慢病毒构建表达人β-防御素3的滑膜间质干细胞及其抑菌活性鉴定

Lentiviral-medicated hβD-3 gene transfer induces chondrogenesis of human synovium derived mesenchymal stem cells and its antibacterial activity

目的 检测利用慢病毒介导人β-防御素3（humanβ-defensin 3, hβD-3）基因转染人滑膜间质干细胞（synovium-derived mesenchymal stem cells, SMSCs）的安全性及体外抗菌活性。方法 通过膝关节镜手术获取人膝关节交叉韧带及滑膜组织以分离、培养SMSCs, 贴块法培养并用磁珠分选仪纯化, 通过显微镜观察、免疫荧光鉴定和细胞表面标记对细胞进行鉴定。通过成脂、成骨和成软骨分化诱导分别对细胞多向分化潜能进行测定。利用质粒构建包含hβD-3基因的慢病毒载体并转染SMSCs, 绘制细胞生长曲线, 采用流式细胞仪确定其DNA含量, 使用NOD/SCID小鼠验证其致瘤性。使用琼脂扩散法对转染后的SMSCs进行抑菌活性检测, 同时用兔膝关节内金黄色葡萄球菌感染模型验证其体内抑菌作用。结果 贴块法获得的SMSCs经分离纯化后表现出间质干细胞应有的结构和表面标志物, 具有多向分化潜能。免疫荧光证实被慢病毒转染的SMSCs能稳定表达hβD-3蛋白。增殖动力学、核型分析、致瘤型分析等证实转染后的SMSCs安全。重组细胞体外和动物模型体内抑菌活性检测显示, 传代后的SMSCs（采用P5代细胞）能稳定表达hβD-3并具有抗菌活性。与阴性、阳性对照组比较, 低浓度组有一定的抑菌活性, 并且随浓度增高, 抑菌圈逐渐增大。结论 经重组慢病毒载体感染的人SMSCs能安全稳定地传代并表达hβD-3, 体外具有明显的抗菌活性。

Objective This article is aim to test the safety and the in vitro antibacterial activity of the Lentiviral-medicated humanβ-defensin 3 （hβD-3） transfecion synovium-derived mesenchymal stem cells （SMSCs）. Methods Tissues with SMSCs were obtained by surgical operations. Cells were explant cultured and purified by magnetic cell separation system. Cells were identified by microscopic observation, immunofluorescence and cell surface markers. Through inducing the cells into fat, osteoblasts and chondroblasts to respectively determine the multi-directional differentiation potential. Construct a hβD-3 contained lentivirus and transfected into SMSCs. Got the cell growth curve and determine the DNA content by using flow cytometry. The NOD/SCID mice were applied to verify the tumorigenicity of SMSCs. Agar diffusion test was applied to doing antibacterial activity test of post-transfecton SMSCs. The rabbit model of knee joint in staphylococcus aureus infections verify its bacteriostatic action in vivo. Results： Purified SMSCs had the structure and surface signatures of MSCs. It had the potential of multi-differentiation. Immunofluorescence had verified that SMSCs transfected by lentivirus could stably express hβD-3. Cell proliferation kinetics, karyotype analysis and Tumorigenic type analysis verified the safety of SMSCs after transfection. The in vivo and vitro antibacterial activity test of the recombinant SMSCs showed that after cell passages, the subcultured SMSCs （P5 cells were used in this article） could express hβD-3 stably and had antibacterial activity.The result of the antibacterial circle showed that low concentration group inhibit bacterial activity while the medium and high group could enlarge compared with the negative and positive control group. Conclusion Lentiviral-medicated hβD-3 gene express SMSCs could successfully subcultured and stably express hβD-3, meanwhile it had apparent in vitro antibacterial activity.