高压氧对小鼠缺血再灌注肾组织血管生成、凋亡相关因子和纤维化因子表达的影响

发布时间：2018-07-11 10:10

本文选题：高压氧 + 肾脏　；参考：《遵义医学院》2017年硕士论文

【摘要】：目的:通过建立同系(C57BL/6J)小鼠肾缺血再灌注损伤(ischemia reperfusion injury,IRI)模型观察血管内皮生长因子(vascular endothelial growth factor,VEGF)、B淋巴细胞瘤-2(B-cell lymphoma-2,BCL-2)、核转录因子(nuclear factor kappa B,NF-κB)、低氧诱导因子(hypoxia inducible factor-lalpha,HIF-1α)和结缔组织生长因子(connective tissue growth factor,CTGF)在缺血再灌注肾组织中的表达变化以及高压氧(hyperbaric oxygenation,HBO)治疗对这些因子表达的影响,进一步研究缺血再灌注后肾组织的病理生理改变以及HBO对缺血再灌注肾损伤的治疗作用和机制。方法:将C57BL/6J小鼠随机分为正常对照组、假手术组、肾缺血组、HBO+肾缺血组、肾IR组、肾IR㧏HBO治疗组,然后再分别将IR组及IR㧏HBO治疗组分为1h、3h、6h、12h、24h组,每小组6只小鼠。正常对照组及切开小鼠腹腔立即处死小鼠取双肾,假手术组仅切开腹腔后便将腹腔关闭;肾缺血组:切开腹腔,双侧肾蒂阻断45min后,立即处死小鼠,采血并切取肾脏标本;缺血+HBO组:在进行肾缺血组相同处理前对小鼠进行1h高压氧治疗;肾IR组:切开腹腔,双侧肾蒂阻断45min后开放血流,分别于再灌注后1、3、6、12、24h切取双肾并采取静脉血;IR㧏HBO治疗组:切开腹腔,双侧肾蒂阻断45min后开放血流,分别于再灌注后0、2、5、11、23h时行HBO治疗,治疗1h后切取双肾并采取静脉血,双侧肾蒂不阻断,于相应时点切取双肾并采取静脉血。采用HE染色观察肾组织的病理改变,免疫组织化学法检测VEGF、BCL-2、NF-κB和HIF-1α在肾组织中的表达,实时荧光定量PCR(real-time PCR)检测VEGF、CTGF的表达以及全自动生化分析仪检测血清肌酐(SCr)。结果:1.肾功能变化:结果显示,假手术组与正常对照组血肌酐无明显差异,肾缺血组和HBO+肾缺血组的血肌酐值明显高于假手术组(P0.05),在HBO治疗后,肾缺血引起的肾功能损害有改善但不明显。再灌注后肾功能损害加重,且随着再灌注时间的延长逐渐加重,在再灌注24h时损伤最重。HBO治疗则明显改善了再灌注后的肾功能,以1h、3h、6h和12h时突出,24小时后高压氧治疗对肾功能的保护作用减弱。2.肾组织病理变化:正常对照组和假手术组可见肾小管细胞无明显病理改变,缺血组及缺血+HBO组可见肾小管细胞明显肿胀,HBO治疗后肾小管细胞肿胀程度较前有轻微好转,IR组于1h时,可见系膜细胞轻度增生,肾间质血管轻度扩张充血,肾小球体积轻度增大,肾小管上皮细胞轻度肿胀;于3h时,可见肾小管上皮细胞肿胀进一步加重,呈颗粒样变性,组织血管充血;于6h时,可见肾肾小管细胞已经逐渐失去细胞原有形态,肾小管上皮细胞肿胀进一步加重,伴少量中性粒细胞浸润,12h及24h可见大量的小管细胞破坏,肾小管细胞出现了相互融合,细胞结构被完全破坏,并出现少量核碎裂核溶解,并出现大量的的中性粒细胞浸润。经过高压氧治疗后各时间点均可见肾小管细胞肿胀明显减轻,细胞破坏明显减少,核溶解现象也有所减少。3.免疫组织化学法检测VEGF、BCL-2、NF-κB和HIF-1α在肾组织中的表达:(1)肾组织VEGF蛋白的表达变化:VEGF蛋白主要表达于肾小管上皮细胞胞质,细胞核少量表达,高压氧治疗后胞核及胞质表达均增加。相对定量结果显示,肾缺血组、HBO+肾缺血组、肾IR组及肾IR+HBO组VEGF蛋白的表达均显著高于正常对照组和假手术组(P0.05),而在肾缺血前运用高压氧治疗可见VEGF表达增加(P0.05)。肾IR后,VEGF的表达较假手术组呈持续性上升并在6h达到峰值(P0.05),12h后开始表现出下降趋势但仍高于假手术组(P0.05);经高压氧治疗后,肾IR+HBO组VEGF对应各时间点的表达相较于肾IR组有明显的增加并且在6h增加最为明显(P0.05)。(2)肾组织HIF-1α蛋白的表达变化:HIF-1α蛋白主要表达于肾小管细胞的胞质,细胞核有少量表达。相对定量结果显示,肾缺血组、HBO+肾缺血组、肾IR组及肾IR+HBO组HIF-1α蛋白的表达均显著高于正常对照组和假手术组(P0.05),而在肾缺血前运用高压氧治疗可见HIF-1α表达增加,但并无统计学意义(P0.05)。肾缺血再灌注损伤发生后,HIF-1α的表达呈持续性上升并在6h达到峰值(P0.05),12h后开始表现出下降趋势但仍高于假手术组(P0.05);经高压氧治疗后,HIF-1α的表达各时间点相比较于肾IR组有明显的增加并且在6h增加最为明显(P0.05)。(3)肾组织Bcl-2蛋白的表达变化:Bcl-2蛋白主要表达于肾小管上皮细胞胞质,细胞核少量表达。相对定量结果显示,肾缺血组、HBO+肾缺血组、肾IR组及肾IR+HBO组Bcl-2蛋白的表达均显著高于正常对照组和假手术组(P0.05),而在肾缺血前运用高压氧治疗可见Bcl-2表达增加,但并无统计学意义(P0.05)。肾缺血再灌注损伤发生后,Bcl-2的表达呈持续性上升并在3-6h达到峰值(P0.05),12h后开始表现出下降趋势但仍高于假手术组(P0.05);经高压氧治疗后,Bcl-2蛋白各时间点的表达相比较于肾IR组均有明显的增加并且在3-6h增加最为明显(P0.05)。(4)肾组织NF-κB蛋白的表达变化:NF-κB蛋白主要表达于肾小管上皮细胞胞质,细胞核少量表达。相对定量结果显示,肾缺血组、HBO+肾缺血组、肾IR组及肾IR+HBO组NF-κB蛋白的表达均显著高于正常对照组和假手术组(P0.05),而在肾缺血后运用高压氧治疗预处理可见NF-κB表达无显著差异(P0.05)。肾缺血再灌注损伤发生后,NF-κB的表达呈持续性上升并在6h达到峰值,12h后开始表现出下降趋势但仍高于假手术组(P0.05);经高压氧治疗后,NF-κB各时间点的表达相比较于肾IR组有明显的减少并且在6h减少最为明显(P0.05)。4.实时荧光定量PCR(real-time PCR)检测肾组织VEGF m RNA、CTGF m RNA的表达变化:(1)肾组织VEGF m RNA的表达变化:肾缺血组、HBO+肾缺血组,肾IR组及肾IR+HBO组VEGF m RNA的表达量较正常对照组和假手术组均增加(P0.05),经高压氧治疗后HBO+肾缺血组VEGF m RNA的表达高于肾缺血组(P0.05)。肾IR时VEGF m RNA的表达随着时间的延长逐渐增加并在6h达到峰值(P0.05),12h后表达开始逐渐下降。当经过高压氧治疗后我们可以发现肾IR+HBO治疗组各时间点VEGF m RNA的表达相比较于肾IR组在1h、3h、6h、12h均有明显增加(P0.05),24h无明显差异(P0.05)。(2)肾组织CTGF m RNA的表达变化:肾缺血组、HBO+肾缺血组、肾IR组及肾IR+HBO组CTGF m RNA的表达均显著高于正常对照组和假手术组(P0.05),而在肾缺血后运用高压氧治疗预处理可见CTGF m RNA表达无显著差异(P0.05)。肾缺血再灌注损伤发生后,CTGF m RNA的表达呈持续性上调(P0.05);经高压氧治疗后,6h后各时间点相比较于肾IR组CTGF m RNA的表达有明显的减少(P0.05)。结论:1.HBO治疗可以通过HIF-VEGF-Notch通路显著改善缺血再灌注肾组织的缺氧状态,促进新生血管的生成,从而减轻肾缺血再灌注损伤。2.HBO治疗可以减少CTGF表达,通过减少致纤维化因子的表达在对于肾缺血再灌注损伤中减轻肾脏损伤,对肾脏起到保护作用。3.HBO治疗可以通过增加Bcl-2的表达并减少NF-κB的表达,对细胞凋亡上游因子的调控,从而对肾脏在缺血再灌注损伤中起到了保护作用。4.HBO治疗在肾缺血再灌注早期便开始发挥保护作用,对于新生血管的生成、纤维化因子的抑制以及对于凋亡因子的抑制都有着明显的作用,随着时间的推移有所减弱,因此在肾缺血再灌注损伤的预防及治疗过程中应尽早采用高压氧治疗,以减轻缺血再灌注对肾脏的损害。
[Abstract]:Objective: To observe the vascular endothelial growth factor (vascular endothelial growth factor, VEGF) and B lymphocytic tumor (ischemia reperfusion injury (IRI) model in C57BL/6J mice. The expression of ible factor-lalpha, HIF-1 alpha) and connective tissue growth factor (connective tissue growth factor, CTGF) in the renal tissue of ischemia and reperfusion and the effect of hyperbaric oxygen (hyperbaric oxygenation, HBO) on the expression of these factors, further study the pathophysiological changes of renal tissue and HBO against ischemia after blood reperfusion. Treatment and mechanism of reperfusion renal injury. Methods: C57BL/6J mice were randomly divided into normal control group, sham operation group, renal ischemia group, HBO+ kidney ischemia group, kidney IR group, kidney IR HBO treatment group, then IR group and IR HBO treatment group were divided into 1H, 3h, 6h, 12h, 6 mice in each group. The dead mice took two kidneys, and the sham operation group closed the abdominal cavity after only incision of the abdominal cavity. In the renal ischemia group, after the incision of the abdominal cavity and the bilateral renal pedicle blocking 45min, the mice were killed and the kidney specimens were harvested. The ischemic +HBO group was treated with 1H high pressure oxygen therapy before the same treatment in the kidney ischemia group; the kidney IR group: after the incision of the abdominal cavity and the bilateral renal pedicle blocking 45min The blood flow was opened and 1,3,6,12,24h was cut into two kidneys and venous blood was taken after reperfusion respectively. IR? HBO treatment group: incision of abdominal cavity, bilateral renal pedicle after blocking 45min to open blood flow, respectively after reperfusion after 0,2,5,11,23h, HBO treatment. After treatment 1H, two kidneys were cut and venous blood was taken. The two sides of kidney did not block, and the two kidneys were cut at the corresponding time points and taken static and static. Pulse blood. The pathological changes of renal tissue were observed by HE staining. Immunohistochemistry was used to detect the expression of VEGF, BCL-2, NF- kappa B and HIF-1 alpha in the renal tissue. Real-time fluorescent quantitative PCR (real-time PCR) was used to detect VEGF, CTGF expression and automatic biochemical analyzer to detect serum creatinine (SCr). Results: 1. renal function changes: the results showed sham operation group and sham operation group. There was no significant difference in serum creatinine in the normal control group. The blood creatinine value of renal ischemia group and HBO+ renal ischemia group was significantly higher than that of the sham operation group (P0.05). After HBO treatment, renal impairment caused by renal ischemia was improved but not obvious. The damage of renal function was aggravated after reperfusion, and with the prolongation of reperfusion time, the damage was the heaviest at 24h reperfusion. .HBO treatment significantly improved renal function after reperfusion, prominent in 1H, 3h, 6h and 12h. After 24 hours, the protective effect of hyperbaric oxygen therapy on renal function weakened the pathological changes of.2. renal tissue: there was no obvious pathological changes in renal tubule cells in normal control group and sham operation group, and obvious swelling of renal tubule cells in ischemic group and ischemic +HBO group, HBO After treatment, the swelling degree of renal tubular cells was slightly better than that before the treatment. When group IR was 1H, the mesangial cells were slightly proliferated, the renal interstitial vessels were slightly dilated and congested, the volume of glomeruli was slightly enlarged, and the renal tubular epithelial cells were slightly swelling. At 3h, the swelling of the tubular epithelial cells was further aggravated, with granular degeneration and hyperemia of the tissue vessels; at 6 At h, the renal tubular cells had gradually lost the original morphology of the cells. The swelling of the renal tubular epithelial cells was further aggravated, with a small amount of neutrophils infiltration. A large number of tubules were destroyed in 12h and 24h. The renal tubular cells were fused with each other, the cell structure was completely destroyed, and a small amount of nuclear disintegration was dissolved, and a large number of them appeared. Neutrophils infiltration. After hyperbaric oxygen therapy, the swelling of renal tubule cells was obviously reduced, cell destruction was significantly reduced, and nuclear dissolution also decreased the expression of VEGF, BCL-2, NF- kappa B and HIF-1 alpha in renal tissue by.3. immunohistochemistry: (1) the expression of VEGF protein in renal tissue: VEGF protein main The expression of the cytoplasm of renal tubular epithelial cells, a small amount of nuclear expression, increased nuclear and cytoplasmic expression after hyperbaric oxygen therapy. The relative quantitative results showed that the expression of VEGF protein in renal ischemia group, HBO+ renal ischemia group, kidney IR group and renal IR+HBO group was significantly higher than that of normal control group and artificial hand group (P0.05), and hyperbaric oxygen therapy was used before renal ischemia. The expression of VEGF increased (P0.05). After IR, the expression of VEGF was higher than that in the sham operation group and reached the peak value at 6h (P0.05). After 12h, the expression of VEGF began to decline but was still higher than that of the sham operation group (P0.05). After hyperbaric oxygen therapy, the expression of VEGF in the renal IR+HBO group was significantly higher than that in the IR group and increased in the 6h. The expression of HIF-1 alpha protein in renal tissue was significantly (P0.05) (2): the expression of HIF-1 alpha protein was mainly expressed in the cytoplasm of renal tubular cells and a small amount of expression in the nucleus. The relative quantitative results showed that the expression of HIF-1 alpha protein in renal ischemia group, HBO+ renal ischemia group, kidney IR group and kidney IR+HBO group were significantly higher than that of normal control group and sham operation group (P0.05), but in kidney, the renal tissue was significantly higher than that of the normal control group and the sham operation group (P0.05). The expression of HIF-1 alpha was increased in hyperbaric oxygen therapy before ischemia, but there was no statistical significance (P0.05). After renal ischemia-reperfusion injury, the expression of HIF-1 alpha increased continuously and reached a peak in 6h (P0.05). After 12h, it began to show a downward trend but still higher than that of the sham operation group (P0.05). After hyperbaric oxygen therapy, the expression of HIF-1 a was at all time points. Compared with the renal IR group, there was a significant increase in the 6h (P0.05). (3) the expression of Bcl-2 protein in the renal tissue: the expression of Bcl-2 protein was mainly expressed in the cytoplasm of renal tubular epithelial cells and a small number of nuclei. The relative quantitative results showed that the expression of Bcl-2 protein in the renal ischemia group, the HBO+ kidney deficiency group, the renal IR group and the renal IR+HBO group were all significant. It was higher than the normal control group and the sham operation group (P0.05), but the expression of Bcl-2 increased with hyperbaric oxygen therapy before renal ischemia, but there was no statistical significance (P0.05). After renal ischemia-reperfusion injury, the expression of Bcl-2 increased continuously and reached the peak value at 3-6h (P0.05). After 12h, the decline trend began to decline but still higher than that of the sham operation group (P0.05). After hyperbaric oxygen therapy, the expression of Bcl-2 protein at all time points was significantly increased compared with that in group IR and increased in 3-6h (P0.05). (4) the expression of NF- kappa B protein in renal tissue: the expression of NF- kappa B protein was mainly expressed in the cytoplasm of renal tubular epithelial cells and a small amount of nuclear expression. The relative quantitative results showed that the renal ischemia group, HBO+ kidney was a relative quantitative result. In the ischemic group, the expression of NF- kappa B protein in the renal IR group and the kidney IR+HBO group was significantly higher than that in the normal control group and the sham operation group (P0.05), but there was no significant difference in the expression of NF- kappa B (P0.05) in the preconditioning with hyperbaric oxygen therapy after renal ischemia. The expression of NF- kappa B increased continuously and reached the peak in 6h after renal ischemia-reperfusion injury. The downward trend was still higher than the sham operation group (P0.05). After the hyperbaric oxygen therapy, the expression of NF- kappa B at each time point was significantly decreased compared to the renal IR group and the most obvious (P0.05).4. real-time quantitative PCR (real-time PCR) was most obvious in 6h (real-time PCR), and the expression changes of renal tissue were observed. (1) the expression of renal tissue was changed. The expression of VEGF m RNA in renal ischemia group, HBO+ renal ischemia group, kidney IR group and renal IR+HBO group increased (P0.05). The expression of VEGF m RNA in HBO+ renal ischemia group was higher than that in renal ischemia group after hyperbaric oxygen therapy. After 12h, the expression of VEGF m RNA in the renal IR+HBO treatment group was significantly increased in 1H, 3h, 6h and 12h in the renal IR+HBO treatment group (P0.05). (2) the renal ischemia group, renal ischemia group, renal ischemia group and renal failure group were in the renal ischemia group. The expression of CTGF m RNA in group O was significantly higher than that in the normal control group and the sham operation group (P0.05), but there was no significant difference in the expression of CTGF m RNA (P0.05). After renal ischemia and reperfusion, the expression of CTGF m RNA was continuously up-regulated after renal ischemia-reperfusion injury, and the time points were compared after hyperbaric oxygen therapy. The expression of CTGF m RNA in group IR was significantly decreased (P0.05). Conclusion: 1.HBO therapy can improve the hypoxia state of renal tissue of ischemia and reperfusion through HIF-VEGF-Notch pathway and promote the formation of new blood vessels, thus reducing the expression of CTGF in renal ischemia reperfusion injury by.2.HBO therapy, and reducing the expression of fibrotic factors by reducing the expression of fibrotic factors in the renal ischemia reperfusion injury. Renal ischemia reperfusion injury alleviates renal injury, and the protective effect of.3.HBO on kidney can be protected by increasing the expression of Bcl-2 and reducing the expression of NF- kappa B, regulating the upstream factors of apoptosis, and thus protecting the kidney from ischemia-reperfusion injury,.4.HBO treatment begins to play in the early stage of renal ischemia and reperfusion. The protective effect has obvious effect on the formation of new blood vessels, the inhibition of fibrotic factors and the inhibition of apoptosis factors, which weaken with time. Therefore, hyperbaric oxygen therapy should be adopted as early as possible in the prevention and treatment of renal ischemia-reperfusion injury, in order to reduce the damage to renal ischemia and reperfusion.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692.5

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