论文部分内容阅读
目的:探讨外泌体在草酸钙晶体所致肾损伤及肾间质纤维化中的作用。方法:C57小鼠30只(6～8周龄，体重24～26 g)，其中24只野生型(WT)小鼠，6只纯合子Rab27an -/-(KO)小鼠。WT小鼠随机分为空白对照(NC)组、造模1周组、造模2周组、造模4周组(WT造模组)，Rab27an -/-小鼠为KO造模组，每组6只，1.25%乙二醇灌胃，分别持续给药0、1、2、4及4周，构建肾结石小鼠动物模型。苏木精-伊红(HE)染色评估肾小管损伤，Von-kossa染色评估结石晶体形成，Masson染色评估肾间质胶原纤维沉积，免疫组织化学染色检测α-平滑肌肌动蛋白(α-SMA)、纤维连接蛋白(Fibronectin)及外泌体标志物白细胞分化抗原63(CD63)表达，两组间比较采用n t检验。n 结果:HE染色显示，在野生型小鼠中，造模1周、造模2周及造模4周肾小管损伤逐渐加重(0.57±0.18比1.30±0.19，n t＝6.248，n P<0.05；1.30±0.19比2.30±0.28，n t＝6.642，n P<0.05)，Von-kossa染色显示，肾脏晶体形成逐渐增加(1.42±0.61比5.24±0.55，n t＝10.410，n P<0.05；5.24±0.55比10.01±0.94，n t＝9.946，n P<0.05)，差异均有统计学意义。免疫组织化学染色显示，造模1周、造模2周及造模4周CD63阳性表达面积逐渐增加(1.33±0.34比4.92±0.71，n t＝10.250，n P<0.05；4.92±0.71比8.55±1.04，n t＝6.448，n P<0.05)差异有统计学意义。WT造模组Masson染色胶原纤维沉积面积高于NC组(8.57±0.84比0.60±0.34，n t＝19.660，n P<0.05)，α-SMA阳性表达面积高于NC组(11.30±1.04比1.63±0.55，n t＝18.430，n P<0.05)，Fibronectin阳性表达面积高于NC组(12.85±0.68比2.78±0.55，n t＝25.810，n P<0.05)，差异均有统计学意义。WT造模组Masson染色胶原纤维沉积面积高于KO造模组(8.57±0.84比5.53±0.74，n t＝6.082，n P<0.05)，α-SMA阳性表达面积高于KO造模组(11.30±1.04比6.63±0.91，n t＝7.553，n P<0.05)，Fibronectin阳性表达面积高于KO造模组(12.85±0.68比8.80±0.93，n t＝7.875，n P<0.05)，差异均有统计学意义。n 结论:肾结石形成过程中，肾小管损伤加重，外泌体分泌增加。同时，抑制外泌体分泌能减轻草酸钙晶体诱导的肾脏纤维化。“,”Objective:To explore the roles of exosomes in renal injury and renal interstitial fibrosis induced by calcium oxalate crystals.Methods:A total of 30 C57 mice, including 24 wild type (WT) mice, and 6 homozygous Rab27an -/- (knockout) mice were enrolled in this study. WT mice were randomly divided into no-treatment control (NC) group, one-week stone model group, 2-week stone model group, 4-week stone model group (WT stone model group), and 6 homozygous Rab27an -/- (knockout) mice served as KO stone model group, n n=6 each group. The model of mice kidney stone formation was established by gavage of 1.25% ethylene glycol for 0, 1, 2, 4 weeks (WT stone model group) and 4 weeks (KO stone model group) respectively. HE staining was used to evaluate renal tubular injury, Von-Kossa staining was used to evaluate the formation of stone crystals, Masson staining was used to evaluate the deposition of collagen fibers in renal interstitium, and immunohistochemical staining was used to detect the expression of α-smooth muscle actin (α-SMA), Fibronectin and exosome marker leukocyte differentiation antigen (CD) 63. n T test was used for comparison between the two groups.n Results:HE staining showed that in WT mice, renal tubule injury aggravated gradually at 1st, 2nd and 4th week (0.57±0.18 vs. 1.30±0.19, n t=6.248, n P<0.05; 1.30±0.19 vs. 2.30±0.28,n t=6.642, n P<0.05); Von-Kossa staining showed a gradual increase in renal crystal formation (1.42±0.61 vs. 5.24±0.55,n t=10.410, n P<0.05; 5.24±0.55 vs. 10.01±0.94,n t=9.946, n P<0.05). Immunohistochemical staining showed that the positive expression area of CD63 increased gradually (1.33±0.34 vs. 4.92±0.71,n t=10.250, n P<0.05; 4.92±0.71 vs. 8.55±1.04,n t=6.448, n P<0.05) at 1st, 2nd and 4th week of modeling. As compared with WT group, the collagen fiber deposition area was significantly increased (0.60±0.34 vs. 8.57±0.84,n t=19.660, n P<0.05), and the positive expression area of α-SMA (1.63±0.55 vs. 11.30±1.04,n t=18.430, n P<0.05) and Fibronectin (2.78±0.55 vs. 12.85±0.68,n t=25.810, n P<0.05) significantly increased in WT stone model group. As compared with WT stone model group, collagen fiber deposition area significantly decreased (5.53±0.74,n t=6.082, n P<0.05), and the positive expression area of α-SMA (6.63±0.91,n t=7.553, n P<0.05) and Fibronectin (8.80±0.93,n t=7.875, n P<0.05) decreased significantly in KO stone model group.n Conclusion:During the formation of kidney stones, renal tubular injury is aggravated and exosome secretion is increased. At the same time, inhibition of exosome secretion can reduce calcium oxalate crystal-induced renal fibrosis.