CXC受体4阳性表达骨髓间充质干细胞的分选及其骨向分化的能力  

The sorting efficiency and osteogenic capacity of CXCR4-positive bone marrow mesenchymal stem cells

作　　者：麦宇芸 叶舒 胡晓莉[1] 权晶晶[1] 张晓磊[1] Mai Yuyun;Ye Shu;Hu Xiaoli;Quan Jingjing;Zhang Xiaolei(Guanghua School of Stomatology,Hospital of Stomatology,Sun Yat-sen University,Guangdong Provincial Key Laboratory of Stomatology,Guangzhou 510055,China)

机构地区：[1]中山大学光华口腔医学院·附属口腔医院,广东省口腔医学重点实验室,广州510055

出　　处：《中华口腔医学研究杂志（电子版）》2018年第5期285-293,共9页Chinese Journal of Stomatological Research(Electronic Edition)

基　　金：国家自然科学基金(11772361、81470731);青年科学基金项目81500839

摘　　要：目的研究磁珠分选法分选表面CXC受体4(CXCR4)阳性表达的大鼠骨髓间充质干细胞(BMSC)的分选效率,以及分选后BMSC的增殖、迁移和成骨能力是否受到影响。方法全骨髓贴壁法分离培养大鼠BMSC,通过生长曲线、成骨成脂诱导分化和流式检测BMSC表面标记蛋白表达,鉴定大鼠BMSC。流式检测细胞表面CXCR4的阳性率,通过生长曲线及克隆形成实验检测细胞的增殖活性及克隆形成能力,Transwell体外趋化实验检测细胞迁移能力,通过检测成骨诱导早期细胞ALP活性和成骨相关基因的表达,分析细胞成骨能力,数据采用单因素方差分析处理。结果通过全骨髓贴壁法获得具有自我更新和多向分化能力的大鼠BMSC。磁珠分选阳性细胞表面CXCR4表达[(55.13±0.67)%]较未分选组[(6.49±0.59)%]显著增加(LSD-t=63.66,P<0.001)。分选阳性细胞的克隆形成能力[(16.22±1.68)%]以及培养后期细胞增殖活性(A_(450(5 d))=1.40±0.04;A_(450(7 d))=1.60±0.01)较未分选细胞[CFU=(17.00±1.76)%;A_(450(5 d))=1.49±0.07;A_(450(7 d))=1.60±0.12]均无明显变化;而培养早期(第1、3天)分选阳性细胞的增殖活性[A_(450(1 d))=0.50±0.01;A_(450(3 d))=0.81±0.05]较未分选细胞[A_(450(1 d))=0.57±0.01;A_(450(3 d))=0.96±0.06]稍低[LSD-t_(1 d)=5.208,P_(1 d)=0.002;LSD-t_(3 d)=3.563,P_(3 d)=0.012]。分选阳性细胞的基质细胞衍生因子1α(SDF-1α)定向迁移能力(71.33±5.69)较未分选细胞(23.33±1.53)显著增强(LSD-t=17.211,P<0.001)。在成骨诱导早期,分选阳性细胞的Runx2 mRNA(0.93±0.12)较未分选组(0.51±0.14)表达上调(LSD-t=4.703,P=0.003),而ALP活性及ALP mRNA、OCN mRNA的表达均无明显变化(P>0.05)。结论磁珠分选法可有效分选CXCR4^+BMSC,显著提高BMSC向SDF-1α的迁移效率,对细胞的增殖活性影响不大。Objective To investigate the sorting efficiency of magnetic activated cell sorting(MACS)for CXCR4-positive bone marrow mesenchymal stem cells(CXCR4+BMSCs),and the influence on the proliferation,migration and osteogenesis of BMSCs.Methods The bone marrow mesenchymal cells from Sprague-Dawley rats were isolated and cultured in vitro by the whole bone marrow adherence method,and identified by growth curve,in vitro differentiation and BMSCs surface markers.BMSCs were sorted and purified using immunomagnetic beads to obtain CXCR4+BMSCs.The unsorted and sorted target cells were compared as follows,the expression of BMSCs surface CXCR4(detected by flow cytometry),proliferative ability(detected by the CCK-8 method),clonogenicity ability(detected by the colony formation assay),in vitro chemotactic capacity towards SDF-1α(detected by Transwell migration assay),and osteogenic capacity(detected by ALP activity and the expression of osteogenesis-related genes).The results were analyzed by One-Way ANOVA.Results In comparison with unsorted cells[(6.49±0.59)%],CXCR4+BMSCs[(55.13±0.67)%]had a higher expression levels of surface CXCR4(LSD-t=63.66,P<0.001).In addition,CXCR4+BMSCs exhibited a slightly lower proliferative ability[A450(unsorted,1 d)=0.57±0.01,A450(positive,1 d)=0.50±0.01;A450(unsorted,3 d)=0.96±0.06,A450(positive,3 d)=0.81±0.05]in early culture stage(LSD-t1 d=5.208,P1 d=0.002;LSD-t3 d=3.563,P3 d=0.012],but a similar clonogenicity ability[CFUpositive=(16.22±1.68)%,CFUunsorted=(17.00±1.76)%;F=0.218,P=0.810],a higher chemotactic activity towards SDF-1α(71.33±5.69 vs 23.33±1.53,LSD-t=17.211,P<0.001),as well as increased expression levels of Runx2 mRNA(0.93±0.12 vs 0.51±0.14,LSD-t=4.703,P=0.003).However,During the period of osteogenic induction,there was no significant difference in the ALP activity or expression levels of ALP and OCN mRNA among these groups(P>0.05).Conclusions CXCR4-positive BMSCs can be sorted efficiently by magnetic activated cell sorting.MACS significantly improved the SDF-1α-induced che

关 键 词：间充质干细胞 骨髓 磁珠分选 基质细胞衍生因子1 CXC受体4 细胞迁移分析 

分 类 号：R78[医药卫生—口腔医学]