主动脉直接插管原位灌注的方式处理供肾,以供、受体肾脏同名血管端端吻合、输尿管端端吻合的方式移植肾脏,受体自体肾采用“肾血管体外延迟结扎”方式处理。
2. Dark Agouti (DA)(RTlavl)大鼠为供体,Wistar-Furth (WF)(RTlu)大鼠为受体建立同种异体肾移植(DA-WF)模型并标记为实验组,WF-WF大鼠肾移植做对照组。术前3d及术后2w内WF受体大鼠接受10mg/kg/d的环孢素皮下注射。于术后第2w、4w、8w、12w测量大鼠体重并测血肌酐和尿素氮,动态监测大鼠一般情况和肾功能变化。取第12w实验组及对照组移植肾脏组织分别行H&E染色. Masson染色、E-cadherin免疫组化. a-SMA免疫组化。采用HPIAS-1000彩色病理图像分析仪分析、SPSS15.0统计软件处理数据。
3.建立DA-WF肾移植模型,随机编号分为两组,一组行CORM-2干预,一组行iCORM-2干预,WF-WF做空白参照。每组内的大鼠选取固定数目在术后第2w、4w、8w、12w分别称重并处死。留取处死受体大鼠移植肾组织及腔静脉血。移植肾组织备行进一步实验,腔静脉血检测血肌酐、尿素氮指标进行分析。使用SPSS15.0统计软件处理数据。
4.将第2w、4w、8w、12w共四批次行CORM-2干预,iCORM-2干预及空白参照移的植肾组织标本分别行H&E染色、Masson染色、E-cadherin免疫组化、α-SMA免疫组化、CD3免疫组化、CD163免疫组化。采用HPIAS-1000彩色病理图像分析仪分析、SPSS15.0统计软件处理数据。
结论
1.通过先期探索及练习,基本掌握了建立大鼠肾移植模型的相关技术,能基本保证模型的成活率。为课题实验提供了技术支持及相关经验。
2.通过建立并对比DA-WF、WF-WF两组肾移植模型的术后一般状况、移植肾功能比较及第12w移植肾组织的HE染色、Masson染色和移植肾组织的E-cadherin和α-SMA的免疫组化染色证实DA-WF大鼠肾移植模型诱导了慢性排斥反应,出现了肾脏间质纤维化改变,并且证实了移植肾脏模型中EMT的存在。
3.在对CORM-2干预,iCORM-2干预、空白参照三组大鼠肾移植模型的移植肾功能的检测结果分析中可以证实一氧化碳释放分子CORM-2可以抑制移植肾脏EMT的进程,预测CORM-2在体内释放的CO应具有某种移植肾保护作用。
4.通过对CORM-2干预,iCORM-2干预及空白参照移植肾组织标本的HE染色、Masson染色及E-cadherin、α-SMA、CD3T细胞和CD-163巨噬细胞的免疫组化研究分析得出结论,CORM-2释放的CO对移植肾小管EMT有显著的抑制或逆转作用,这种作用的可能机制为CO在肾移植背景下对T淋巴细胞、巨噬细胞具有确切的抑制功能。
Background:
Kidney transplantation is an effective and final treatment of patients with renal failure. Now, The1-year graft survival rate is more than90percent, but its long-term survival has no significant improvement. In nearly half of transplant patients'kindney will be in fibrosis and failure within10years, which is mainly attributed to chronic kidney transplant rejection. Although the specific molecular and cellular mechanisms are unclear, but the non-immunological factors and immune factors plays an important role are certainly.
The main pathological features of chronic renal rejection are the excessive accumulation of parenchymal cells and the progressive reduction of extracellular matrix (ECM). ECM is mainly generated from the fibroblasts. The over-expression fibroblasts derived from the phenotypic transition of tubular epithelial cells. In certain conditions, the renal tubular epithelial cells change it's morphological and functional, and transformate into the activity of mesenchymal cells, named the epithelial-mesenchymal transition (epithelial-mesenchymal transition EMT). EMT molecular manifestations of decreased expression of epithelial cell marker E-cadherin (E-cadherin) and continuous myofibroblast cell marker a-SMA.
The TGF-β1is a key cytokine in the regulation of renal tubular EMT. Its role is inhibit the expression of epithelial cell marker E-cadherin, raising the expression of myofibroblast marker α-SMA, and increase the ECM accumulation. The physiological expression of TGF-β1is a potent immunosuppressive and anti-inflammatory factor, it can inhibit acute rejection and induce the immune tolerance significantly. So, it is not wise to block its expression. Studies have indicated that the overexpressed TGF-β1is considerablly part from the T cells and monocytes-macrophage cells. The EMT progression should be limited by inhibiting the infiltration of immune cells.
Heme oxygenase-1(HO-1) was found to have the action of suppress of immune cell infiltration. It can catalysis heme to generate biliverdin, carbon monoxide (CO) and free iron in body. CO can completely or partly replace the protective effect of HO-1in almost all of the injury and disease models. On this basis, we speculate that the CO should have the exact suppression of immune cells action in the context of renal transplantation. Further speculated that the CO have the significantly action of inhibit or reverse the renal tubular EMT.
Objective:
1. Exploration and optimization of the operative techniques to establish a stable DA-WF rats transplanted kidney animal model.
2. Identify the graft interstitial fibrosis appears by relevant stained test on the basis of established renal transplantation model, and confirm the presence of epithelial-mesenchymal transition (EMT) in transplanted kidney.
3. Interference the DA-WF model with carbon monoxide released molecule (CORM-2), and gain the tissue.
4. Study CO inhibit or reverse the transplanted kidney's EMT, significantly delay the process of renal allograft fibrosis is in the mechanism of suppress the immune cell infiltration by testing this tissues.
Method
This topic is divided into four parts:
1. Exploration and optimization of the operative techniques of kidney transplantation surgery with SD rats. Chloral hydrate anesthetize the rats, the donor kidney perfusion with abdominal aortic intubation in situ. The donor kidney are transplant to the receptor in the same name renal vascular and ureteral. The receptor's another kidney is deal with the manner of blood vessels in vitro delayed ligation.
2. To establishe the model of kidney transplantation with Dark Agouti (DA)(RTlavl) rats for the donor, Wistar-Furth (WF)(RTlu) rats for the receptor, and marked as the experimental group. The WF-WF rats kidney transplantation marked as the control group. The WF receptors receive10mg/kg/d of cyclosporine subcutaneous injection preoperative3d and postoperative2w. Measure the body weight of rats, blood serum creatinine and blood urea nitrogen at postoperative2w,4w,8w,12w, to dynamic monitor the general situation of rats and kidney function. Take the12w experimental group and control group transplanted kidney tissue to perform H&E staine, Masson staine, E-cadherin immunohistochemistry, α-SMA immunohistochemistry. Analysis with HPIAS-1000analyzer, and SPSS15.0statistical software processing data.
3. Establishe the DA-WF kidney transplant model, random numbers and divide into two groups, one group of CORM-2intervention, another of iCORM-2intervention. The WF-WF are blank reference. Select a fixed number of rats in each group to weigh and sacrifice at postoperative2w,4w,8w,12w. Remain the sacrificed receptors's kidney and cavity blood. The kidney allograft tissues are equipped for further experiments, the cavity blood is to detect blood serum creatinine and urea nitrogen. Analysis the data with SPSS15.0statistical software.
4. Take the kidney allograft tissues intervention CORM-2, iCORM-2and blank reference at postoperative2w,4w.8w,12w, to perform H&E staine, Masson staine, E-cadherin immunohistochemistry, a-SMA immunohistochemistry, CD3immunohistochemistry, CD163immunohistochemistry. Analysis with HPIAS-1000analyzer, and SPSS15.0statistical software processing data.
Conclusion
1. Through the early exploration and practice, grasp the technic of establish the rat kidney transplant model, to guarantee the survival rate of the model, and to provide technical support and relevant experience for the subject of experiments
2. To establish and compare the two groups of kidney transplantation of DA-WF, WF-WF postoperative's general condition, graft function, and the12w transplanted kidney tissue HE staine, Masson staine, E-cadherin immunohistochemical, α-SMA immunohistochemical to confirm that of DA-WF rat renal transplantation model induce chronic rejection, a renal interstitial fibrosis, and confirm the presence of the EMT in the kidney transplant model.
3. By the results of detection of the three groups'(CORM-2intervention, iCORM-2intervention, blank reference) renal function, we can confirm that the carbon monoxide release molecule (CORM-2) can inhibit the process EMT of kidney transplant, prediction that the CO released by CORM-2in vivo should have some sort of transplant renal protective effects.
4. By the analysis of CORM-2intervention, iCORM-2intervention and blank reference tissue's H&E staine, Masson staine, E-cadherin immunohistochemistry, α-SMA immunohistochemistry, CD3immunohistochemistry, CD163immunohistochemistry. We can confirm that the CO released by CORM-2should significantly inhibit or reverse the transplant renal tubular EMT. and the possible mechanism of this effect should the exact suppression of T lymphocytes, macrophages in the context of renal transplantation.
引文
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