CAJ | 학술논문

目的：探讨慢病毒载体介导降钙素基因相关肽（CGRP）基因体外转染间充质干细胞（MSC）及其对MSC生物学特性的影响。方法大鼠MSC进行分离、培养及鉴定。CGRP重组慢病毒转染MSC后，采用荧光显微镜和流式细胞技术测定其转染率。实时定量PCR、免疫荧光细胞化学及ELISA法检测MSC中CGRP的表达。慢病毒转染后通过MTT、β‐半乳糖苷酶染色和诱导分化评价MSC的增殖、衰老及分化能力。结果慢病毒载体介导CGRP基因转染 MSC后48 h可稳定表达，MOI＝30时，转染率达80％以上。与MSC组和空载病毒转染（MSC‐EGFP组）比较，CGRP修饰的MSC（MSC‐CGRP组）中CGRP的mR‐NA和蛋白表达水平增高（均 P＜0．01）。MTT、β‐半乳糖苷酶染色和诱导分化结果显示，病毒转染后对MSC增殖、衰老及向内皮分化基本没有影响。结论 MSC是一种理想的基因载体细胞，可作为CGRP基因转染的靶细胞用于基因治疗，为后续体内、体外实验奠定基础。
목적：탐토만병독재체개도강개소기인상관태（CGRP）기인체외전염간충질간세포（MSC）급기대MSC생물학특성적영향。방법대서MSC진행분리、배양급감정。CGRP중조만병독전염MSC후，채용형광현미경화류식세포기술측정기전염솔。실시정량PCR、면역형광세포화학급ELISA법검측MSC중CGRP적표체。만병독전염후통과MTT、β‐반유당감매염색화유도분화평개MSC적증식、쇠로급분화능력。결과만병독재체개도CGRP기인전염 MSC후48 h가은정표체，MOI＝30시，전염솔체80％이상。여MSC조화공재병독전염（MSC‐EGFP조）비교，CGRP수식적MSC（MSC‐CGRP조）중CGRP적mR‐NA화단백표체수평증고（균 P＜0．01）。MTT、β‐반유당감매염색화유도분화결과현시，병독전염후대MSC증식、쇠로급향내피분화기본몰유영향。결론 MSC시일충이상적기인재체세포，가작위CGRP기인전염적파세포용우기인치료，위후속체내、체외실험전정기출。
Objective To explore in vitro mesenchymal stem cell (MSC) transfection of lentiviral vector mediated calcitonin gene‐related peptide(CGRP) gene and its effects on biological properties of MSC .Methods MSC were isolated ,cultured and identi‐fied .MSC were infected by lentivirus encoding recombinant enhanced green fluorescent protein (EGFP) gene and CGRP (Lv‐EG‐FP‐CGRP) .The transfection efficiency was determined by the inverted fluorescence microscope and flow cytometry .The expression levels of CGRP were detected in CGRP‐modified MSC by using real‐time PCR ,immunocytochemistry and enzyme‐linked immu‐nosorbent assay (ELISA) .The proliferation ,aging and differentiation ability of MSC were evaluated by MTT ,β‐galactosidase stai‐ning and inducing differentiation respectively .Results After 48 h of MSC transfection by Lv‐EGFP‐CGRP ,EGFP/CGRP could be expressed stably .When multiplicity of infection (MOI) was 30 ,the transfection efficiency reached more than 80% .Compared with the MSC group and the MSC‐EGFP group ,the mRNA and protein expression levels of CGRP in CGRP‐modified MSC(MSC‐CGRP group) were markedly increased(all P<0 .01) .The results of MTT ,β‐galactosidase staining and inducing differentiation assay dem‐onstrated that the transfected CGRP basically had no effect on the proliferation ,aging and endotheliocyte differerntiation of MSC . Conclusion MSC is a kind of ideal genetic vector cell ,which can serve as the target cell of CGRP gene transduction for the applica‐tion of gene therapy and lays the foundation for follow‐up in vitro and vivo experiments .