Department of Anatomy, Capital Medical University, Beijing, China.
PLoS One. 2013 May 22;8(5):e64389. doi: 10.1371/journal.pone.0064389. Print 2013.
Gene regulation remains one of the major challenges for gene therapy in clinical trials. In the present study, we first generated a binary tetracycline-on (Tet-On) system based on two lentivirus vectors, one expressing both human glial cell line-derived neurotrophic factor (hGDNF) and humanized recombinant green fluorescent protein (hrGFP) genes under second-generation tetracycline response element (TRE), and the other expressing the advanced reverse tetracycline-controlled transactivator--rtTA2S-M2 under a human minimal cytomegalovirus immediate early (CMV-IE) promoter. This system allows simultaneous expression of hGDNF and hrGFP genes in the presence of doxycycline (Dox). Human bone marrow-derived mesenchymal stem cells (hMSCs) were transduced with the binary Tet-On lentivirus vectors and characterized in vitro in the presence (On) or absence (Off) of Dox. The expression of hGDNF and hrGFP transgenes in transduced hMSCs was tightly regulated as determined by flow cytometry (FCM), GDNF enzyme-linked immunosorbent assay (ELISA) and quantitative real time-polymerase chain reaction (qRT-PCR). There was a dose-dependent regulation for hrGFP transgene expression. The levels of hGDNF protein in culture medium were correlated with the mean fluorescence intensity (MFI) units of hrGFP. The levels of transgene background expression were very low in the absence of Dox. The treatment of the conditioned medium from cultures of transduced hMSCs in the presence of Dox protected SH-SY5Y cells against 6-hydroxydopamine (6-OHDA) toxicity as determined by cell viability using 3, [4,5-dimethylthiazol-2-yl]-diphenyltetrazolium bromide (MTT) assay. The treatment of the conditioned medium was also found to improve the survival of dopaminergic (DA) neurons of ventral mesencephalic (VM) tissue in serum-free culture conditions as assessed by cell body area, the number of neurites and dendrite branching points, and proportion of tyrosine hydroxylase (TH)-immunoreactive (IR) cells. Our inducible lentivirus-mediated hGDNF gene delivery system may provide useful tools for basic research on gene therapy for chronic neurological disorders such as Parkinson's disease (PD).
基因调控仍然是临床试验中基因治疗的主要挑战之一。在本研究中，我们首先基于两个慢病毒载体生成了一个二元四环素激活（Tet-On）系统，一个载体表达人胶质细胞源性神经营养因子（hGDNF）和人源化重组绿色荧光蛋白（hrGFP）基因，受第二代四环素反应元件（TRE）调控，另一个载体表达受先进的反式四环素调控转录激活子 rtTA2S-M2，受人类最小巨细胞病毒立即早期（CMV-IE）启动子调控。该系统允许在多西环素（Dox）存在的情况下同时表达 hGDNF 和 hrGFP 基因。人骨髓间充质干细胞（hMSCs）被二元 Tet-On 慢病毒载体转导，并在 Dox 存在（On）或不存在（Off）的情况下进行体外特征分析。通过流式细胞术（FCM）、GDNF 酶联免疫吸附测定（ELISA）和实时定量聚合酶链反应（qRT-PCR）测定，转导的 hMSCs 中转基因 hGDNF 和 hrGFP 的表达受到严格调控。hrGFP 转基因表达呈剂量依赖性调节。培养基中 hGDNF 蛋白的水平与 hrGFP 的平均荧光强度（MFI）单位相关。在没有 Dox 的情况下，转基因背景表达水平非常低。在 Dox 存在的情况下，处理转导的 hMSCs 培养物的条件培养基可通过 3, [4,5-二甲基噻唑-2-基]-二苯基四唑溴盐（MTT）测定法保护 SH-SY5Y 细胞免受 6-羟基多巴胺（6-OHDA）毒性。还发现该处理可改善无血清培养条件下腹侧中脑（VM）组织中多巴胺能（DA）神经元的存活，通过细胞体面积、神经突和树突分支点的数量以及酪氨酸羟化酶（TH）免疫反应性（IR）细胞的比例进行评估。我们的诱导型慢病毒介导的 hGDNF 基因传递系统可为慢性神经退行性疾病（如帕金森病（PD））的基因治疗基础研究提供有用的工具。
