CAJ | 학술논문

目的：探讨Let‐7b过表达和Let‐7b抑制慢病毒载体在体外诱导大鼠骨髓间充质干细胞（MSCs）向神经细胞分化中的作用。方法构建Let‐7b过表达和Let‐7b抑制慢病毒载体并感染大鼠MSCs ，筛选最适感染复数（MOI）；实验分未感染组、感染组1（感染LV‐rno‐Let‐7b‐up）、感染组2（感染LV‐rno‐Let‐7b‐inhibition）、阴性对照组（感染空病毒）；采用法舒地尔诱导感染后大鼠M SCs分化为神经元细胞。倒置荧光显微镜下观察M SCs感染后荧光表达情况；采用免疫细胞化学染色检测神经元烯醇化酶（NSE）、神经微管结合蛋白（MAP‐2）的表达变化；PT‐PCR检测MAP‐2 mRNA的表达变化；MTT 法检测细胞存活率。结果倒置显微镜下观察大鼠LV‐rno‐Let‐7b‐up和LV‐rno‐Let‐7b‐inhibition慢病毒载体感染成功，MOI值为10，感染3 d时大鼠LV‐rno‐Let‐7b‐up慢病毒感染率最高，细胞存活率较高，感染率可达（92.1±4.3）％；MOI为20，感染5 d时大鼠LV‐rno‐Let‐7b‐inhibition慢病毒感染率最高，细胞存活率较高，感染率可达（90.3±4.2）％。法舒地尔可以诱导MSCs向神经细胞分化，感染组1诱导MSCs为神经细胞显著高于阴性对照组和感染组2，NSE、MAP‐2表达率明显高于阴性对照组和感染组，差异有统计学意义（P＜0.05）。结论结论 LV‐rno‐Let‐7b‐up可更高效感染大鼠 MSCs ，感染LV‐rno‐Let‐7b‐up后大鼠MSCs经法舒地尔诱导向神经细胞分化比率增加。
목적：탐토Let‐7b과표체화Let‐7b억제만병독재체재체외유도대서골수간충질간세포（MSCs）향신경세포분화중적작용。방법구건Let‐7b과표체화Let‐7b억제만병독재체병감염대서MSCs ，사선최괄감염복수（MOI）；실험분미감염조、감염조1（감염LV‐rno‐Let‐7b‐up）、감염조2（감염LV‐rno‐Let‐7b‐inhibition）、음성대조조（감염공병독）；채용법서지이유도감염후대서M SCs분화위신경원세포。도치형광현미경하관찰M SCs감염후형광표체정황；채용면역세포화학염색검측신경원희순화매（NSE）、신경미관결합단백（MAP‐2）적표체변화；PT‐PCR검측MAP‐2 mRNA적표체변화；MTT 법검측세포존활솔。결과도치현미경하관찰대서LV‐rno‐Let‐7b‐up화LV‐rno‐Let‐7b‐inhibition만병독재체감염성공，MOI치위10，감염3 d시대서LV‐rno‐Let‐7b‐up만병독감염솔최고，세포존활솔교고，감염솔가체（92.1±4.3）％；MOI위20，감염5 d시대서LV‐rno‐Let‐7b‐inhibition만병독감염솔최고，세포존활솔교고，감염솔가체（90.3±4.2）％。법서지이가이유도MSCs향신경세포분화，감염조1유도MSCs위신경세포현저고우음성대조조화감염조2，NSE、MAP‐2표체솔명현고우음성대조조화감염조，차이유통계학의의（P＜0.05）。결론결론 LV‐rno‐Let‐7b‐up가경고효감염대서 MSCs ，감염LV‐rno‐Let‐7b‐up후대서MSCs경법서지이유도향신경세포분화비솔증가。
Objective To investigate the role of Let‐7b in inducing bone marrow mesenchymal stem cell(MSCs) differenti‐ation into neurons.Methods The lentiviral vector of Let‐7b up (LV‐rno‐Let‐7b‐up) and inhibition (LV‐rno‐Let‐7b‐inhibition) was constructed and transfected into rat MSCs.The cells were divided into non‐transfected group ,transfected group 1(trans‐fected with LV‐rno‐Let‐7b‐up) ,transfected group 2 (transfected with LV‐rno‐Let‐7b‐inhibition) and negative control group (transfected with empty virus ).MSCs were treated with Fashudier as an inducer for triggering the cells to differentiate into neurons.The fluorescence expressed by transfected MSCs were observed under inverted fluorescence microscope.The mRNA expression of microtublin‐associated protein 2 (MAP‐2) was detected by RT‐PCR.The expression of neuron‐specific markers , neuron‐specific enolase(NSE) and MAP‐2 were measured by immunocytochemical method.The viability of MSCs was deter‐mined by MTT method.Results In LV‐rno‐Let‐7b‐up the best transfection efficiency(up to 92.1% ± 4.3% ) and survival rate appeared when multiply of infection (MOI) was 10 and on 3rd day.In LV‐rno‐Let‐7b‐inhibition the best transfection efficiency (up to 90.3% ± 4.2% ) and survival rate appeared when MOI was 20 and on 5th day.Fashudier induced MSCs to differentiate into neurons and the best efficiency of the induction was observed in transfected group 1.The expression levels of NSE and MAP‐2 in transfected cells were higher than those in the cells of other group ( P<0.05).Conclusion LV‐rno‐Let‐7b‐up has high transfection efficiency in rat MSCs.Higher differentiation rate from rat MSCs to neurons is induced by Fashudier after the cells are transfected with LV‐rno‐Let‐7b‐up.