目的

探讨胶原/盘状结构域受体2 （DDR2）信号活化对肾间质纤维化进程中肾成纤维细胞增殖和迁移功能的影响。

方法

采用SPF 级雄性C57BL/6 小鼠，通过单侧输尿管梗阻 （UUO）术建立肾小管间质纤维化模型，并收集病理样本和新鲜组织，进行免疫组化、Western blot 等检测。通过培养人肾成纤维细胞，将shDDR2 基因慢病毒及其阴性对照慢病毒、OE-DDR2 慢病毒及其阴性对照慢病毒分别转染至KFB 细胞，经5 μg/mL 嘌呤霉素筛选后，构建稳转细胞株，并使用浓度为10 μg/mL 胶原处理48 h。对照组1：shCtrl+胶原组 （shDDR2 阴性对照，未下调DDR2 表达但添加胶原处理）；对照组2：Control+胶原组 （OE-DDR2 阴性对照，未过表达DDR2 但添加胶原处理）；实验组1：shDDR2+胶原组 （DDR2 基因敲低并添加胶原处理）；实验组2：OE-DDR2+胶原组 （DDR2 基因过表达并添加胶原处理）；实验组3：在实验组2基础上，使用浓度为1 μmol/L 的达沙替尼处理，所有组别处理时间均为48 h。采用qRT-PCR 法检测DDR2 mRNA 的表达水平，采用Western blot 法检测DDR2 蛋白的水平。采用CCK-8 和BrdU 实验进行细胞增殖检测。采用HE 染色和免疫组织化学染色方法观察细胞定位和病理变化。两组间比较采用独立样本 t 检验，方差不齐性采用 t' 检验；多组间比较采用单因素方差分析，两两比较组间方差齐时采用 LSD-t 检验，方差不齐时采用 Tamhane's T2 检验。

结果

DDR2在小鼠肾组织中定位于肾间质成纤维细胞中，且与胶原呈共定位关系。与shCtrl+胶原组相比，sh-DDR2+胶原组的细胞增殖速度 （0.28 ± 0.00 比0.38 ± 0.01）减缓 （P ＜ 0.05），Transwell 转移［ （6.00 ± 1.00 比21.66 ± 0.57）个］降低 （P ＜ 0.05）。与Control+胶原组相比，OE-DDR2+胶原组的细胞增殖速度 （0.43 ± 0.01 比0.39 ± 0.01）加快 （P ＜ 0.05），Transwell 迁移［ （32.66 ± 0.57比22.33 ± 0.57）个］增加 （P ＜ 0.05），而经达沙替尼处理后，细胞增殖速度 （0.36 ± 0.01 比0.39 ±0.01）降低。

结论

胶原/DDR2 信号活化显著促进KFB 细胞的增殖及迁移，最终促进肾间质纤维化的发生发展。

Objective

To investigate the effect of collagen/DDR2 signaling on the proliferation and migration of renal fibroblasts during renal interstitial fibrosis.

Methods

A renal tubulointerstitial fibrosis model was established in SPF male C57BL/6 mice via unilateral ureteral obstruction （UUO）， and pathological samples and fresh tissues were obtained for Western blotting and immunohistochemistry. Human kidney fibroblasts were cultivated， followed by the transfection of KFB cells with shDDR2 and its negative control lentivirus， OE-DDR2 and its negative control lentivirus， and screening with 5 μg/mL puromycin. A stable transfection cell line was then created and exposed to 10 μg/mL collagen for 48 hours. Control Group 1： shCtrl + Collagen group （shDDR2 negative control， without downregulation of DDR2 expression but with collagen treatment）； Control Group 2： Control + Collagen group （OE-DDR2 negative control， without overexpression of DDR2 but with collagen treatment）； Experimental Group 1： shDDR2 + Collagen group （DDR2 gene knockdown with collagen treatment）； Experimental Group 2： OE-DDR2 + Collagen group （DDR2 gene overexpression with collagen treatment）； Experimental Group 3： Based on Experimental Group 2， treated with 1 μmol/L dasatinib， with all groups being treated for 48 hours. The expression level of DDR2 mRNA was detected by RT-qPCR， and the level of DDR2 protein was assessed by Western blot. Cell proliferation was evaluated using the CCK-8 assay and the BrdU assay. Cell localization and pathological changes were observed by HE staining and immunohistochemical staining methods. Comparisons between two groups were performed using independent samples t-test， and the t' test was used when variances were unequal. For comparisons among multiple groups，one-way ANOVA was applied， with LSD-t test used for pairwise comparisons when variances were homogeneous， and Tamhane's T2 test was used when variances were heterogeneous.

Results

DDR2 is localized in renal stromal fibroblasts in mouse kidney tissue， and has a colocalization relationship with collagen. Compared with the shCtrl+collagen group， the cell proliferation rate （0.28 ± 0.00 vs 0.38 ± 0.01） in the sh-DDR2+ collagen group slowed down （P＜ 0.05）， and the transwell transfer rate （6.00 ± 1.00 vs 21.66 ± 0.57） decreased （P＜ 0.05）. Compared with the Control+collagen group， the cell proliferation rate of the OE-DDR2+ collagen group （0.43 ± 0.01 vs 0.39 ± 0.01） was accelerated （P＜ 0.05）， and the transwell mobility rate （32.66 ± 0.57 vs 22.33 ± 0.57） was increased（P＜ 0.05）. After the addition of dasatinib， the cell proliferation rate （0.36 ± 0.00 vs 0.39 ± 0.01）was significantly reduced.

Conclusion

Collagen/DDR2 signaling activation significantly promoted the proliferation of KFB cells and inhibited apoptosis， ultimately promoting the occurrence and development of renal interstitial fibrosis.