摘要
目的通过构建重组双荧光素酶报告基因质粒Myh6-Luc,筛选诱导小鼠畸胎瘤细胞P19向心肌细胞分化的最佳中药有效成分,并对诱导效果进行验证。方法将Myh6基因的启动子区域(~1507 bp)插入pGL6报告质粒荧光素酶基因的上游,构建心肌细胞特异启动子荧光素酶报告基因系统。通过阳性药物0.005%二甲基亚砜对该荧光素酶报告基因系统进行验证。通过双荧光素酶检测方法筛选诱导P19细胞向心肌细胞分化的最佳中药有效成分,经心肌细胞标志物心肌肌钙蛋白T(cTnT)免疫荧光染色对该有效成分的诱导作用进行验证,并通过RT-qPCR检测miR-1和miR-25的表达。结果经双酶切鉴定质粒构建成功,并通过阳性药物诱导证实该系统的有效性。一定浓度的三七皂苷Fe、三七皂苷R1(NGR1)和人参皂苷Rh1均能对P19细胞产生诱导作用,其中1μmol/L NGR1诱导效果最明显。1μmol/L NGR1对P19细胞进行诱导后,大部分细胞可表达cTnT,且miR-1表达上调,miR-25表达下调。结论基于Myh6启动子的药物筛选系统可用于发现诱导干细胞向心肌细胞分化的中药有效成分,1μmol/L NGR1是诱导P19细胞向心肌细胞分化的有效药物,其机制可能与上调miR-1、下调miR-25的表达有关。
Objective To screen the best effective components of Chinese materia medica to induce mouse teratoma cell P19 to differentiate into cardiomyocytes by constructing recombinant dual luciferase reporter gene plasmid Myh6-Luc,and verified the induction effect.Methods The promoter region of Myh6 gene(~1507 bp)was inserted into the upstream of luciferase gene of pGL6 reporter plasmid to construct the luciferase reporter gene system of cardiomyocyte specific promoter.The luciferase reporter gene system was verified by positive drug 0.005% dimethyl sulfoxide.Double luciferase detection method was used to screen the best active components of Chinese materia medica for inducing P19 cells to cardiomyocytes.The induction effect of the active components was verified by immunofluorescence staining of cTnT,a marker of cardiomyocytes.The expressions of miR-1 and miR-25 were detected by RT-qPCR.Results The plasmid was constructed successfully which identified by double enzyme digestion,and the effectiveness of the system was confirmed by the induction of positive drugs.Notoginsenoside Fe,Notoginsenoside R1(NGR1)and Ginsenoside Rh1 with a certain concentration could induce P19 cells,and the effects of 1μmol/L NGR1 were the most obvious.After the induction of P19 cells with 1μmol/L NGR1,most of the cells could express cTnT,and miR-1 was up-regulated and miR-25 was down-regulated.Conclusion The drug screening system based on Myh6 promoter can be used to find the effective components in Chinese materia medica to induce stem cells to differentiate into cardiomyocytes.1μmol/L NGR1 is an effective drug to induce P19 cells to differentiate into cardiomyocytes,and its mechanism may be related to up-regulating the expression of miR-1 and down-regulating the expression of miR-25.
作者
李涵
邵长乐
王亚超
邵水金
国海东
LI Han;SHAO Changle;WANG Yachao;SHAO Shuijin;GUO Haidong(Department of Anatomy,School of Basic Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)
出处
《中国中医药信息杂志》
CAS
CSCD
2021年第9期79-84,共6页
Chinese Journal of Information on Traditional Chinese Medicine
基金
国家自然科学基金(81673729)
上海市人才发展资金(2019090)
上海市自然科学基金(21ZR1463100)。
