ICAM-1基因转染对小鼠间充质干细胞成脂分化功能的影响

Effects of ICAM-1 gene transfection on the adipogenic differentiation of MSCs

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MIGRl-ICAM-1重组逆转录病毒质粒，连同空质粒MIGRl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C3H10T1／2细胞，荧光倒置显微镜下观察及real．timePCR法和流式细胞术检测转染效率，将获得稳定过表达ICAM-1的C3H10T1／2细胞（C3H10T1／2-ICAM-1细胞）和转入空质粒的C3H10T1／2细胞（C3H10T1／2-MIGRl细胞）向脂肪细胞诱导分化，以原位油红O染色和real—timePCR检测成脂分化能力及其关键转录因子C／EBPoL和PPARy mRNA的表达水平。结果成功构建MIGRl-ICAM一1逆转录病毒载体；感染C3H10T1／2细胞获得稳定过表达ICAM-1的C3H10T1／2-ICAM-1细胞和空载体对照C3H10T1／2一MIGRl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，C3H10T1／2-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）；C3H10T1／2一ICAM一1细胞自分化组和诱导分化组C／EBPoLmRNA表达水平分别为1．2±0．7和2．9±0．9）；PPAR-,／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±2．3：2453．04-215．6和9856．3±542．2下调（P〈0．05）。结论细胞表面ICAM一1过表达可抑制小鼠MSC的成脂分化。

Objective To explore the effects of ICAM-1 gene transfection on the differentiation of MSCs to adipocytes. Methods The recombinant retroviral expression plasmid MIGRI-ICAM-1 containing full length of mouse ICAM-1 gene was constructed. The constructed plasmid MIGRI-ICAM-1, empty plasmid MIGR1 and packaging plasmid ECOS were transfected into T293 cell lines and then the superuatant 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/EBPct and PPARy, 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 transfeetion 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/EBPa [（1.2±0. 7）, （2.9 ±0.9）1 and PPARy [（1557.6 ±70.2）,（7547.0 ±442.2）] when compared with C3H10T 1/2- MIGR1 E（5.8±0.5）,（23.0±2.3） and （2453.0±215.6）,（9856.3 ±542.2） I （P 〈 0.05 ）. Moreover, little lipid droplet and decreased quantity of adipocytes were detected in C3 H 10T 1/2-ICAM-1 [ （ 3.2 ± 0.5 ）/well, （ 12.2 ±3.8 ）/well than that in C3 H10T 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 adipocytie differentiation.