丙泊酚对肾小管上皮细胞系缺血再灌注损伤后纤维化和TAK1的影响

发布时间：2018-03-14 00:27

本文选题：丙泊酚　切入点：缺血再灌注损伤　出处：《广东医学》2017年20期 　论文类型：期刊论文

【摘要】：目的探讨丙泊酚对人肾小管上皮细胞(HK-2细胞)缺血再灌注损伤后纤维化的作用及转化生长因子β激活激酶(TAK1)在其中的可能作用机制。方法采用随机数字表法将HK-2细胞分为6组(n=36):空白对照组(C组)、缺血再灌注组(IR组)、丙泊酚组(IRP组)、脂肪乳剂组(IRF组)、TAK1过表达组(IRPT组)、TAK1过表达阴性病毒对照组(IRPTI组)。采用抗霉素A耗竭HK-2细胞ATP后更换正常培养液恢复补充代谢底物的方法制备ATP耗竭再恢复损伤模型模拟体内细胞缺血再灌注损伤状态。C组正常培养,不作任何处理;IR组:正常培养的HK-2细胞更换为含10μmol/L抗霉素A及1μmol/L钙离子通道载体A23187的无血清DMEM培养基孵育1 h,PBS清洗之后再换成正常含血清培养基继续培养12、24、48 h;IRP组于ATP耗竭前1 h,正常培养基中加入丙泊酚(终浓度20μmol/L)预孵育1 h;IRPT组于正常培养基中加入丙泊酚(终浓度20μmol/L)及TAK1慢病毒(滴度为2×107TU/mL)预孵育1 h;IRF组正常培养基中加入10%脂肪乳剂预孵育1 h;IRPTI组正常培养基中加入丙泊酚(终浓度20μmol/L)及TAK1阴性慢病毒预孵育1 h,其余处理均同IR组。于ATP再恢复12 h时,采用流式细胞仪检测细胞凋亡,MTT法测定细胞活力;于ATP再恢复12、24和48 h时,采用Western blot法测定TAK1的表达水平,于ATP再恢复48 h时Western blot法检测α平滑肌肌动蛋白(α-SMA)、纤维粘连蛋白(FN)、胶原蛋白1(COL 1)的表达水平。结果与C组比较,IR组细胞凋亡率增加,细胞活力降低,ATP再恢复后TAK1、COL1、α-SMA和FN表达均上调(P0.05);与IR组比较,IRP组细胞凋亡率减少,细胞活力增强,ATP再恢复后TAK1、COL1、α-SMA和FN表达均下调(P0.05);与IRP组比较,IRPT组细胞凋亡率增加,细胞活力降低,再恢复后TAK1、COL1、α-SMA和FN表达均上调(P0.05)。结论丙泊酚预处理可减轻ATP耗竭再恢复诱导的HK-2细胞纤维化,其机制可能是通过下调TAK1表达,进而抑制了早期细胞凋亡,从而减轻了HK-2细胞纤维化程度。
[Abstract]:Objective to investigate the effect of propofol on fibrosis after ischemia-reperfusion injury in human renal tubular epithelial cells (HK-2) and the possible mechanism of transforming growth factor 尾 -activated kinase (TGF- 尾 -activated kinase 1) in the process. The cells were divided into 6 groups: control group C, ischemia reperfusion group, propofol group IRP group, fat emulsion group IRF group, IRF group IRF group, HK-2 group with negative expression of TAK1. The HK-2 cells were depleted by anti-mycin A. Methods the model of ATP depletion and recovery injury was made by replacing the normal culture medium after ATP. The model was used to simulate the ischemia reperfusion injury of cells in vivo. Group C was cultured in normal condition. No treatment: normal cultured HK-2 cells were replaced by serum-free DMEM medium containing 10 渭 mol/L anti-mycin A and 1 渭 mol/L calcium channel carrier A23187, incubated for 1 hour, then washed, then replaced with normal serum-containing medium for 12244h. One hour before ATP depletion, propofol (final concentration 20 渭 mol / L) was preincubated in normal culture medium for 1 hour. In IRPT group, propofol (final concentration 20 渭 mol / L) and TAK1 lentivirus (titer was 2 脳 10 7 Tu / mL) preincubated for 1 h were added to normal culture medium. 10% lipid emulsion preincubated for 1 hour in normal culture medium with propofol (final concentration 20 渭 mol / L) and TAK1 negative lentivirus preincubated for 1 hour. The cell viability was detected by flow cytometry and the expression of TAK1 was detected by Western blot method at 1224 and 48 h after ATP was restored. The expression of 伪 -smooth muscle actin (伪 -SMAN), fibronectin (FN) and collagen 1 (Col 1) were detected by Western blot assay at 48 h after the recovery of ATP. Results compared with group C, the apoptosis rate of IR group was higher than that of C group. Compared with IR group, the apoptotic rate of IRP group decreased, and the expression of 伪 -SMA and FN decreased after the recovery of ATP. Compared with IRP group, the apoptosis rate of IRP group increased and the cell viability decreased, and the expression of 伪 -SMA and FN decreased in IRP group compared with that in IRP group. Conclusion Propofol pretreatment can reduce the fibrosis of HK-2 cells induced by ATP depletion and recovery, and its mechanism may be by down-regulating the expression of TAK1, and then inhibiting the early apoptosis of HK-2 cells by down-regulating the expression of TAK1, 伪 -SMA and FN. Thus, the degree of HK-2 cell fibrosis was alleviated.
【作者单位】：佛山市第一人民医院麻醉科;
【基金】：佛山市自筹经费类科技计划项目(编号:2016AB002761)
【分类号】：R614

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