慢病毒介导重组质粒转染兔下颌骨骨髓间充质干细胞

摘要

BACKGROUND:Central nerve damage and peripheral nerve injury are common clinical problems that have no ideal treatment. Nerve growth factor has an important role in neuronal repairing and growth. But its local injections may have shorts of inactivation and loss. OBJECTIVE:To construct human nerve growth factor beta recombinant plasmids, which are transfected into bone marrow mesenchymal stem cells from the rabbit mandible by lentiviral vectors, and to investigate the bioactivity of human nerve growth factor beta. METHODS:pDC316-hNGFβ-mCMV-EGFP plasmids were constructed via lentiviral vectors using Hind III+Not I digestion. Bone marrow mesenchymal stem cells from the rabbit mandible were isolated and cultured, and then transfected by recombinant plasmids. The expression of human nerve growth factor beta in transfected cells was detected by ELISA method. RESULTS AND CONCLUSION:pDC316-hNGFβ-mCMV-EGFP plasmids were proved to be constructed successful y by gene sequencing and enzyme identification. The transfected cells under a fluorescence microscope emitted green fluorescence, and the fluorescence intensity had no change with incubation time. The expression of human nerve growth factor beta was maintained at a level of 25μg/L at 7 days after celltransfection, and the bioacitivty was increased significnalty.%背景：中枢或周围神经系统损伤是临床中常见的问题，且治疗效果尚不理想。神经生长因子在神经元细胞损伤修复和生长发育方面具有重要作用，但局部应用的神经生长因子存在易于失活、流失的缺点。目的：旨在通过慢病毒载体构建人神经生长因子β重组质粒，转染荧光兔下颌骨骨髓间充质干细胞并研究其生物学活性。方法：采用慢病毒作为载体，经 Hind Ⅲ+NotⅠ双酶切法构建含目的基因的 pDC316-hNGFβ-mCMV-EGFP质粒。分离和培养兔下颌骨骨髓间充质干细胞，经重组质粒转染后，应用酶联免疫吸附法检测骨髓间充质干细胞分泌人神经生长因子β的情况。结果与结论：成功构建了pDC316-hNGFβ-mCMV-EGFP真核表达载体，经酶切鉴定和测序均证明质粒构建完整、正确。质粒转染后兔下颌骨骨髓间充质干细胞在荧光显微镜下可以发出绿色荧光，且荧光强度不随培养时间延长而衰退。转染后兔下颌骨骨髓间充质干细胞表达的人神经生长因子β可以在第7天仍维持在25μg/L水平，生物学活性显著提升。