Chen Ting, Huang Dangsheng, Chen Guanghui, Yang Tingshu, Yi Jun, Tian Miao
Department of Cardiology, First Affiliated Hospital of Chinese People's Liberation Army General Hospital, Beijing, 100048, People's Republic of China.
J Mol Histol. 2015 Feb;46(1):1-11. doi: 10.1007/s10735-014-9599-7. Epub 2014 Dec 3.
The adipose tissue-derived stem cells (ADSCs) represent a significant area of the cell therapy. Genetic modification of ADSCs may further improve their therapeutic potential. Here, we aimed to generate a lentiviral vector expressing insulin-like growth factor-I (IGF-1) and investigate the impact of IGF-1 transduction on the properties of cultured ADSCs. Isolated rat ADSCs were assessed by flow cytometric analysis. IGF-1 was cloned and inserted into the pLenO-DCE plasmid to acquire pLenO-DCE-IGF-1 plasmid. Lentivirus was enveloped with pRsv-REV, pMDlg-pRRE and pMD2G plasmids in 293T cells. The ADSCs were transfected with the vectors. And then IGF-1-induced anti-apoptosis was evaluated by annexin V-FITC. Besides, proliferation of cells was detected by MTT assay and EdU. Moreover, Akt phosphorylation was evaluated by Western blotting analysis. Stable expression of IGF-1 in ADSCs was confirmed. ADSCs were positive for CD90 and CD29, but negative for CD31, CD34 and CD45. The transduction of IGF-1 to the ADSCs caused a dramatic increase in P-Akt expression. Over-expression of IGF-1 in ADSCs could improve the paracine of IGF-1 in a time-dependent manner, but could not promote the proliferation of ADSCs. This study indicated that lentiviral vectors offered a promising mean of delivering IGF-1 to the ADSCs. Lentiviral-mediated over-expression of therapeutic IGF-1 gene in ADSCs could prolong the anti-apoptosis effect of IGF-1, which might be induced by the activation of the PI3K/Akt pathway. And our data would improve the efficacy of ADSC-based therapies.
脂肪组织来源的干细胞（ADSCs）是细胞治疗的一个重要领域。对ADSCs进行基因改造可能会进一步提高其治疗潜力。在此，我们旨在构建一种表达胰岛素样生长因子-I（IGF-1）的慢病毒载体，并研究IGF-1转导对培养的ADSCs特性的影响。通过流式细胞术分析评估分离的大鼠ADSCs。将IGF-1克隆并插入pLenO-DCE质粒以获得pLenO-DCE-IGF-1质粒。在293T细胞中用pRsv-REV、pMDlg-pRRE和pMD2G质粒包装慢病毒。用这些载体转染ADSCs。然后通过膜联蛋白V-FITC评估IGF-1诱导的抗凋亡作用。此外，通过MTT法和EdU检测细胞增殖。而且，通过蛋白质印迹分析评估Akt磷酸化。证实了IGF-1在ADSCs中的稳定表达。ADSCs对CD90和CD29呈阳性，但对CD31、CD34和CD45呈阴性。IGF-1转导至ADSCs导致P-Akt表达显著增加。ADSCs中IGF-1的过表达可随时间依赖性方式改善IGF-1的旁分泌，但不能促进ADSCs的增殖。本研究表明慢病毒载体为将IGF-1递送至ADSCs提供了一种有前景的手段。慢病毒介导的治疗性IGF-1基因在ADSCs中的过表达可延长IGF-1的抗凋亡作用，这可能是由PI3K/Akt途径的激活所诱导。并且我们的数据将提高基于ADSC的治疗的疗效。
