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目的考察肾组织PHD锌指蛋白14(PHF14)的表达变化,探讨PHF14在急性损伤后肾纤维化中的作用。方法选用IVC级C57BL/6小鼠20只,随机分为2组:对照组(n=5)和叶酸刺激实验组(n=15),实验组又分为叶酸作用后2、14和28d3个亚组,每个亚组5只小鼠。实验组单次给予叶酸腹腔注射(250mg/kg,溶媒为0.3mol/L碳酸氢钠溶液),对照组单次给予溶媒腹腔注射。每组小鼠在预定的时间点处死,留取血清检测肌酐(SCr)和尿素氮(BUN);留取肾组织,行Masson染色验证模型,蛋白质免疫印迹法研究PHF14在急性肾损伤纤维化进程中的表达变化。利用PHF14基因敲除鼠,制作与上述相同的急性肾损伤模型,比较PHF14敲除组和未敲除组肾脏在第2、14和28天的病理变化,并行α-平滑肌肌动蛋白(α-SMA)和collagen 1免疫组织化学染色,蛋白质免疫印迹法检测转化生长因子β(TGF-β)、α-SMA、collagen 1的表达规律。结果与对照组相比,实验组各时间点小鼠SCr与BUN变化曲线提示急性肾损伤病程;肾脏病理变化显示,随叶酸作用时间的增加,肾脏逐渐进展到纤维化,提示模型制作成功。在急性肾损伤至慢性纤维化过程中,PHF14持续高表达。与野生型小鼠相比,PHF14敲除小鼠TGF-β、α-SMA、collagen 1表达较高,肾纤维化程度更重。结论叶酸腹腔注射可成功建立急性肾损伤及损伤后肾小管间质纤维化模型,PHF14的表达在叶酸促纤维化刺激后持续上调,而PHF14敲除会加重急性损伤后肾纤维化的发生,提示PHF14对肾脏纤维化进程起抑制作用,可能是内源性肾纤维化保护因子。
Objective To investigate the expression of PHF14 in renal tissue and to explore the role of PHF14 in renal fibrosis after acute injury. Methods Twenty Twenty-four C57BL / 6 mice were randomly divided into two groups: control group (n = 5) and folic acid stimulation group (n = 15). The experimental group was divided into 2 groups Subgroup, 5 mice per subgroup. The experimental group was given intraperitoneal injection of folic acid (250mg / kg, 0.3mol / L sodium bicarbonate solution). The control group was intraperitoneally injected with vehicle. Each group of mice was killed at a predetermined time point, serum creatinine (SCr) and urea nitrogen (BUN) were collected. Renal tissues were collected and subjected to Masson staining. Western blotting was used to investigate the effect of PHF14 on fibrosis progression of acute kidney injury In the expression of change. PHF14 knockout mice were used to make the same model of acute kidney injury as above. The pathological changes of the kidneys in PHF14 knockout and untreated groups were compared on the 2nd, 14th and 28th days. The α-smooth muscle actin (α- SMA) and collagen 1 were detected by immunohistochemistry. The expressions of TGF-β, α-SMA and collagen 1 were detected by Western blotting. Results Compared with the control group, the changes of SCr and BUN in mice at each time point showed the course of acute kidney injury. The pathological changes of kidney showed that the kidney gradually progressed to fibrosis with the increase of folic acid time, which suggested that the model was successfully made. During acute kidney injury to chronic fibrosis, PHF14 persistently high expression. Compared with wild-type mice, PHF14-knockout mice had higher expression of TGF-β, α-SMA and collagen-1 and more severe renal fibrosis. Conclusion Intraperitoneal injection of folic acid can successfully establish a model of acute renal injury and tubulointerstitial fibrosis after injury, the expression of PHF14 continues to increase after folic acid stimulation and fibrosis, while PHF14 knockout aggravates the occurrence of renal fibrosis after acute injury, suggesting that PHF14 plays an inhibitory role in the process of renal fibrosis and may be an endogenous protective factor of renal fibrosis.