Lee Hsiao-Ping, Kaul Gunter, Cucchiarini Magali, Madry Henning
Center of Experimental Orthopaedics, Saarland University, 66421, Homburg, Germany.
Int Orthop. 2014 Sep;38(9):1923-30. doi: 10.1007/s00264-014-2410-2. Epub 2014 Jun 25.
Our aim was to evaluate whether nonviral vectors can genetically modify primary human juvenile and adult meniscal fibrochondrocytes at low toxicity in vitro and to test the hypothesis that transfected human meniscal fibrochondrocytes transplanted into longitudinal defects and onto human medial meniscus explant cultures are capable of expressing transgene products in vitro.
Eighteen nonviral gene transfer systems were examined to identify the best suited method for an efficient transfection of primary cultures of juvenile and adult human meniscal fibrochondrocytes using luciferase and lacZ reporter gene constructs and then transplanted to meniscus explant cultures.
Gene transfer systems FuGENE 6, GeneJammer, TurboFectin 8, calcium phosphate co-precipitates and GeneJuice led to minimal toxicity in both cell types. Nanofectin 2 and JetPEI resulted in maximal luciferase activity in both cell types. Maximal transfection efficiency based on X-gal staining following lacZ gene transfer was achieved using Lipofectamine 2000, revealing a mean transfection efficiency of 8.6 % in human juvenile and of 8.4 % in adult meniscal fibrochondrocytes. Transfected, transplanted meniscal fibrochondrocytes adhered to the meniscal tissue and continued to express the transgene for at least five days following transfection.
Nonviral gene transfer systems are safe and capable of transfecting both juvenile and adult human meniscal fibrochondrocytes, which, when transplanted to meniscal tissue in vitro, permit the expression of selected transgenes to be maintained. These results are of value for combining gene therapy and cell transplantation approaches as a means to enhance meniscal repair.
我们的目的是评估非病毒载体能否在体外以低毒性对原代人幼年和成年半月板纤维软骨细胞进行基因改造，并检验以下假设：将转染的人半月板纤维软骨细胞移植到纵向缺损处以及人内侧半月板外植体培养物上，能够在体外表达转基因产物。
检测了18种非病毒基因转移系统，以确定使用荧光素酶和lacZ报告基因构建体对原代幼年和成年人人半月板纤维软骨细胞进行高效转染的最适合方法，然后将其移植到半月板外植体培养物中。
基因转移系统FuGENE 6、GeneJammer、TurboFectin 8、磷酸钙共沉淀物和GeneJuice对两种细胞类型的毒性均最小。Nanofectin 2和JetPEI在两种细胞类型中均导致最大的荧光素酶活性。使用Lipofectamine 2000在lacZ基因转移后基于X-gal染色实现了最大转染效率，显示在人幼年半月板纤维软骨细胞中的平均转染效率为8.6%，在成年半月板纤维软骨细胞中的平均转染效率为8.4%。转染并移植的半月板纤维软骨细胞粘附于半月板组织，并在转染后至少五天持续表达转基因。
非病毒基因转移系统安全且能够转染幼年和成年人人半月板纤维软骨细胞，当将其移植到体外半月板组织时，能够维持所选转基因的表达。这些结果对于将基因治疗和细胞移植方法结合作为增强半月板修复的手段具有价值。
