聚氨酯介导TRIB2-siRNA转染人源脂肪干细胞的体外成脂分化  

Adipogenesis differentiation of human adipose-derived stem cells in vitro induced by cationic polyurethane-mediated TRIB2-siRNA transfection

作　　者：刘娟 林超[2] 庄志刚 LIU Juan;LIN Chao;ZHUANG Zhi-gang(Dept.of Breast Surgery,Shanghai First Maternity and Infant Hospital,School of Medicine,Tongji University,Shanghai 201204,China;School of Medicine,Tongji University,Shanghai 200092,China)

机构地区：[1]同济大学附属第一妇婴保健院乳腺外科,上海201204 [2]同济大学医学院,上海200092

出　　处：《同济大学学报（医学版）》2021年第3期306-312,共7页Journal of Tongji University(Medical Science)

摘　　要：目的使用阳离子型聚氨酯(cationic polyurethane,CPU)介导TRIB2-siRNA体外转染人源脂肪干细胞沉默TRIB2基因表达,研究基因沉默效率及基因沉默对干细胞成脂分化的影响。方法在氮/磷(N/P)为10∶1的条件下,混合CPU与TRIB2-siRNA或阴性对照siRNA(NC)制备复合物,并转染人源脂肪干细胞。将CPU/TRIB2-siRNA作为实验组,CPU/NC作为阴性对照组,Lipofectamine^(TM)2000/TRIB2-siRNA作为阳性对照组,未处理的干细胞作为空白对照组,使用实时荧光定量PCR检测干细胞内TRIB2的mRNA表达;使用Alamar Blue法检测干细胞的存活率;应用激光共聚焦显微镜观察复合物的细胞吞噬情况;用油红O染色法在光学显微镜下观察干细胞的成脂情况。结果与空白对照组比,CPU/TRIB2-siRNA实验组有效沉默了TRIB2 mRNA,差异有统计学意义(P<0.001);实验组的沉默效果较阳性对照组更好(P<0.001);实验组的细胞存活率接近100%。CPU能介导TRIB2-siRNA在8 h后逃逸溶酶体。油红O染色及定量分析表明,实验组在诱导28 d后出现较多的脂滴。结论聚氨酯作为载体可有效递送TRIB2-siRNA进入脂肪干细胞并降低TRIB2 mRNA的表达,进而促进脂肪干细胞向脂肪的分化。Objective To induce adipogenesis differentiation of human adipose-derived stem cells(hADSCs)by cationic polyurethane(CPU)-mediated transfection of TRIB2-siRNA.Methods At an nitrogen/phosphorus(N/P)ratio of 10∶1,CPU was mixed with TRIB2-siRNA to yield the complexes for the transfection against hADSCs.CPU/TRIB2-siRNA,CPU/NC(negative control),and Lipo-fectamine^(TM)2000/TRIB2-siRNA were set as the study group,negative control group,and positive control group;and untreated stem cells were served as the blank control group.The real-time fluorescent quantitative PCR(qPCR)was used to test TRIB2 mRNA expression in the cells.Alamar Blue assay was applied to probe survival rate of the cells.Confocal laser microscope was used to observe cell uptake of the complexes.The adipogenesis of the stem cells as stained with Oil red O was observed under an optical microscopy.Results Compared to the blank control and negative groups,the study group showed a significant decrease in mRNA expression of TRIB2(P<0.001),and silencing efficacy was better(P<0.001)as compared to that yielded by the positive control group.Besides,the CPU group elicited nearly 100%of the cell survival rate.CPU facilitated TRIB2-siRNA to escape endosomes after 8 h.Oil red O staining and quantitative analysis indicated that,after adipo-genesis induction of 28 days,more lipid droplets were observed in the study group compared to the blank control group.Conclusion Cationic polyurethane as a carrier can effectively deliver TRIB2-siRNA into hADSCs for silencing TRIB2 mRNA,thereby promoting their adipogenesis differentiation.

关 键 词：人源脂肪干细胞 成脂分化 TRIB2基因 聚氨酯 

分 类 号：R318[医药卫生—生物医学工程]