Han Li-ying, Ye Ming-zhu, Li Ya-ping, Wang Bo-wei, Wang Qiang, Zhao Shu-hua, Li He-lian
Department of Obstetrics and Gynecology, Second Hospital, Jilin University, Changchun, Jilin, China.
Chin Med J (Engl). 2008 May 5;121(9):800-5.
Most of gynecologic malignancies are sensitive to chemotherapy. Myelosuppression is the main dose-related toxicity of many chemotherapeutic drugs. The human multidrug resistance (mdr1) gene is well known for its ability to confer drug resistance. This study aimed to explore the feasibility of expression and resistance of mdr1 gene transduction into human placenta mesenchymal stem cells (P-MSCs) by retrovirus vector.
Human P-MSCs were isolated from trypsin-digested term placentas, and their immunophenotypes and differentiation potential were evaluated. Human P-MSCs were transduced by reconstructed retroviral vector containing the mdr1 gene and green fluorescent protein (GFP) reporter gene. The integration and expression of the mdr1 gene were observed indirectly by the expression of GFP, and fluorescence-activated cell sorter was used to evaluate the functional activity of permeability glycoprotein (P-gp) encoded by the mdr1 gene. The stimulating test was made in vitro to show pleiotropic drug resistance of transfected cells.
The isolated, cultured and expanded P-MSCs expressed stem cell markers such as CD29, CD44 and CD73, and showed osteogenic and adipogenic differentiation potentials under appropriate conditions. The expression of P-gp in the non-transfected P-MSCs cells was (0.4 +/- 0.1)%, but increased to (28.1 +/- 4.7)% after gene transfection (P < 0.01). And positive staining of P-gp located mainly at cell membrane and cytoplasm. Accumulation and extrusion assays showed that P-gp expressed by the transfected cells had pump-functional activity and could efflux daunomycin out of cells. The analysis of cell survival confirmed that transfected P-MSCs had a characteristic of multidrug resistance with a significant increase in the resistance to anticancer agents.
Transfer and expression of human mdr1 gene mediated by retrovirus vector conferred P-MSCs drug resistance. It might provide a new alternative to chemoprotection strategies.
大多数妇科恶性肿瘤对化疗敏感。骨髓抑制是许多化疗药物主要的剂量相关毒性。人类多药耐药（mdr1）基因因其赋予耐药性的能力而广为人知。本研究旨在探讨通过逆转录病毒载体将mdr1基因转导至人胎盘间充质干细胞（P-MSCs）中表达及耐药的可行性。
从经胰蛋白酶消化的足月胎盘中分离出人P-MSCs，并对其免疫表型和分化潜能进行评估。用含有mdr1基因和绿色荧光蛋白（GFP）报告基因的重组逆转录病毒载体转导人P-MSCs。通过GFP的表达间接观察mdr1基因的整合和表达，并用荧光激活细胞分选仪评估mdr1基因编码的通透糖蛋白（P-gp）的功能活性。进行体外刺激试验以显示转染细胞的多药耐药性。
分离、培养和扩增的P-MSCs表达干细胞标志物如CD29、CD44和CD73，并在适当条件下表现出成骨和成脂分化潜能。未转染的P-MSCs细胞中P-gp的表达为（0.4±0.1）%，但基因转染后增加至（28.1±4.7）%（P<0.01）。P-gp的阳性染色主要位于细胞膜和细胞质。蓄积和外排试验表明，转染细胞表达的P-gp具有泵功能活性，可将柔红霉素排出细胞。细胞存活分析证实，转染的P-MSCs具有多药耐药特性，对抗癌药物的耐药性显著增加。
逆转录病毒载体介导的人mdr1基因转移和表达赋予P-MSCs耐药性。这可能为化学保护策略提供一种新的选择。
