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 adipocytes, 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.
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。