人淋巴细胞-NT3基因工程细胞的建立及鉴定

Construction and Assessment of the Model of NT3 Genetically Engineered Lymphocyte

目的建立能够存活较长时间并稳定表达神经营养素-3(neurotrophin-3,NT3)的人淋巴细胞株,为后续的动物和人体耳蜗内基因转染实验奠定基础。方法从正常人全血中通过Ficoll液提取人淋巴细胞,并在其完全血清培养基中加入白细胞介素-2(IL-2)使其良好生长。通过RT-PCR得到人胎肝组织的NT3cDNA,以T4DNA连接酶和真核表达载体pIRES-DsRed2相连接。以阳离子脂质体作为载体,将pIRES-DsRed2-NT3转染人淋巴细胞,进行RT-PCR及Western Blot分析鉴定。结果建立了一种能使体外培养淋巴细胞成活时间延长的新方法,成功转染NT3基因至淋巴细胞中,并能继续存活相对较长时间,Western Blot证明细胞上清液中有NT3,构建了稳定表达NT3的人基因工程淋巴细胞。结论构建重组质粒pIRES-DsRed2-NT3,利用淋巴细胞作为中介宿主,进而表达、分泌NT3,为进一步NT3基因转染致聋动物耳蜗实验奠定了基础,并有可能为人体基因转染治疗耳聋找到很好的中介细胞。

Obstact To estabhsh a lymphocyte line capable of surviving longer time and to expresses human NT3. Those will serve as a model for subsequent experiments on animal and human gene transfeetion in cochlea. Methods Human lymphocytes were extracted from the normal human blood via Ficoll fluid. IL- 2 was added into the serum culture medium to make the lymphocyte in good growth condition. The NT3 cDNA was obtained by RT- PCR and was transfected into lymphocyte hne by using cationic hposome （LP2000）. The lymphocyte transfected with NT3 - cDNA was examined by RT - PCR, Western Blot methods. Results We estabhshed a new method to extend the lymphocytes survival time in vitro and successfully transfected NT3 into lymphocyte. The genetically engineered lymphocytes could survive a longer time. The positive signals of protein were obtained by Western Blot. Conclusion By constructing recombined plasmid pIRES - DsRed2 - NT3 by means of lymphocytes as the intermediary, we are able to get a lymphocyte line which can express and secrete NT3. This can estabhsh the foundation for the subsequent animal gene transfection experiment in the cochlea, thus leading to the possibility of finding suitable intermediary cell for the treatment of deafness through gene therapy.