褐藻多糖硫酸酯对TGF-β_1诱导HK-2间质转分化过程中Smad2/3、Smad7表达的影响

Effects of fucoidan on expression of Smad2 /3 and Smad7 during the course of epithelial mesenchymal transition induced by TGF-β_1 of HK-2 cells

目的观察褐藻多糖硫酸酯对转化生长因子β1(TGF-β1)诱导肾小管上皮细胞(HK-2)间质转分化过程中细胞信号转导分子(Smad)2/3和Smad7表达的影响,并探讨其机制。方法 HK-2细胞复苏后用RPMI1640培养基加10%胎牛血清在37℃、5%CO2培养箱中培养。待细胞贴壁后分为3组,对照组加入等量RPMI1640细胞培养基,诱导组予5μg/L TGF-β1,药物组予5μg/L TGF-β1+50μg/mL褐藻多糖硫酸酯。采用倒置相差显微镜观察细胞形态变化,免疫细胞化学法检测Smad2/3、Smad7表达,图像分析技术分析Smad2/3、Smad7表达阳性的平均光密度值。结果培养第3天,对照组HK-2细胞呈铺路石样上皮细胞形态特点;诱导组细胞呈长梭形,类似纤维细胞;药物组细胞以上皮细胞为主,少数细胞呈梭形。与对照组比较,诱导组细胞Smad2/3表达增强,Smad7表达减弱;与诱导组比较,药物组细胞Smad2/3表达减弱,Smad7表达增强。与对照组比较,诱导组细胞Smad2/3阳性细胞平均光密度值显著升高,Smad7阳性细胞平均光密度值显著下降;与诱导组相比较,药物组细胞Smad2/3阳性细胞平均光密度值显著下降,Smad7阳性细胞平均光密度值显著升高(P均<0.05)。结论褐藻多糖硫酸酯可能通过影响TGF-β1信号转导通路中Smad2/3和Smad7的表达发挥抑制HK-2间质转分化作用。

Objective To observe the effects of fueoidan on the expression of cell signal transduction molecule （Smad）2/ 3 and Smad7 during the course of epithelial mesenehymal transition induced by TGF-β1 of human renal tubular epithelial cells （HK-2 cells） in vitro and explore its possible molecular mechanisms. Methods Normal HK-2 cells were cultured in the RP- MI1640 medium combined with 10% fetal bovine serum at the temperature of 37 -12 and 5%CO2 incubator. After the cells adher- ence, the cells were divided into 3 groups. The control group was added the equal amount of RPMI1640 cell culture medium, the induction group was treated with 5 μg/L TGF-β1 , and the intervention group was treated with 5 μg/L TGF-β1 ＋50 μg/mL fu- coidan. Morphological changes of cells were observed by using an inverted phase contrast microscope; immunocytochemieal meth- od was used to detect the expression of Smad2/3 and Smad7, and image analysis was used to detect the average optical density values of Smad2/3 and Srnad7-positive cells. Results After three days of culture, HK-2 cells showed cobblestone morphology characteristics of epithelial cells in the control group, the induction group showed fusiform cells, similar to fibroblasts, and the intervention group mainly had epithelial cells, but a few cells were fusiform. Compared with the control group, the expression of Smad2/3 was enhanced, Smad7 expression decreased in the induction group; compared with the induction group, Smad2/3 ex- pression decreased, Smad7 expression increased in the intervention group. Compared with the control group, the average optical density values in Smad7-positive cells were significantly decreased and the average optical density values in Smad2/3-positive cells increased in the induction group; compared with the induction group,the average optical density values in Smad7-positive cells were significantly increased and the average optical density values in Smad2/3-positive cells were significantly decreased in the intervention group （all P 〈 0.05）. Conclusion Fucoidan may play an important role in inhibiting the course of epithelial mesenchymal transition induced by TGF-β1 through altering the expression of Smad2/3 and Smad7.