CAJ | 학술논문

目的探讨细胞间黏附分子1(ICAM-1)基因转染对小鼠间充质干细胞(MSC)向脂肪细胞分化的影响.方法构建含小鼠ICAM-1基因全长DNA片段的MIGR1-ICAM-1重组逆转录病毒质粒,连同空质粒MIGR1及包装质粒ECOS分别转染T293细胞,收集相关病毒上清并感染小鼠MSC细胞系C3H10T 1/2细胞,荧光倒置显微镜下观察及real-time PCR法和流式细胞术检测转染效率,将获得稳定过表达ICAM-1的C3H10T 1/2细胞(C3H10T 1/2-ICAM-1细胞)和转入空质粒的C3H10T 1/2细胞(C3H10T 1/2-MIGR1细胞)向脂肪细胞诱导分化,以原位油红O染色和real-time PCR检测成脂分化能力及其关键转录因子C/EBPα和PPARγ mRNA的表达水平.结果成功构建MIGR1-ICAM-1逆转录病毒载体；感染C3H10T 1/2细胞获得稳定过表达ICAM-1的C3H10T 1/2-ICAM-1细胞和空载体对照C3H10T 1/2-MIGR1细胞.成脂细胞诱导分化结果显示,无论是在自分化还是诱导分化组中,C3H10T 1/2-ICAM-1细胞与转染空载体的细胞相比脂滴变小,其脂肪细胞数量分别为[(3.2±0.5)/孔、(12.2±3.8)/孔],显著少于转染空载体的细胞[(11.2±0.4)/孔、(51.3±2.8)/孔](P＜0.05)；C3H10T 1/2-ICAM-1细胞自分化组和诱导分化组C/EBPα mRNA表达水平分别为1.2±0.7和2.9±0.9)；PPARγ mRNA表达水平分别为1557.6±70.2和7547.0±442.2,均较转染空载体细胞的5.8±0.5和23.0±2.3;2453.0±215.6和9856.3±542.2下调(P＜0.05).结论细胞表面ICAM-1过表达可抑制小鼠MSC的成脂分化.
목적 탐토세포간점부분자1(ICAM-1)기인전염대소서간충질간세포(MSC)향지방세포분화적영향.방법 구건함소서ICAM-1기인전장DNA편단적MIGR1-ICAM-1중조역전록병독질립,련동공질립MIGR1급포장질립ECOS분별전염T293세포,수집상관병독상청병감염소서MSC세포계C3H10T 1/2세포,형광도치현미경하관찰급real-time PCR법화류식세포술검측전염효솔,장획득은정과표체ICAM-1적C3H10T 1/2세포(C3H10T 1/2-ICAM-1세포)화전입공질립적C3H10T 1/2세포(C3H10T 1/2-MIGR1세포)향지방세포유도분화,이원위유홍O염색화real-time PCR검측성지분화능력급기관건전록인자C/EBPα화PPARγ mRNA적표체수평.결과 성공구건MIGR1-ICAM-1역전록병독재체；감염C3H10T 1/2세포획득은정과표체ICAM-1적C3H10T 1/2-ICAM-1세포화공재체대조C3H10T 1/2-MIGR1세포.성지세포유도분화결과현시,무론시재자분화환시유도분화조중,C3H10T 1/2-ICAM-1세포여전염공재체적세포상비지적변소,기지방세포수량분별위[(3.2±0.5)/공、(12.2±3.8)/공],현저소우전염공재체적세포[(11.2±0.4)/공、(51.3±2.8)/공](P＜0.05)；C3H10T 1/2-ICAM-1세포자분화조화유도분화조C/EBPα mRNA표체수평분별위1.2±0.7화2.9±0.9)；PPARγ mRNA표체수평분별위1557.6±70.2화7547.0±442.2,균교전염공재체세포적5.8±0.5화23.0±2.3;2453.0±215.6화9856.3±542.2하조(P＜0.05).결론 세포표면ICAM-1과표체가억제소서MSC적성지분화.
Objective To explore the effects of ICAM-1 gene transfection on the differentiation of MSCs to adipocytes.Methods The recombinant retroviral expression plasmid MIGR1-ICAM-1 containing full length of mouse ICAM-1 gene was constructed.The constructed plasmid MIGR1-ICAM-1,empty plasmid MIGR1 and packaging plasmid ECOS were transfected into T293 cell lines and then the supernatant generated from T293 cells were used to infect mouse MSCs cell line C3H10T 1/2.The transfective efficiency was determined by inverted fluorescence microscope,real-time PCR and flow cytometry.Furthermore,ICAM-1 overexpressing MSCs (C3H10T 1/2-ICAM-1) and empty vector transfection MSCs (C3H10T 1/2-MIGR1)were cultured in medium with or without induction reagents,Oil-red-O staining was used to detect the lipid accumulation,and the expression of transcriptional factors C/EBPα and PPARγ,which were key factors in the differentiation of MSCs to adipoeytes,were tested by real-time-PCR.Results The recombinant retrovirus vector containing mouse ICAM-1 gene was successful constructed.After transfection into MSCs cell line C3H10T 1/2,the overexpression ICAM-1 MSCs cell line (C3H10T 1/2-ICAM-1) and control cell line (C3H10T 1/2-MIGR1) were obtained.Furthermore,these two cell lines were treated without or with adipocytic induction reagents,C3H10T 1/2-ICAM-1 showed significantly lower mRNA expression level for C/EBPα [(1.2 ±0.7),(2.9 ±0.9)] and PPARγ [(1557.6 ±70.2),(7547.0 ±442.2)] when compared with C3H10T 1/2-MIGR1 [(5.8 ± 0.5),(23.0 ± 2.3) and (2453.0 ± 215.6),(9856.3 ±542.2)] (P ＜ 0.05).Moreover,little lipid droplet and decreased quantity of adipocytes were detected in C3H10T 1/2-ICAM-1 [(3.2 ±0.5)/well,(12.2 ±3.8)/well] than that in C3H10T 1/2-MIGR1 [(11.2 ±0.4)/well,(51.3 ±2.8)/well] (P ＜0.05).Conclusion Overexpression of ICAM-1 in MSCs can inhibit its adipocytic differentiation.