过表达Ctnnb1慢病毒及其稳转大鼠间充质干细胞株的构建  

Construction of overexpressing Ctnnb1 lentivirus and its stable transgenic rat mesenchymal stem cell line

作　　者：徐康乔 韦雅芹 周长喜[2] 夏世金 Xu Kangqiao;Wei Yaqin;Zhou Changxi;Xia Shijin(Shanghai Institute of Geriatrics,Huadong Hospital Affiliated to Fudan University,Shanghai,200040,P.R.China;Department of Geriatricst Second Medical Centert PLA General Hospital,Beijing,100853,P.R.China)

机构地区：[1]复旦大学附属华东医院上海市老年医学研究所,上海200040 [2]解放军总医院第二医学中心老年医学科,北京100853

出　　处：《老年医学与保健》2022年第1期69-74,共6页Geriatrics & Health Care

基　　金：国家重点研发计划课题(2020YFC2008900);国家自然科学基金(81870044);解放军总医院第二医学中心课题(NLBJ-2019005)。

摘　　要：目的构建过表达大鼠Ctnnb1 (catenin beta1)慢病毒载体,建立过表达Ctnnb1的大鼠间充质干细胞(MSC)稳转株,为后续研究过表达Ctnnb1的MSC产生的外泌体对老年慢性阻塞性肺疾病相关肺动脉高压(COPD-PH)的作用和机制奠定基础。方法 PCR扩增Ctnnb1基因全序列,将序列插入PGMLV-18844:PGMLV-CMV-MCS-3×Flag-PGK-Puro载体,构建PGMLV-CMV-Rat;tnnb1-3×Flag-PGK-Puro慢病毒表达质粒,质粒瞬转293细胞后,使用Western Blot验证其过表达效率。再将构建的慢病毒载体和包装质粒共转染293细胞,包装病毒。包装的过表达慢病毒和阴性对照病毒感染大鼠MSC,再次使用Western Blot测定其过表达效果。结果经测序分析,本研究成功构建了PGMLV-CMV-Rat;tnnb1-3×Flag-PGK-Puro慢病毒载体。Western Blot结果提示,该慢病毒载体成功转染293细胞,构建的载体能高效过表达Ctnnb1;过表达Ctnnb1的慢病毒感染大鼠MSC后,得到过表达Ctnnb1基因的大鼠MSC稳转株。结论本研究成功构建过表达Ctnnb1慢病毒及其稳转大鼠MSC株。Objective To construct a rat catenin beta1(Ctnnb1)overexpressing lentivirus and establish a stable transgenic line of rat mesenchymal stem cells(MSC)overexpressing Ctnnb1,and lay a foundation for the subsequent study of the effect of exosomes produced by Ctnnb1 overexpressing MSC on elderly patients with chronic obstructive pulmonary disease-related pulmonary hypertension(COPD-PH). Methods The full-length of Ctnnb1 cDNA was amplified by PCR and inserted into PGMLV-18844:PGMLV-CMV-MCS-3 ×Flag-PGK-Puro vector to constructPGMLV-CMV-Rat_Ctnnb1-3 ×Flag-PGK-Purolentiviralplasmid. After the plasmid was transiently transfected into293 cells,the overexpression efficiency was verified by Western blot. The constructed lentiviral vector and packaging plasmid were then co-transfected into293 cells to package the virus. The packaged overexpressing lentivirus and negative control virus were used to infect rat MSC,and the overexpression effect was determined by Western blot again. Results The sequencing analysis showed that the PGMLVCMV-Rat_Ctnnb1-3 ×Flag-PGK-Puro lentiviral vector was successfully constructed in this study. The results of Western blot indicated that the lentiviral vector was successfully transfected into293 cells,and the constructed vector could overexpress Ctnnb1 efficiently. After rat MSC were infected with lentivirus overexpressing CTNNB1,a stable transgenic rat MSC strain overexpressing CTNNB1 gene was obtained. Conclusion The overexpressing Ctnnb1 lentivirus and its stable transgenic rat MSC line were successfully constructed.

关 键 词：老年 慢性阻塞性肺疾病相关肺动脉高压 Β-连环蛋白 慢病毒载体 间充质干细胞 

分 类 号：R563.9[医药卫生—呼吸系统]