目的:系统分析黄甲软肝颗粒及其中间体的化学成分,阐明其复方新药的化学物质基础。方法:采用超高效液相色谱-飞行时间串联质谱(UHPLC-Q-TOF-MS)技术,离子源为ESI,色谱条件:Agilent Zorbax Eclipse Plus C_(18)色谱柱(2.1mm×150mm,...目的:系统分析黄甲软肝颗粒及其中间体的化学成分,阐明其复方新药的化学物质基础。方法:采用超高效液相色谱-飞行时间串联质谱(UHPLC-Q-TOF-MS)技术,离子源为ESI,色谱条件:Agilent Zorbax Eclipse Plus C_(18)色谱柱(2.1mm×150mm,1.8μm),流动相为0.1%甲酸水溶液(A)-0.1%甲酸乙腈(B),洗脱梯度:0~20min,3%~60%B;20min~25min,60%~95%B;25min~30min,95%B;30min~31min,95%~3%B;31min~35min,3%B,流速0.35mL·min^(-1),柱温35℃,进样量1μL。质谱分析采用正、负离子模式扫描,扫描范围m/z100~1700。通过与自建化学成分数据库比对(部分对照品验证)后对各离子峰进行鉴定。结果:从黄甲软肝颗粒中共鉴定出126个化学成分,黄甲软肝中间体中共鉴定出141个化学成分,包括有机酸类、酚酸类、黄酮类、三萜类、皂苷类等。结论:建立的UHPLC-TOF-MS分析方法,结合数据库匹配,可全面快速地分析黄甲软肝颗粒及中间体中的化学成分,为其组方药效物质基础及质量控制研究提供科学依据。展开更多
Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells （iPSCs） derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by ...Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells （iPSCs） derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX （F IX） gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay.Results The cell line bore a missense mutation in the 6th coding exon （c.676 C〉T） of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% （10/45） and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.展开更多
文摘目的:系统分析黄甲软肝颗粒及其中间体的化学成分,阐明其复方新药的化学物质基础。方法:采用超高效液相色谱-飞行时间串联质谱(UHPLC-Q-TOF-MS)技术,离子源为ESI,色谱条件:Agilent Zorbax Eclipse Plus C_(18)色谱柱(2.1mm×150mm,1.8μm),流动相为0.1%甲酸水溶液(A)-0.1%甲酸乙腈(B),洗脱梯度:0~20min,3%~60%B;20min~25min,60%~95%B;25min~30min,95%B;30min~31min,95%~3%B;31min~35min,3%B,流速0.35mL·min^(-1),柱温35℃,进样量1μL。质谱分析采用正、负离子模式扫描,扫描范围m/z100~1700。通过与自建化学成分数据库比对(部分对照品验证)后对各离子峰进行鉴定。结果:从黄甲软肝颗粒中共鉴定出126个化学成分,黄甲软肝中间体中共鉴定出141个化学成分,包括有机酸类、酚酸类、黄酮类、三萜类、皂苷类等。结论:建立的UHPLC-TOF-MS分析方法,结合数据库匹配,可全面快速地分析黄甲软肝颗粒及中间体中的化学成分,为其组方药效物质基础及质量控制研究提供科学依据。
基金Supported by the National Science and Technology Major Project(2011ZX09102-010-04)
文摘Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells （iPSCs） derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX （F IX） gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay.Results The cell line bore a missense mutation in the 6th coding exon （c.676 C〉T） of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% （10/45） and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.
