西罗莫司对大鼠肾脏缺血再灌注损伤早期的保护及对肾小管上皮细胞修复的影响
发布时间：2018-08-27 19:58
【摘要】：缺血再灌注损伤是实体器官移植不可避免的一个阶段,在肾移植中,因肾脏缺血再灌注损伤的存在,可显著增加肾移植术后急性排斥反应发生率,同样肾小管上皮细胞的修复需要相对较长一段时间,从而导致肾移植术后移植肾功能延迟恢复,从长远考虑可能会影响移植肾和受者的长期存活。肾小管细胞培养模型、动物实验和临床研究均提示肾脏缺血再灌注损伤介导的肾小管上皮细胞死亡有坏死和凋亡两种形式。早期即出现大量肾小管上皮细胞凋亡和坏死,肾小管上皮细胞受累后主要发生去分化,细胞骨架的完整性和极性受到损害,最终导致肾组织结构破坏,从而影响肾功能。晚期在各种修复机制的作用下发生去分化的肾小管上皮细胞增殖、移行,并进行再分化,肾小管上皮细胞增殖的逐步启动后,经历一段时间后受损的部分肾功能逐渐恢复。作为实体器官移植与缺血再灌注损伤并存的现状,我们应采取积极有效手段和方法,最大程度上抑制肾小管上皮细胞的凋亡,减轻肾脏缺血再灌注损伤带来的不利影响,保护肾功能,对于减少肾移植术后移植肾功能延迟恢复以及急性排斥反应发生率,最终达到延长移植肾和受者的存活时间的目的,具有深远的临床意义。 西罗莫司(SRL)是哺乳类SRL靶分子(mammalian target of rapamycin, mTOR)抑制剂,动物实验和多年的临床应用结果均显示,以SRL为主而不含CNI的免疫抑制方案或将CNI类免疫抑制剂转换为以西罗莫司为主的免疫抑制方案后与以CNI为基础的免疫抑制方案相比,能显著降低因CNI类肾毒性造成的肾移植术后血肌酐呈爬行性上升的趋势,肾功能明显好转。因西罗莫司几乎无肾脏毒性,肝毒性较低,且与CNI类为基础的免疫抑制方案相比,患者的1年移植物存活率、急性排斥反应发生率均无明显差异,肾功能却明显优于CNI组。对肾移植术后血肌酐呈阶梯性、爬行性慢性上升的患者,并经病理确诊的因CNI类肾毒性和慢性移植肾病的患者,适时的进行SRL转换治疗可明显改善移植肾功能,延长移植肾和受者的存活时间。 目前,关于SRL对缺血再灌注损伤的作用仍存在争议。研究发现SRL可能通过以下机制明显减轻大鼠肾脏缺血再灌注损伤和改善肾功能：(1)通过非直接通路激活T细胞凋亡程序,阻止了树突状细胞(DC)的抗原呈递,并进一步抑制其成熟；(2)减少细胞因子的分泌,阻止DC在肾组织迁移聚集；周四桂等用SRL处理经缺氧复氧的血管内皮细胞,通过抑制ROS、JNK、NF-κB的信号传导途径,抑制血管内皮细胞与中性粒细胞的粘附。Yang B等发现使用SRL干预肾脏缺血再灌注损伤,主要是激活了24KDCaspase-3,减少凋亡的发生,同时减少远曲小管中Fas的表达,通过减轻Fas信号途径引起的免疫损伤,起到保护缺血肾脏再灌注损伤的作用。Bohmova R等在转基因高血压大鼠肾缺血再灌注损伤中观察发现,小剂量的SRL能够减轻由于肾素产生过多引起的肾脏再灌注损伤。但是也有一些学者持相反的观点,在体外,SRL在1ng/ml时抑制鼠的近曲小管细胞的增生,同时通过抑制肾小管细胞中70kDaS6蛋白激酶来发挥促进凋亡作用。在体内SRL通过抑制肾小管细胞再生和增加肾小管细胞的凋亡,抑制肾缺血再灌注后肾小管的再生,影响损伤的修复,从而延缓肾功能的恢复。Lui SL等报道SRL在1天、3天时降低增殖细胞核抗原(PCNA)的表达,但是第7天时与对照组没有差别,表明SRL会加重肾脏的损害主要是通过抑制肾小管细胞的再生而延缓了肾功能的恢复,但并不是一直抑制肾小管细胞的再生。 在缺血再灌注损伤中,不同剂量的SRL对不同时期大鼠肾缺血再灌注损伤及肾小管上皮细胞凋亡后增殖、修复作用尚不明确。我们推测,在缺血再灌注损伤早期,SRL的抗凋亡作用可能会抑制肾小管上皮细胞的坏死,起到对损伤肾脏组织的保护作用,但在后期随着肾小管上皮细胞修复程序的逐步启动,其抗增殖作用可能会抑制肾小管上皮细胞的细胞增殖、移行、再分化以及骨架的重建,进而影响肾功能恢复并存在剂量的依赖性和可逆性。我们研究发现给于大鼠SRL3mg/(kg.d),其谷浓度为肾移植术后早期治疗浓度,以此剂量为标准我们建立大鼠肾缺血再灌注损伤模型,通过检测肾脏损伤敏感标志物NGAL、IL-18;凋亡基因Fas、Bcl-2和PCNA蛋白表达,肾小管上皮细胞修复因子HGF和BMP-7mRNA表达等方面,研究其对肾缺血再灌注损伤及肾小管上皮细胞修复的综合影响。 第一部分西罗莫司对大鼠肾脏缺血再灌注损伤早期的保护 摘要 目的：探讨西罗莫司(SRL)对不同时期大鼠肾缺血再灌注损伤的影响。 方法： 1.选用雄性SD(Sprague-Dawley)大鼠共90只,随机均匀分为五组：sham、I/RI/R+SRL1mg/(kg.d)、I/R+SRL3mg/(kg.d)、 I/R+SRL5mg/(kg.d)。 2.建立大鼠缺血再灌注损伤模型。 3.药物各组术前3天及术后每日给予对应剂量SRLlmg.3mg.5mg与10ml生理盐水灌胃至各个观察日。 4.sham、I/R给予等量生理盐水灌胃至各个观察日。 5.术后1、3、7天每组各处死6只大鼠取全血及肾组织,检测血清肌酐、p2-MG； 6. ELISA法测定中性粒细胞明胶酶相关脂质运载蛋白(NGAL)、IL-18； 7.HE染色评价组织病理学改变； 8.免疫组化SABC法检测肾组织Fas、Bcl-2蛋白的表达； 9. Western-blot技术检测Fas、Bcl-2蛋白表达强度； 10.脱氧核糖核苷酸末端转移酶介导的缺口末端标记(TUNEL)法检测肾组织细胞凋亡率。 结果： 1.术后1、3天血Cr、p2-MG、IL-18、Fas和Bcl-2水平、病理学评分、Fas和Bcl-2蛋白阳性表达率、凋亡指数均升高,除Bcl-2水平外shamI/R+SRL5mg/(kg.d)I/R+SRL3mg/(kg.d)I/R+SRL1mg/(kg.d)I/R(P0.05)。 2.Bc1-2水平：][/R+SRL5mg/(kg.d)I/R+SRL3mg/(kg.d)I/R+SRL1mg/(kg.d)I/Rsham(P0.05)。 3.术后7天Fas、Bcl-2水平：I/R+SRL5mg/(kg.d)与sham和I/R+SRLlmg/(kg.d)比较有差异(P0.05)； 4. NGAL水平术后1天高于sham(P0.05)。凋亡指数I/R+SRL5mg/(kg.d)与余四组比较有差异(P0.05)。 结论： 1.成功建立大鼠肾脏缺血再灌注损伤模型。 2.西罗莫司在缺血再灌注损伤早期通过其显著抑制肾小管上皮细胞细胞凋亡的作用,起到保护肾功能的作用。 3.西罗莫司在缺血再灌注损伤早期对肾功能保护具有剂量依赖性。 第二部分西罗莫司对大鼠肾小管上皮细胞修复的影响 摘要 目的：探讨西罗莫司(SRL)对不同时期大鼠肾小管上皮细胞修复的影响。 方法： 1.选用雄性SD(Sprague-Dawley)大鼠共90只,随机均匀分为五组：sham、I/R、I/R+SRLlmg/(kg.d)、I/R+SRL3mg/(kg.d)、 I/R+SRL5mg/(kg.d)。 2.建立大鼠缺血再灌注损伤模型。 3.药物各组术前3天及术后每日给予对应剂量SRLlmg、3mg、5mg与10ml生理盐水灌胃至各个观察日。； 4.sham、I/R给予等量生理盐水灌胃至各个观察日。 5.术后1、3、7天每组各处死6只大鼠取全血及肾组织,检测血清肌酐； 6. ELISA法测定p2-MG、肝细胞生长因子(HGF)水平； 7.HE染色评价组织病理学改变； 8.免疫组化SABC法检测肾组织增殖细胞核抗原(PCNA)蛋白的表达； 9. RT-PCR技术检测修复因子HGF的mRNA、骨形态发生蛋白-7(BMP-7) mRNA表达。 结果： 1.术后1、3天血Cr、β2-MG、病理学评分均升高：shamI/R+SRL5mg/(kg.d)I/R+SRL3mg/(kg.d)I/R+SRL1mg/(kg.d)I/R(P0.05)； 2.术后1、3天血HGF值、HGFmRNA、BMP-7mRNA水平和1、3、7天PCNA水平：shamI/R+SRL5mg/(kg.d)I/R+SRL3mg/(kg.d)I/R+SRLlmg/(kg.d)I/R(P0.05); 3.Sham术后各时期均未见增殖细胞,余四组均有大量细胞增殖。 结论： 1.成功建立大鼠缺血再灌注损伤模型。 2.西罗莫司在缺血再灌注损伤中其抗增殖作用阻碍了肾小管上皮细胞的移行、骨架重建等修复过程。 3.西罗莫司在缺血再灌注损伤中其抗增殖作用具有剂量依赖性。
[Abstract]:Ischemia-reperfusion injury is an unavoidable stage in solid organ transplantation. In renal transplantation, the presence of ischemia-reperfusion injury can significantly increase the incidence of acute rejection after kidney transplantation. Similarly, the repair of renal tubular epithelial cells takes a relatively long time, resulting in delayed graft function after kidney transplantation. Renal tubular cell culture models, animal experiments and clinical studies suggest that renal tubular epithelial cell death induced by renal ischemia-reperfusion injury has two forms of necrosis and apoptosis. In the late stage, the dedifferentiated renal tubular epithelial cells proliferate, migrate and redifferentiate under the action of various repair mechanisms, and the proliferation of renal tubular epithelial cells starts gradually. As the coexistence of solid organ transplantation and ischemia-reperfusion injury, we should adopt active and effective methods to inhibit the apoptosis of renal tubular epithelial cells to the greatest extent, alleviate the adverse effects of renal ischemia-reperfusion injury, protect renal function and reduce it. The delayed recovery of graft function and the incidence of acute rejection after oligokidney transplantation can ultimately prolong the survival time of transplanted kidney and recipients, which has far-reaching clinical significance.
Sirolimus (SRL) is a mammalian target of rapamycin (mTOR) inhibitor. Animal experiments and many years of clinical application have shown that the CNI-based immunosuppressive regimen or the CNI-based immunosuppressive regimen after switching from SRL-based to sirolimus-based immunosuppressive regimens are CNI-based immunosuppressive. Compared with CNI-based immunosuppressive regimen, sirolimus significantly reduced the creeping increase of serum creatinine and improved renal function after renal transplantation. The renal function was significantly better than that of the CNI group. For patients with chronic creeping creatinine elevation and CNI nephrotoxicity after renal transplantation, timely SRL conversion therapy can significantly improve renal transplant function and prolong the survival time of renal transplant recipients.
At present, the role of SRL in ischemia-reperfusion injury is still controversial. It has been found that SRL can significantly alleviate renal ischemia-reperfusion injury and improve renal function in rats through the following mechanisms: (1) activating T cell apoptosis through indirect pathway, preventing the antigen presentation of dendritic cells (DC) and further inhibiting its maturation; (2) On Thursday, Gui et al. treated hypoxic-reoxygenated vascular endothelial cells with SRL, inhibited the adhesion of vascular endothelial cells to neutrophils by inhibiting the signal transduction pathways of ROS, JNK and NF-kappa B. Yang B et al. found that SRL interfered with renal ischemia-reperfusion injury, mainly activating. 24KDCaspase-3 can reduce the occurrence of apoptosis and the expression of Fas in distal convoluted tubules. It can protect the ischemic kidney from reperfusion injury by alleviating the immune injury induced by Fas signal pathway. Bohmova R et al. Observation of renal ischemia-reperfusion injury in transgenic hypertensive rats showed that small doses of SRL can alleviate the production of renin. In vitro, SRL inhibits the proliferation of proximal convoluted tubular cells and promotes apoptosis by inhibiting 70 kDaS6 protein kinase in renal tubular cells. In vivo, SRL inhibits tubular cell regeneration and enhances tubular fineness. Lui SL reported that SRL decreased the expression of proliferating cell nuclear antigen (PCNA) on day 1 and day 3, but on day 7 there was no difference between the control group, suggesting that SRL could aggravate renal damage mainly by inhibiting renal tubular cells. The regeneration of renal tubular cells delayed the recovery of renal function, but did not always inhibit the regeneration of renal tubular cells.
In ischemia-reperfusion injury, the effects of different doses of SRL on the proliferation and repair of renal tubular epithelial cells after ischemia-reperfusion injury and apoptosis of renal tubular epithelial cells in rats at different stages are not clear. Protective effect, however, may inhibit the proliferation, migration, redifferentiation and skeleton remodeling of renal tubular epithelial cells in the later stage as the repair procedure of renal tubular epithelial cells is initiated step by step, which may affect the recovery of renal function in a dose-dependent and reversible manner. Rat renal ischemia-reperfusion injury model was established based on the valley concentration as the early treatment concentration after renal transplantation. The expression of apoptotic gene Fas, Bcl-2 and PCNA, the expression of tubular epithelial cell repair factor HGF and BMP-7 mRNA, and the expression of NGAL, IL-18, Fas, Bcl-2 and PCNA were detected. The combined effects of perfusion injury and repair of renal tubular epithelial cells.
Part one: protective effects of sirolimus on renal ischemia-reperfusion injury in early stage
abstract
Objective: To investigate the effects of sirolimus (SRL) on renal ischemia-reperfusion injury in rats at different stages.
Method:
1. Ninety male SD rats were randomly divided into five groups: sham, I/RI/R+SRL 1mg/(kg.d), I/R+SRL 3mg/(kg.d), I/R+SRL 5mg/(kg.d).
2. a rat model of ischemia-reperfusion injury was established.
3. The corresponding dosage of SRLlmg.3mg.5mg and 10ml normal saline were given to each group 3 days before operation and daily after operation.
4.sham and I/R were given normal saline to each observation day.
5. after 1,3,7 days, 6 rats in each group were sacrificed to get whole blood and kidney tissue, and serum creatinine was detected, p2-MG.
Neutrophil gelatinase associated lipid carrier protein (NGAL) and IL-18 were measured by 6. ELISA.
7.HE staining was used to evaluate histopathological changes.
8. immunohistochemical SABC method was used to detect the expression of Fas and Bcl-2 protein in renal tissue.
9. Western-blot technology was used to detect the expression intensity of Fas and Bcl-2 protein.
10. Deoxyribonucleotide terminal transferase mediated nick end labeling (TUNEL) was used to detect the apoptosis rate of renal cells.
Result:
1. The levels of serum Cr, p2-MG, IL-18, Fas and Bcl-2, pathological score, positive expression rate of Fas and Bcl-2 protein and apoptosis index were all increased at 1 and 3 days after operation. Except for Bcl-2, I/R+SRL5 mg/(kg.d) I/R+SRL3 mg/(kg.d) I/R+SRL1 mg/(kg.d) I/R (P 0.05).
2.Bc1-2 level:][/R+SRL5mg/ (kg.d) I/R+SRL3mg/ (kg.d) I/R+SRL1mg/ (kg.d) I/Rsham (P0.05).
3. Fas, Bcl-2 level 7 days after operation: I/R+SRL5mg/ (kg.d) was different from sham and I/R+SRLlmg/ (kg.d) (P0.05).
4. The level of NGAL was higher than that of sham (P 0.05) one day after operation. The apoptosis index I/R+SRL5 mg/(kg.d) was different from the other four groups (P 0.05).
Conclusion:
1. a rat model of renal ischemia-reperfusion injury was successfully established.
2. Sirolimus can protect renal function by inhibiting apoptosis of renal tubular epithelial cells in the early stage of ischemia-reperfusion injury.
3. sirolimus dose dependently protects renal function at the early stage of ischemia-reperfusion injury.
The second part is the effect of sirolimus on the repair of rat renal tubular epithelial cells.
abstract
Objective: To investigate the effect of sirolimus (SRL) on the repair of renal tubular epithelial cells in different periods.
Method:
1. Ninety male SD (Sprague-Dawley) rats were randomly divided into five groups: sham, I/R, I/R + SRLlmg / (kg.d), I/R + SRL3mg / (kg.d), I/R + SRL5mg / (kg.d).
2. a rat model of ischemia-reperfusion injury was established.
3. Each group was given the corresponding dose of SRLlmg, 3mg, 5mg and 10ml normal saline 3 days before operation and every day after operation.
4.sham and I/R were given normal saline to each observation day.
5. after 1,3,7 days, 6 rats in each group were sacrificed to get whole blood and kidney tissues, and serum creatinine was detected.
The level of p2-MG and hepatocyte growth factor (HGF) was determined by 6. ELISA.
7.HE staining was used to evaluate histopathological changes.
8. immunohistochemical SABC method was used to detect the expression of proliferating cell nuclear antigen (PCNA) protein in renal tissue.
9. RT-PCR technology was used to detect the expression of mRNA, bone morphogenetic protein -7 (BMP-7) mRNA in repair factor HGF.
Result:
1. The levels of serum Cr, beta 2-MG and pathological score were all increased at 1 and 3 days after operation: sham I/R + SRL 5 mg / (kg.d) I/R + SRL 3 mg / (kg.d) I/R + SRL 1 mg / (kg.d) I/R (P 0.05);
2. Blood HGF value, HGF mRNA, BMP-7 mRNA level and PCNA level at 1,3,7 days after operation: shamI/R+SRL5mg/(kg.d) I/R+SRL3mg/(kg.d) I/R+SRLlmg/(kg.d) I/R (P 0.05);
No proliferating cells were found in all stages after 3.Sham.
Conclusion:
1. a rat model of ischemia-reperfusion injury was successfully established.
2. The antiproliferative effect of sirolimus on renal tubular epithelial cells in ischemia-reperfusion injury hinders the process of renal tubular epithelial cell migration and skeletal reconstruction.
3. sirolimus has a dose-dependent anti proliferative effect in ischemia-reperfusion injury.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965
本文编号：2208298
[Abstract]:Ischemia-reperfusion injury is an unavoidable stage in solid organ transplantation. In renal transplantation, the presence of ischemia-reperfusion injury can significantly increase the incidence of acute rejection after kidney transplantation. Similarly, the repair of renal tubular epithelial cells takes a relatively long time, resulting in delayed graft function after kidney transplantation. Renal tubular cell culture models, animal experiments and clinical studies suggest that renal tubular epithelial cell death induced by renal ischemia-reperfusion injury has two forms of necrosis and apoptosis. In the late stage, the dedifferentiated renal tubular epithelial cells proliferate, migrate and redifferentiate under the action of various repair mechanisms, and the proliferation of renal tubular epithelial cells starts gradually. As the coexistence of solid organ transplantation and ischemia-reperfusion injury, we should adopt active and effective methods to inhibit the apoptosis of renal tubular epithelial cells to the greatest extent, alleviate the adverse effects of renal ischemia-reperfusion injury, protect renal function and reduce it. The delayed recovery of graft function and the incidence of acute rejection after oligokidney transplantation can ultimately prolong the survival time of transplanted kidney and recipients, which has far-reaching clinical significance.
Sirolimus (SRL) is a mammalian target of rapamycin (mTOR) inhibitor. Animal experiments and many years of clinical application have shown that the CNI-based immunosuppressive regimen or the CNI-based immunosuppressive regimen after switching from SRL-based to sirolimus-based immunosuppressive regimens are CNI-based immunosuppressive. Compared with CNI-based immunosuppressive regimen, sirolimus significantly reduced the creeping increase of serum creatinine and improved renal function after renal transplantation. The renal function was significantly better than that of the CNI group. For patients with chronic creeping creatinine elevation and CNI nephrotoxicity after renal transplantation, timely SRL conversion therapy can significantly improve renal transplant function and prolong the survival time of renal transplant recipients.
At present, the role of SRL in ischemia-reperfusion injury is still controversial. It has been found that SRL can significantly alleviate renal ischemia-reperfusion injury and improve renal function in rats through the following mechanisms: (1) activating T cell apoptosis through indirect pathway, preventing the antigen presentation of dendritic cells (DC) and further inhibiting its maturation; (2) On Thursday, Gui et al. treated hypoxic-reoxygenated vascular endothelial cells with SRL, inhibited the adhesion of vascular endothelial cells to neutrophils by inhibiting the signal transduction pathways of ROS, JNK and NF-kappa B. Yang B et al. found that SRL interfered with renal ischemia-reperfusion injury, mainly activating. 24KDCaspase-3 can reduce the occurrence of apoptosis and the expression of Fas in distal convoluted tubules. It can protect the ischemic kidney from reperfusion injury by alleviating the immune injury induced by Fas signal pathway. Bohmova R et al. Observation of renal ischemia-reperfusion injury in transgenic hypertensive rats showed that small doses of SRL can alleviate the production of renin. In vitro, SRL inhibits the proliferation of proximal convoluted tubular cells and promotes apoptosis by inhibiting 70 kDaS6 protein kinase in renal tubular cells. In vivo, SRL inhibits tubular cell regeneration and enhances tubular fineness. Lui SL reported that SRL decreased the expression of proliferating cell nuclear antigen (PCNA) on day 1 and day 3, but on day 7 there was no difference between the control group, suggesting that SRL could aggravate renal damage mainly by inhibiting renal tubular cells. The regeneration of renal tubular cells delayed the recovery of renal function, but did not always inhibit the regeneration of renal tubular cells.
In ischemia-reperfusion injury, the effects of different doses of SRL on the proliferation and repair of renal tubular epithelial cells after ischemia-reperfusion injury and apoptosis of renal tubular epithelial cells in rats at different stages are not clear. Protective effect, however, may inhibit the proliferation, migration, redifferentiation and skeleton remodeling of renal tubular epithelial cells in the later stage as the repair procedure of renal tubular epithelial cells is initiated step by step, which may affect the recovery of renal function in a dose-dependent and reversible manner. Rat renal ischemia-reperfusion injury model was established based on the valley concentration as the early treatment concentration after renal transplantation. The expression of apoptotic gene Fas, Bcl-2 and PCNA, the expression of tubular epithelial cell repair factor HGF and BMP-7 mRNA, and the expression of NGAL, IL-18, Fas, Bcl-2 and PCNA were detected. The combined effects of perfusion injury and repair of renal tubular epithelial cells.
Part one: protective effects of sirolimus on renal ischemia-reperfusion injury in early stage
abstract
Objective: To investigate the effects of sirolimus (SRL) on renal ischemia-reperfusion injury in rats at different stages.
Method:
1. Ninety male SD rats were randomly divided into five groups: sham, I/RI/R+SRL 1mg/(kg.d), I/R+SRL 3mg/(kg.d), I/R+SRL 5mg/(kg.d).
2. a rat model of ischemia-reperfusion injury was established.
3. The corresponding dosage of SRLlmg.3mg.5mg and 10ml normal saline were given to each group 3 days before operation and daily after operation.
4.sham and I/R were given normal saline to each observation day.
5. after 1,3,7 days, 6 rats in each group were sacrificed to get whole blood and kidney tissue, and serum creatinine was detected, p2-MG.
Neutrophil gelatinase associated lipid carrier protein (NGAL) and IL-18 were measured by 6. ELISA.
7.HE staining was used to evaluate histopathological changes.
8. immunohistochemical SABC method was used to detect the expression of Fas and Bcl-2 protein in renal tissue.
9. Western-blot technology was used to detect the expression intensity of Fas and Bcl-2 protein.
10. Deoxyribonucleotide terminal transferase mediated nick end labeling (TUNEL) was used to detect the apoptosis rate of renal cells.
Result:
1. The levels of serum Cr, p2-MG, IL-18, Fas and Bcl-2, pathological score, positive expression rate of Fas and Bcl-2 protein and apoptosis index were all increased at 1 and 3 days after operation. Except for Bcl-2, I/R+SRL5 mg/(kg.d) I/R+SRL3 mg/(kg.d) I/R+SRL1 mg/(kg.d) I/R (P 0.05).
2.Bc1-2 level:][/R+SRL5mg/ (kg.d) I/R+SRL3mg/ (kg.d) I/R+SRL1mg/ (kg.d) I/Rsham (P0.05).
3. Fas, Bcl-2 level 7 days after operation: I/R+SRL5mg/ (kg.d) was different from sham and I/R+SRLlmg/ (kg.d) (P0.05).
4. The level of NGAL was higher than that of sham (P 0.05) one day after operation. The apoptosis index I/R+SRL5 mg/(kg.d) was different from the other four groups (P 0.05).
Conclusion:
1. a rat model of renal ischemia-reperfusion injury was successfully established.
2. Sirolimus can protect renal function by inhibiting apoptosis of renal tubular epithelial cells in the early stage of ischemia-reperfusion injury.
3. sirolimus dose dependently protects renal function at the early stage of ischemia-reperfusion injury.
The second part is the effect of sirolimus on the repair of rat renal tubular epithelial cells.
abstract
Objective: To investigate the effect of sirolimus (SRL) on the repair of renal tubular epithelial cells in different periods.
Method:
1. Ninety male SD (Sprague-Dawley) rats were randomly divided into five groups: sham, I/R, I/R + SRLlmg / (kg.d), I/R + SRL3mg / (kg.d), I/R + SRL5mg / (kg.d).
2. a rat model of ischemia-reperfusion injury was established.
3. Each group was given the corresponding dose of SRLlmg, 3mg, 5mg and 10ml normal saline 3 days before operation and every day after operation.
4.sham and I/R were given normal saline to each observation day.
5. after 1,3,7 days, 6 rats in each group were sacrificed to get whole blood and kidney tissues, and serum creatinine was detected.
The level of p2-MG and hepatocyte growth factor (HGF) was determined by 6. ELISA.
7.HE staining was used to evaluate histopathological changes.
8. immunohistochemical SABC method was used to detect the expression of proliferating cell nuclear antigen (PCNA) protein in renal tissue.
9. RT-PCR technology was used to detect the expression of mRNA, bone morphogenetic protein -7 (BMP-7) mRNA in repair factor HGF.
Result:
1. The levels of serum Cr, beta 2-MG and pathological score were all increased at 1 and 3 days after operation: sham I/R + SRL 5 mg / (kg.d) I/R + SRL 3 mg / (kg.d) I/R + SRL 1 mg / (kg.d) I/R (P 0.05);
2. Blood HGF value, HGF mRNA, BMP-7 mRNA level and PCNA level at 1,3,7 days after operation: shamI/R+SRL5mg/(kg.d) I/R+SRL3mg/(kg.d) I/R+SRLlmg/(kg.d) I/R (P 0.05);
No proliferating cells were found in all stages after 3.Sham.
Conclusion:
1. a rat model of ischemia-reperfusion injury was successfully established.
2. The antiproliferative effect of sirolimus on renal tubular epithelial cells in ischemia-reperfusion injury hinders the process of renal tubular epithelial cell migration and skeletal reconstruction.
3. sirolimus has a dose-dependent anti proliferative effect in ischemia-reperfusion injury.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965
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