外泌体与Wnt3a联合诱导间充质干细胞归巢及定向分化  

Homing and Directional Differentiation of MSCs Induced by Exosome Combined with Wnt3a

作　　者：叶涛 李永忠[1] 付爱华 曹华平 喻璐[1] 胡华明 刘晓雯 林华[1] Ye Tao;Li Yongzhong;Fu Aihua(Department of Osteology,The Second People’s Hospital of Yichang(The Second People’s Hospital of the China Three Gorges University),Yichang 443000,China)

机构地区：[1]湖北省宜昌市第二人民医院(三峡大学第二人民医院)骨外科,宜昌443000 [2]三峡大学医学院,肿瘤微环境与免疫治疗湖北省重点实验室,宜昌443000

出　　处：《华中科技大学学报（医学版）》2019年第6期637-643,共7页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金青年科学基金资助项目(No.81402568)

摘　　要：目的观察外泌体(exosome)联合Wnt3a蛋白刺激机体内源性干细胞归巢并定向分化后促进大鼠皮肤软组织缺损修复的过程。方法常规提取大鼠骨髓间充质干细胞(BMSCs)来源外泌体,将外泌体联合Wnt3a蛋白作用于间充质干细胞,运用Western blot及qRT-PCR方法检测血管形成相关细胞表面标志物的表达,明确外泌体联合Wnt3a蛋白在体外对BMSCs的诱导分化作用。随后,制作大鼠大面积皮肤软组织缺损模型,组织创面注射外泌体和Wnt3a蛋白,采用免疫荧光染色、Western blot及qRT-PCR方法观察外泌体联合Wnt3a刺激机体内源性干细胞后产生的干细胞归巢效应,并对创面愈合率进行分析,以观察该联合方案对组织创面的修复效果。结果 qRT-PCR检测结果显示,与对照组BMSCs比较,Exosome+Wnt3a组中Klf4、Oct4表达明显下降(均P<0.01),周细胞标记物NG2、血管内皮细胞标志物VEGF和CD31、血管平滑肌细胞标志物α-SMA的mRNA表达均升高,证实BMSCs产生分化。在大鼠大面积皮肤软组织缺损模型中,Exosome+Wnt3a组大鼠伤口创面在第16天愈合率达(93.05±2.72)%,明显高于其它组(P<0.01)。肉芽组织和伤口成熟度评分结果显示,Exosome+Wnt3a组在术后第11天的评分高于假手术组(P<0.01),而且第9天出现了大量的新生血管,与假手术组比较差异有统计学意义[(48.260±1.821)个/mm^2 vs.(8.484±1.291)个/mm^2,P<0.01]。免疫荧光染色结果显示,大鼠组织创面成熟血管数量在外泌体联合Wnt3a治疗后明显多于假手术组[(28.536±1.882)个/mm^2vs.(4.712±1.883)个/mm^2,P<0.01]。结论外泌体及Wnt3a蛋白可定向诱导BMSCs分化,促进成熟肉芽组织沉积,加速创面血管形成,进一步促进组织创面修复,为临床上慢性创面的细胞治疗提供了新的思路。Objective To explore the specific role of Wnt3 a combined with exosome in the homing and directional differentiation of mesenchymal stem cells and the accelerated cutaneous tissue reconstruction in rats. Methods The exosome derived from BMSCs was collected and further utilized to induce the differentiation of BMSCs in the combination with the Wnt3 a.In vitro,the specific markers related to the angiogenesis in the BMSCs were evaluated by using Western blotting and qRT-PCR.In vivo,the full cutaneous defects in the rats were created and Wnt3 a combined with exosome were injected around the wounds,and then the accelerated effect of this combinatory therapy towards the wounds was assessed by using the immunofluorescent staining,Western blotting and qRT-PCR.Results The results of RT-PCR showed the increased expression of Klf4 and Oct4,NG2,VEGF and CD31,as well as the α-SMA in the exosome combined with Wnt3 a group compared with control group,demonstrating the differentiation of BMSCs.In vivo,the rats treated with exosome combined with Wnt3 a showed accelerated repair of the wounds on day 16[(93.05±2.72)%]compared with control group(P<0.01).The results of the maturity score of the granulation tissue and wounds showed the enhanced wound repair in the exosome combined with Wnt3 a group compared with control group(P<0.01),furthermore,more new formed blood vessels were observed on day 9 compared with control group[(48.260±1.821)/mm^2vs.(8.484±1.291)/mm^2,P<0.01].In addition,the immunofluorescent staining showed that the number of the mature blood vessels in the wounds of the rats treated with exosome combined with Wnt3 a were increased compared with the control group[(28.536±1.882)/mm^2vs.(4.712± 1.883)/mm^2,P<0.01]. Conclusion Our study demonstrated that exosome combined with Wnt3 a is able to induce the direct differentiation of BMSCs,leading to the deposit of mature granulation tissue and enhanced formation of blood vessels to accelerate the wound repair.

关 键 词：骨髓间充质干细胞 外泌体 WNT3A 归巢 定向分化 创面再生 

分 类 号：R329.21[医药卫生—人体解剖和组织胚胎学]