Wnt/β-catenin信号通路及骨髓间充质干细胞源性外泌体在温和灸联合骨髓间充质干细胞移植促进大鼠肛门括约肌修复中的作用

Wnt/β-catenin Signaling Pathway and BMSC-derived Exosomes in Mild Moxibustion Combination Role of BMSC Transplantation in Promoting Anal Sphincter Repair in Rats

目的基于前期研究证实温和灸联合骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMSC)移植可协同促进大鼠损伤肛门括约肌结构和功能修复的基础,探索协同修复效果是涉及Wnt/β-catenin通路,及BMSC源性外泌体对C212成肌细胞的影响。方法将16只SD大鼠随机等分4组,所有大鼠均采用Zutshi大鼠肛门括约肌复合体损伤模型,造模成功后分别采用温和灸30 min、BMSC移植及生理盐水治疗,连续14 d。治疗结束后剥离肛门括约肌后采用q PCR和Western blot检测组间Wnt4、Wnt5a、Wnt5b蛋白表达差异。将BMSC源性外泌体与小鼠成肌细胞C2C12共培养后采用EDU及CCK8检测BMSC外泌体对C2C12细胞增殖作用,探索不同浓度外泌体对BMSC增殖的差异。流式细胞术检测BMSC源性外泌体对C2C12细胞周期的影响。结果免疫印迹和qPCR分析结果提示温和灸联合BMSC移植可促进Wnt4、Wnt5A、Wnt5B高表达。CCK8结果显示BMSC源性外泌体可促进C2C12细胞的增殖,在浓度为25μg/mL时疗效最佳。共培养结果显示BMSC源性外泌体可抑制Cleaved Caspase3和BAX表达,并显著上调BCL2表达。结论温和灸联合BMSC移植可促进Wnt4、Wnt5A、Wnt5B高表达,BMSC源性外泌体可调控成肌细胞C2C12的细胞周期从而促进增殖并抑制凋亡,推测温和灸联合BMSC移植可能通过激活Wnt/β-catenin信号通路促进对大鼠损伤肛门括约肌的修复作用。

Objective Based on the previous study that mild moxibustion combined with BMSC transplantation can synergically promote the repair of the structure and function of the injured anal sphincter in rats,further explore the effect of the synergistic repair involves the Wnt/β-catenin pathway and the effect of BMSC-derived exosomes on C212 myoblasts.Methods Sixteen SD rats were randomly divided into 4 groups.All rats were treated with Zutshi rat anal sphincter complex injury model.After modeling,they were treated with mild moxibustion for 30min,BMSC transplantation and normal saline for 14 days.After treatment,the anal sphincter was stripped and the protein expression of Wnt4,Wnt5a and Wnt5b was detected by qPCR and Western blot.After co-culture of BMSC-derived exosomes with mouse myoblasts C2C12,EDU and CCK8 were used to detect the proliferation effect of BMSC exosomes on C2C12 cells,and to explore the difference of BMSC proliferation with different concentrations of exosomes.Flow cytometry was used to detect the effect of BMSC exosomes on C2C12 cell cycle.Results Western blot and qPCR analysis showed that mild moxibustion combined with BMSC transplantation could promote the high expression of Wnt4,Wnt5A and Wnt5B.CCK8 results showed that BMSC-derived exosomes could promote the proliferation of C2C12 cells,and the optimal concentration was 25μg/mL.Co-culture results showed that Cleaved Caspase3 and BAX expression were inhibited by BMSC-derived exosomes,and BCL2 expression was significantly up-regulated.Conclusion Mild moxibustion combined with BMSC transplantation can promote the high expression of Wnt4,Wnt5A and Wnt5B,and BBMSC exosomes can regulate the cell cycle of myoblast C2C12 to promote proliferation and inhibit apoptosis.It is speculated that mild moxibustion combined with BMSC transplantation may promote the repair effect of injured anal sphincter in rats by activating Wnt/β-catenin signaling pathway.