Wnt经典通路诱导间充质干细胞向肺上皮细胞分化对自噬的影响

Effects of Wnt Classical Pathway on Autophagy Induced Differentiation of Mesenchymal Stem Cells into Lung Epithelial Cells

摘要: 目的：证实细胞自噬参与Wnt经典通路诱导小鼠骨髓来源间充质干细胞(mice bone marrow derived mesenchymal stem cells, mMSCs)向肺泡上皮(type II alveolar epithelial, AT II)细胞的分化。方法：mMSCs与MLE-12结合小气道培养基(SAGM)共培养构建体外诱导MSC向肺上皮细胞分化的模型，通过LiCl或DKK-1分别激活或抑制经典Wnt通路，实验分组如下：a. MSC组：MSC单独培养、b. MSC + MLE组：MSC与MLE-12共培养、c. MSC + MLE + LiCl组：MSC与MLE-12共培养并给予4 mL LiCl激活经典Wnt通路、d. MSC + MLE + DKK-1组：MSC与MLE-12共培养并给予200 ng/mL DKK-1抑制Wnt经典通路。通过Western blot法检测诱导分化的第7天II型肺泡上皮细胞标志表面活性蛋白(surfactant protein, SP) C评估MSC向II型肺泡上皮细胞分化情况，并检测诱导分化第1、3、7天自噬相关蛋白(LC3B及Beclin-l)的表达。结果：与MSC单独培养及与MSC、MLE-12共培养相比较，共培养中加入LiCl时SP-C表达增加(p < 0.05)；与MSC单独培养、与MLE-12共培养及与共培养中加入4 mL LiCl相比，加入DKK-1后SP-C表达下降(p < 0.05)。通过Western blot法检测诱导分化第1、3、7天自噬相关蛋白(LC3B及Beclin-l)的表达发现：与MSC单独培养相比较，共培养中加入LiCl时Beclin-l、LC3-II/I表达增加(p < 0.05)；而加入DKK-1后与MSC、MLE-12共培养相比较，Beclin-l、LC3-II/I表达下降(p < 0.05)。结论：激活经典Wnt通路促进MSC向肺上皮细胞分化，激活经典Wnt通路增加MSC自噬水平。

Abstract: Objective: It was confirmed that autophagy is involved in the induction of mouse bone marrow-derived mesenchymal stem cells (MMSCs) into the alveolar epithelium (type II alveolar epithelial, AT II). Methods: MMSCs and MLE-12 combined with small airway medium (SAGM) were co-cultured to establish a model of in vitro induction of differentiation of MSCs into lung epithelial cells. The classical Wnt pathway was activated or inhibited by LiCl or DKK-1, respectively. The experimental groups were as follows: a. MSC group: MSC was cultured alone; b. MSC+MLE group: MSC was co-cultured with MLE-12; c. MSC + MLE + LiCl group: MSCs were co-cultured with MLE-12 and given 4 mL LiCl to activate the classical Wnt pathway; d. MSC + MLE + DKK-1 group: MSCs were co-cultured with MLE-12 and given 200 ng/mL DKK-1 to inhibit the Wnt classical pathway. Western blot was used to detect II type alveolar epithelial cell marker surfactant protein (SP) C on day 7 of induced differentiation to evaluate the differentiation of MSC to type II alveolar epithelial cells. The expressions of autophagy-related proteins (LC3B and Beclin-L) on the 1st, 3rd and 7th day of differentiation induction were detected. Results: Compared with MSC alone culture and co-culture with MSC and MLE-12, the expression of SP-C was increased when LICL was added in co-culture (p < 0.05). Compared with MSC alone culture, MLE-12 co-culture and co-culture with the addition of 4 mL LiCl, SP-C expression decreased after the addition of DKK-1 (p < 0.05). The expression of autophagy related proteins (LC3B and Beclin-L) at the 1st, 3rd and 7th day after induction of differentiation was detected by Western blot. Compared with MSC alone culture, the expressions of Beclin-L and LC3-II/I were increased when LiCl was added in co-culture (p < 0.05). The expression of Beclin-L and LC3-II/I decreased after DKK-1 co-culture compared with MSC and MLE-12 (p < 0.05). Conclusion: Activation of classical Wnt pathway promotes differentiation of MSCs into lung epithelial cells, and activation of classical Wnt pathway increases autophagy level of MSCs.