内质网源性转录因子CHOP在肾脏缺血再灌注损伤中的作用和机制研究
发布时间：2018-09-03 09:45
【摘要】：急性肾损伤（acute kidney injury,AKI）是由多种疾病引起的一种常见的临床综合征，是指由导致肾脏结构或功能变化的损伤引起的肾脏功能突然恶化。缺血再灌注损伤（ischemia reperfusion injury, IRI）是导致急性肾损伤的主要原因之一，具有较高的发病率和死亡率，伴随着一系列的细胞事件发生，包括细胞坏死、细胞凋亡、炎症细胞的浸润和活性介质的释放，从而导致组织损伤。 内质网（endoplasmic reticulum，ER）是真核细胞中具有重要作用的细胞器，它的主要作用是参与蛋白质的合成及转运，各种蛋白的糖基化修饰以及钙离子的储存与分布等。细胞在多种理化因素（缺氧、饥饿、钙离子平衡失调、化学毒物等）的刺激下，内质网腔内会出现蛋白质的错误折叠及未折叠蛋白质的聚集等现象，从而引起内质网功能紊乱，这种状态称为内质网应激（endoplasmic reticulum stress，ERS）。 肾脏缺血再灌注损伤是由于肾脏供血障碍引起的一种常见的应激性疾病，20世纪70年代由Glaumann等提出在恢复缺血后的肾脏的血液供应，可加重原有的单纯由缺血所造成的损伤，并且指出缺血再灌注损伤的发病基础是局部能量代谢障碍，发病的重要环节则是自由基产生和钙超载。已经证实，肾脏缺血再灌注损伤与内质网应激密切相关。在此过程中，组织的缺血缺氧、葡萄糖/营养物质匮乏、ATP耗竭、大量自由基的产生及钙离子稳态破坏等均可引起内质网功能障碍，触发内质网应激。过度内质网应激通过破坏钙离子稳态、诱导细胞凋亡而加重缺血再灌注损伤，但其具体的机制一直没有得到详细阐述。最新研究发现，肾脏缺血再灌注损伤模型中，内质网源性转录因子CHOP，又名生长抑制DNA损伤基因153抗原（growth arrest and DNAdamage inducible153，GADDl53）表达升高，同时caspase-11的表达升高，造成肾脏功能的损害。据此，我们推测CHOP介导细胞凋亡在肾脏缺血再灌注损伤中起到重要作用。 本课题通过对野生型小鼠和CHOP敲除小鼠建立肾脏缺血再灌注损伤模型，探明CHOP在肾脏缺血再灌注损伤中的具体作用和机制，并进一步在体外实验中培养人肾小管上皮细胞（HK-2）和人脐静脉内皮细胞（HUVEC）并建立缺氧复氧损伤模型，验证CHOP的重要作用及其分子机制，为阐明急性缺血性肾损伤发生机制以及寻找新的治疗靶点提供科学依据。 1.实验方法 1.1实验动物 CHOP敲除小鼠由美国Jackson实验室引进。体重22~26克的成年雄性野生型小鼠和CHOP敲除小鼠应用于本次试验。实验动物给于12小时昼夜交替饲养和自由的饮水、饮食。 1.2肾脏缺血再灌注损伤模型 实验动物经戊巴比妥钠（60毫克/公斤）腹腔注射麻醉后，放置于安装有控温仪器的手术台上，整个实验过程中控制体温在36℃左右。取腹正中切口，切除右肾，用防损伤微血管夹夹闭左侧肾脏动、静脉25分钟。假手术组只分离肾蒂，切除右肾，未夹闭左侧肾动静脉。实验动物在缺血再灌注24小时后经腹主动脉穿刺取血及留取肾脏组织做进一步检查分析。 1.3骨髓移植 供者、受者均选择8~10周的雄鼠，受者经钴源60照射2次，总剂量10.5Gy，间隔4小时。在照射后2小时，由供者提取骨髓细胞按1×107个/只尾静脉注射给受者。在骨髓移植后30天建立肾脏缺血再灌注损伤模型。 1.4细胞培养及缺氧复氧（HR）模型 人肾小管上皮细胞（HK-2）和人脐静脉内皮细胞（HUVEC）培养在含有10%胎牛血清的DMEM中，放置于37°C，5%CO2的培养箱内。实验铺盘于6孔板内，按照5×105个细胞/孔。根据实验方案，细胞放置于正常环境（5%CO2,21%O2和74%N2）和缺氧环境（5%CO2，1%O2和94%N2）。 1.5siRNA干扰的构建 CHOP的siRNA是由美国英杰生物技术公司合成、纯化、退火。siRNA合成的具体序列为：GCUAGCUGAAGAGAAUGAATT；UUCAUUCUCUUCAGCUAGCTT。HK-2细胞和HUVECs按照80nM的质粒浓度运用Lipofectamin2000进行转染干扰。 2.实验结果 2.1CHOP在小鼠肾脏缺血再灌注损伤中介导细胞凋亡 2.1.1肾脏IR后CHOP、cleaved caspase-3蛋白表达情况 与对照组相比，肾脏IR后3小时开始CHOP、cleaved caspase-3蛋白表达升高，于再灌注6小时达高峰，并持续至24小时，均明显高于对照组（p0.05）。 2.1.2CHOP敲除后cleaved caspase-3蛋白表达情况及生存率、肾功、病理的改变 cleaved caspase-3蛋白表达变化：CHOP敲除小鼠IR后6小时的cleaved caspase-3蛋白表达较野生型小鼠降低（p0.05）。 生存率的比较：CHOP敲除小鼠IR后生存率（80%）较野生型小鼠（0%）显著提高（p0.05）。 肾功的比较：CHOP敲除小鼠IR后24小时血肌酐、血尿素氮均较野生型小鼠显著降低（p0.05）。 病理的比较：CHOP敲除小鼠IR后24小时病理改变均较野生型小鼠显著减轻（p0.05）。 2.1.3CHOP敲除通过影响肾脏微循环灌注减轻IR损伤 与野生型小鼠IR后相比，CHOP敲除能显著改善缺血后肾脏早期的微循环灌注，从而减轻小鼠肾脏缺血再灌注损伤。 2.2骨髓移植证实肾脏固有细胞而不是骨髓来源的免疫细胞中CHOP介导IR中的细胞凋亡 2.2.1四组骨髓移植小鼠IR后生存率的改变 生存率的比较：四组骨髓移植小鼠IR后观察7天，骨髓移植WT→WT小鼠生存率（0%）与骨髓移植CHOP-/-→WT小鼠生存率（0%）相比，，无统计学差异（p0.05）；骨髓移植WT→CHOP-/-小鼠生存率（80%）与骨髓移植CHOP-/-→CHOP-/-小鼠生存率（70%）相比，无统计学差异（p0.05）。 2.2.2四组骨髓移植小鼠IR后肾功的改变 肾功的比较：骨髓移植WT→WT小鼠与骨髓移植CHOP-/-→WT小鼠相比，IR后24小时的血肌酐、血尿素氮无统计学差异（p0.05）；骨髓移植WT→CHOP-/-小鼠与骨髓移植CHOP-/-→CHOP-/-小鼠，IR后24小时的血肌酐、血尿素氮无统计学差异（p0.05）。 2.2.3四组骨髓移植小鼠IR后病理的改变 病理的比较：骨髓移植WT→WT小鼠与骨髓移植CHOP-/-→WT小鼠相比，IR后24小时的病理改变无统计学差异（p0.05）；骨髓移植WT→CHOP-/-小鼠与骨髓移植CHOP-/-→CHOP-/-小鼠，IR后24小时的病理改变无统计学差异（p0.05）。 2.3肾小管上皮细胞及内皮细胞中CHOP介导细胞凋亡 2.3.1缺氧复氧引起肾小管上皮细胞损伤 LDH水平：与对照组相比，细胞上清LDH水平于HR后6小时增加，持续增加至复氧24小时（p0.05）。 CHOP蛋白表达：与对照组相比，HR后6小时升高，持续增加至24小时（p0.05）。 cleaved caspase-3蛋白表达：与对照组相比，HR后6小时升高，持续增加至24小时（p0.05）。 2.3.2缺氧复氧引起内皮细胞损伤 LDH水平：与对照组相比，细胞上清LDH水平于HR后6小时增加，持续至复氧24小时未见明显下降（p0.05）。 CHOP蛋白表达：与对照组相比，HR后6小时升高，持续至复氧24小时未见明显下降（p0.05）。 cleaved caspase-3蛋白表达：与对照组相比，HR后6小时升高，持续至复氧24小时未见明显下降（p0.05）。 2.3.3CHOP基因沉默显著减轻HR诱导的肾小管上皮细胞损伤 LDH水平：与HR24小时组比较，CHOP siRNA显著减轻HR后肾小管上皮细胞损伤，细胞培养上清中LDH水平明显下降（p0.05）。 CHOP siRNA显著抑制HR诱导的肾小管上皮细胞CHOP蛋白的表达（p0.05）。 CHOP基因沉默可显著抑制HR后24小时肾小管上皮细胞cleaved caspase-3的蛋白的表达（p0.05）。 2.3.4CHOP基因沉默显著减轻HR诱导的内皮细胞损伤 LDH水平：与HR6小时组比较，CHOP siRNA显著减轻HR内皮细胞损伤，细胞培养上清中LDH水平明显下降（p0.05）。 CHOPsiRNA显著抑制HR诱导的内皮细胞CHOP蛋白的表达（p0.05）。 CHOP基因沉默可显著抑制HR后6小时内皮细胞cleaved caspase-3蛋白的表达（p0.05）。 3.结论 肾脏固有细胞中的CHOP的激活在介导肾脏IR损伤中的细胞凋亡、肾脏功能损害起到重要作用，而敲除固有细胞中的CHOP后细胞凋亡减少，肾脏功能得到一定的保护。
[Abstract]:Acute kidney injury (AKI) is a common clinical syndrome caused by a variety of diseases. It refers to the sudden deterioration of renal function caused by the damage of kidney structure or function. Ischemia reperfusion injury (IRI) is one of the main causes of acute kidney injury and has a high incidence. Morbidity and mortality are accompanied by a series of cellular events, including cell necrosis, apoptosis, infiltration of inflammatory cells and release of active mediators, leading to tissue damage.
Endoplasmic reticulum (ER) is an important organelle in eukaryotic cells. It plays an important role in the synthesis and transport of proteins, glycosylation modification of proteins and the storage and distribution of calcium ions. Endoplasmic reticulum stress (ERS) is a state of endoplasmic reticulum stress (ERS) in which misfolded and unfolded proteins accumulate in the endoplasmic reticulum.
Renal ischemia-reperfusion injury is a common stress disease caused by renal dysfunction of blood supply. Glaumann et al. proposed in the 1970s that restoring the blood supply of the kidney after ischemia could aggravate the original injury caused by ischemia alone, and pointed out that the pathogenesis of ischemia-reperfusion injury is based on the impairment of local energy metabolism. It has been proved that renal ischemia-reperfusion injury is closely related to endoplasmic reticulum stress. In this process, tissue ischemia-hypoxia, glucose/nutrient deficiency, ATP depletion, large amount of free radicals production and calcium homeostasis damage can cause endoplasmic reticulum dysfunction, triggering. Endoplasmic reticulum stress. Excessive endoplasmic reticulum stress aggravates ischemia-reperfusion injury by destroying calcium homeostasis and inducing apoptosis, but its specific mechanism has not been elaborated in detail. The expression of owth arrest and DNA damage inducible 153 (GADDl53) was elevated, while the expression of caspase-11 was elevated, resulting in damage to renal function.
In this study, we established a renal ischemia-reperfusion injury model in wild type mice and CHOP knockout mice to explore the specific role and mechanism of CHOP in renal ischemia-reperfusion injury, and further cultured human renal tubular epithelial cells (HK-2) and human umbilical vein endothelial cells (HUVEC) in vitro and established a hypoxia-reoxygenation injury model. The important role of CHOP and its molecular mechanism provide scientific basis for elucidating the pathogenesis of acute ischemic renal injury and searching for new therapeutic targets.
1. experimental method
1.1 experimental animals
CHOP knockout mice were imported from Jackson Laboratory of USA. Adult male wild type mice weighing 22-26 g and CHOP knockout mice were used in this experiment. The experimental animals were fed day and night for 12 hours and fed free water and diet.
1.2 renal ischemia-reperfusion injury model
After anesthesia with sodium pentobarbital (60 mg/kg), the animals were placed on the operating table equipped with a thermostat. During the whole experiment, the body temperature was controlled at about 36 C. The right kidney was removed through a median abdominal incision, and the left renal artery and vein were clamped with a protective microvascular clip for 25 minutes. Left renal artery and vein were not clamped. Blood samples were taken from abdominal aorta and kidney tissues were taken for further examination 24 hours after ischemia and reperfusion.
1.3 bone marrow transplantation
The recipients were irradiated with cobalt 60 twice at a total dose of 10.5 Gy at intervals of 4 hours. Two hours after irradiation, bone marrow cells were extracted from the donor and injected into the recipient by 1 *107 cells per caudal vein. A renal ischemia-reperfusion injury model was established 30 days after bone marrow transplantation.
1.4 cell culture and anoxia reoxygenation (HR) model
Human renal tubular epithelial cells (HK-2) and human umbilical vein endothelial cells (HUVEC) were cultured in DMEMs containing 10% fetal bovine serum and placed in incubators with 37 C and 5% CO2. The cells were placed in 6-well plates with 5 105 cells/pores. According to the experimental scheme, the cells were placed in normal (5% CO2, 21% O2 and 74% N2) and hypoxic (5% CO2, 1% O2 and 94% N2).
Construction of 1.5siRNA interference
The siRNA of CHOP was synthesized, purified and annealed by Yingjie Biotechnology Company, USA. The specific sequence of siRNA synthesis was GCUAGGAAUGAATT, UUCUCUUCAGCUAGCTT.HK-2 cells and HUVECs were transfected with Lipofectamin 2000 at 80nM plasmid concentration.
2. experimental results
2.1CHOP mediates apoptosis in renal ischemia-reperfusion injury in mice
Expression of CHOP and cleaved caspase-3 protein in 2.1.1 kidneys after IR
Compared with the control group, the expression of cleaved caspase-3 protein increased at the beginning of CHOP 3 hours after IR, reached the peak at 6 hours after reperfusion, and lasted for 24 hours, which was significantly higher than that of the control group (p0.05).
Cleaved caspase-3 protein expression and survival rate, renal function and pathological changes after 2.1.2CHOP knockout
Changes of cleaved caspase-3 protein expression: The expression of cleaved caspase-3 protein in CHOP knockout mice 6 hours after IR was lower than that in wild type mice (p0.05).
Survival rate: the survival rate of CHOP knockout mice after IR (80%) was significantly higher than that of wild type mice (0%) (P0.05).
Comparison of renal function: CHOP knockout mice 24 hours after IR serum creatinine, blood urea nitrogen were significantly lower than wild type mice (p0.05).
Pathological comparison: the pathological changes of CHOP knockout mice were significantly reduced at 24 hours after IR (P0.05).
2.1.3CHOP knockout reduces IR damage by affecting renal microcirculation perfusion
CHOP knockout significantly improved the early microcirculatory perfusion of ischemic kidneys, thereby reducing the renal ischemia-reperfusion injury in mice compared with wild-type mice after IR.
2.2 Bone marrow transplantation confirms that CHOP mediates apoptosis in IR in renal innate cells rather than in bone marrow-derived immune cells
Changes in survival rate after IR in 2.2.1 four groups of bone marrow transplant mice
Survival rate of four groups of bone marrow transplantation mice after IR observation 7 days, bone marrow transplantation WT WT mice survival rate (0%) and bone marrow transplantation CHOP -/- WT mice survival rate (0%) compared with no significant difference (p0.05); bone marrow transplantation WT CHOP -/- mice survival rate (80%) and bone marrow transplantation CHOP -/- CHOP /- mice survival rate (70%) compared with no significant difference. Difference (P0.05).
Changes in renal function after 2.2.2 IR in four groups of bone marrow transplant mice
Comparison of renal function: There was no significant difference in serum creatinine and blood urea nitrogen between bone marrow transplanted WT WT mice and bone marrow transplanted CHOP -/- WT mice 24 hours after IR (p0.05); there was no significant difference in serum creatinine and blood urea nitrogen between bone marrow transplanted WT CHOP -/- mice and bone marrow transplanted CHOP -/ CHOP -/- mice 24 hours after IR (p0.05).
Pathological changes of 2.2.3 four groups of bone marrow transplantation mice after IR
Pathological comparison: BMT WT WT mice and BMT CHOP -/- WT mice had no significant difference in pathological changes 24 hours after IR (p0.05); BMT WT CHOP -/- mice and BMT CHOP -/- mice, there was no significant difference in pathological changes 24 hours after IR (p0.05).
2.3, CHOP mediated apoptosis in renal tubular epithelial cells and endothelial cells.
Injury of renal tubular epithelial cells induced by 2.3.1 hypoxia reoxygenation
LDH level: Compared with the control group, the level of LDH in cell supernatant increased 6 hours after HR and continued to increase to 24 hours after reoxygenation (p0.05).
CHOP protein expression: compared with the control group, HR increased after 6 hours, and increased to 24 hours (P0.05).
Cleaved caspase-3 protein expression: compared with the control group, 6 hours after HR increased, continued to increase to 24 hours (p0.05).
Endothelial cell injury induced by 2.3.2 hypoxia reoxygenation
LDH level: Compared with the control group, the level of LDH in cell supernatant increased 6 hours after HR and did not decrease significantly until 24 hours after reoxygenation (p0.05).
CHOP protein expression: Compared with the control group, HR increased at 6 hours and remained unchanged until 24 hours after reoxygenation (p0.05).
Cleaved caspase-3 protein expression: compared with the control group, HR increased at 6 hours and did not decrease significantly until 24 hours after reoxygenation (p0.05).
2.3.3CHOP gene silencing significantly alleviated HR induced renal tubular epithelial cell injury
LDH level: Compared with HR 24 hours group, CHOP siRNA significantly reduced the injury of renal tubular epithelial cells after HR, and the LDH level in cell culture supernatant decreased significantly (p0.05).
CHOP siRNA significantly inhibited the expression of CHOP protein in renal tubular epithelial cells induced by HR (P0.05).
CHOP gene silencing significantly inhibited the expression of cleaved caspase-3 protein in renal tubular epithelial cells 24 hours after HR (p0.05).
2.3.4CHOP gene silencing significantly alleviated endothelial cell injury induced by HR
LDH level: Compared with HR6-hour group, CHOP siRNA significantly reduced the injury of HR endothelial cells, and LDH level in cell culture supernatant decreased significantly (p0.05).
CHOPsiRNA significantly inhibited HR induced CHOP protein expression in endothelial cells (P0.05).
CHOP gene silencing significantly inhibited the expression of cleaved caspase-3 protein in endothelial cells 6 hours after HR (P0.05).
3. conclusion
The activation of CHOP in intrinsic cells of kidney plays an important role in mediating apoptosis and impairment of renal function in IR injury of kidney.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692.5
本文编号：2219616
[Abstract]:Acute kidney injury (AKI) is a common clinical syndrome caused by a variety of diseases. It refers to the sudden deterioration of renal function caused by the damage of kidney structure or function. Ischemia reperfusion injury (IRI) is one of the main causes of acute kidney injury and has a high incidence. Morbidity and mortality are accompanied by a series of cellular events, including cell necrosis, apoptosis, infiltration of inflammatory cells and release of active mediators, leading to tissue damage.
Endoplasmic reticulum (ER) is an important organelle in eukaryotic cells. It plays an important role in the synthesis and transport of proteins, glycosylation modification of proteins and the storage and distribution of calcium ions. Endoplasmic reticulum stress (ERS) is a state of endoplasmic reticulum stress (ERS) in which misfolded and unfolded proteins accumulate in the endoplasmic reticulum.
Renal ischemia-reperfusion injury is a common stress disease caused by renal dysfunction of blood supply. Glaumann et al. proposed in the 1970s that restoring the blood supply of the kidney after ischemia could aggravate the original injury caused by ischemia alone, and pointed out that the pathogenesis of ischemia-reperfusion injury is based on the impairment of local energy metabolism. It has been proved that renal ischemia-reperfusion injury is closely related to endoplasmic reticulum stress. In this process, tissue ischemia-hypoxia, glucose/nutrient deficiency, ATP depletion, large amount of free radicals production and calcium homeostasis damage can cause endoplasmic reticulum dysfunction, triggering. Endoplasmic reticulum stress. Excessive endoplasmic reticulum stress aggravates ischemia-reperfusion injury by destroying calcium homeostasis and inducing apoptosis, but its specific mechanism has not been elaborated in detail. The expression of owth arrest and DNA damage inducible 153 (GADDl53) was elevated, while the expression of caspase-11 was elevated, resulting in damage to renal function.
In this study, we established a renal ischemia-reperfusion injury model in wild type mice and CHOP knockout mice to explore the specific role and mechanism of CHOP in renal ischemia-reperfusion injury, and further cultured human renal tubular epithelial cells (HK-2) and human umbilical vein endothelial cells (HUVEC) in vitro and established a hypoxia-reoxygenation injury model. The important role of CHOP and its molecular mechanism provide scientific basis for elucidating the pathogenesis of acute ischemic renal injury and searching for new therapeutic targets.
1. experimental method
1.1 experimental animals
CHOP knockout mice were imported from Jackson Laboratory of USA. Adult male wild type mice weighing 22-26 g and CHOP knockout mice were used in this experiment. The experimental animals were fed day and night for 12 hours and fed free water and diet.
1.2 renal ischemia-reperfusion injury model
After anesthesia with sodium pentobarbital (60 mg/kg), the animals were placed on the operating table equipped with a thermostat. During the whole experiment, the body temperature was controlled at about 36 C. The right kidney was removed through a median abdominal incision, and the left renal artery and vein were clamped with a protective microvascular clip for 25 minutes. Left renal artery and vein were not clamped. Blood samples were taken from abdominal aorta and kidney tissues were taken for further examination 24 hours after ischemia and reperfusion.
1.3 bone marrow transplantation
The recipients were irradiated with cobalt 60 twice at a total dose of 10.5 Gy at intervals of 4 hours. Two hours after irradiation, bone marrow cells were extracted from the donor and injected into the recipient by 1 *107 cells per caudal vein. A renal ischemia-reperfusion injury model was established 30 days after bone marrow transplantation.
1.4 cell culture and anoxia reoxygenation (HR) model
Human renal tubular epithelial cells (HK-2) and human umbilical vein endothelial cells (HUVEC) were cultured in DMEMs containing 10% fetal bovine serum and placed in incubators with 37 C and 5% CO2. The cells were placed in 6-well plates with 5 105 cells/pores. According to the experimental scheme, the cells were placed in normal (5% CO2, 21% O2 and 74% N2) and hypoxic (5% CO2, 1% O2 and 94% N2).
Construction of 1.5siRNA interference
The siRNA of CHOP was synthesized, purified and annealed by Yingjie Biotechnology Company, USA. The specific sequence of siRNA synthesis was GCUAGGAAUGAATT, UUCUCUUCAGCUAGCTT.HK-2 cells and HUVECs were transfected with Lipofectamin 2000 at 80nM plasmid concentration.
2. experimental results
2.1CHOP mediates apoptosis in renal ischemia-reperfusion injury in mice
Expression of CHOP and cleaved caspase-3 protein in 2.1.1 kidneys after IR
Compared with the control group, the expression of cleaved caspase-3 protein increased at the beginning of CHOP 3 hours after IR, reached the peak at 6 hours after reperfusion, and lasted for 24 hours, which was significantly higher than that of the control group (p0.05).
Cleaved caspase-3 protein expression and survival rate, renal function and pathological changes after 2.1.2CHOP knockout
Changes of cleaved caspase-3 protein expression: The expression of cleaved caspase-3 protein in CHOP knockout mice 6 hours after IR was lower than that in wild type mice (p0.05).
Survival rate: the survival rate of CHOP knockout mice after IR (80%) was significantly higher than that of wild type mice (0%) (P0.05).
Comparison of renal function: CHOP knockout mice 24 hours after IR serum creatinine, blood urea nitrogen were significantly lower than wild type mice (p0.05).
Pathological comparison: the pathological changes of CHOP knockout mice were significantly reduced at 24 hours after IR (P0.05).
2.1.3CHOP knockout reduces IR damage by affecting renal microcirculation perfusion
CHOP knockout significantly improved the early microcirculatory perfusion of ischemic kidneys, thereby reducing the renal ischemia-reperfusion injury in mice compared with wild-type mice after IR.
2.2 Bone marrow transplantation confirms that CHOP mediates apoptosis in IR in renal innate cells rather than in bone marrow-derived immune cells
Changes in survival rate after IR in 2.2.1 four groups of bone marrow transplant mice
Survival rate of four groups of bone marrow transplantation mice after IR observation 7 days, bone marrow transplantation WT WT mice survival rate (0%) and bone marrow transplantation CHOP -/- WT mice survival rate (0%) compared with no significant difference (p0.05); bone marrow transplantation WT CHOP -/- mice survival rate (80%) and bone marrow transplantation CHOP -/- CHOP /- mice survival rate (70%) compared with no significant difference. Difference (P0.05).
Changes in renal function after 2.2.2 IR in four groups of bone marrow transplant mice
Comparison of renal function: There was no significant difference in serum creatinine and blood urea nitrogen between bone marrow transplanted WT WT mice and bone marrow transplanted CHOP -/- WT mice 24 hours after IR (p0.05); there was no significant difference in serum creatinine and blood urea nitrogen between bone marrow transplanted WT CHOP -/- mice and bone marrow transplanted CHOP -/ CHOP -/- mice 24 hours after IR (p0.05).
Pathological changes of 2.2.3 four groups of bone marrow transplantation mice after IR
Pathological comparison: BMT WT WT mice and BMT CHOP -/- WT mice had no significant difference in pathological changes 24 hours after IR (p0.05); BMT WT CHOP -/- mice and BMT CHOP -/- mice, there was no significant difference in pathological changes 24 hours after IR (p0.05).
2.3, CHOP mediated apoptosis in renal tubular epithelial cells and endothelial cells.
Injury of renal tubular epithelial cells induced by 2.3.1 hypoxia reoxygenation
LDH level: Compared with the control group, the level of LDH in cell supernatant increased 6 hours after HR and continued to increase to 24 hours after reoxygenation (p0.05).
CHOP protein expression: compared with the control group, HR increased after 6 hours, and increased to 24 hours (P0.05).
Cleaved caspase-3 protein expression: compared with the control group, 6 hours after HR increased, continued to increase to 24 hours (p0.05).
Endothelial cell injury induced by 2.3.2 hypoxia reoxygenation
LDH level: Compared with the control group, the level of LDH in cell supernatant increased 6 hours after HR and did not decrease significantly until 24 hours after reoxygenation (p0.05).
CHOP protein expression: Compared with the control group, HR increased at 6 hours and remained unchanged until 24 hours after reoxygenation (p0.05).
Cleaved caspase-3 protein expression: compared with the control group, HR increased at 6 hours and did not decrease significantly until 24 hours after reoxygenation (p0.05).
2.3.3CHOP gene silencing significantly alleviated HR induced renal tubular epithelial cell injury
LDH level: Compared with HR 24 hours group, CHOP siRNA significantly reduced the injury of renal tubular epithelial cells after HR, and the LDH level in cell culture supernatant decreased significantly (p0.05).
CHOP siRNA significantly inhibited the expression of CHOP protein in renal tubular epithelial cells induced by HR (P0.05).
CHOP gene silencing significantly inhibited the expression of cleaved caspase-3 protein in renal tubular epithelial cells 24 hours after HR (p0.05).
2.3.4CHOP gene silencing significantly alleviated endothelial cell injury induced by HR
LDH level: Compared with HR6-hour group, CHOP siRNA significantly reduced the injury of HR endothelial cells, and LDH level in cell culture supernatant decreased significantly (p0.05).
CHOPsiRNA significantly inhibited HR induced CHOP protein expression in endothelial cells (P0.05).
CHOP gene silencing significantly inhibited the expression of cleaved caspase-3 protein in endothelial cells 6 hours after HR (P0.05).
3. conclusion
The activation of CHOP in intrinsic cells of kidney plays an important role in mediating apoptosis and impairment of renal function in IR injury of kidney.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692.5
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