Ad-hNGFβ转染原代培养的大鼠脊髓星形胶质细胞及其生物活性研究

Infection of Astrocyte from Rat Spinal Cord with Recombinant Adenovirus Containing Human NGF-beta Gene and the Biological Activity Research

目的:体外培养新生SD大鼠脊髓星形胶质细胞,观察携带人β神经生长因子基因的重组腺病毒(Ad-hNGFβ)对其存活及毒性作用,为Ad-hNGFβ在体研究打下基础。方法:分离新生SD大鼠的脊髓星形胶质细胞(Astrocyte,Ast),应用DMEM/F-121∶1培养基进行原代培养扩增鉴定。重组Ad-hNGFβ(MOI=25,50,100)转染Ast后,噻唑蓝(MTT)法检测病毒感染滴度与细胞存活的关系。ELISA方法测定培养3,6,9,12,15d时培养上清中hNGFβ的含量;用流式细胞仪对转染细胞进行倍体分析,观察转染Ad-hNGFβ对细胞周期和凋亡的影响;Western blot印迹法测定转染后表达产物的存在形式。结果:从新生SD大鼠的脊髓组织中成功分离培养出星形胶质细胞,GFAP免疫荧光阳性。经MTT法测定,当MOI=25,50,100时试验组的吸光值OD490与对照组比无显著差异,P>0.05。转染Ad-hNGFβ后试验组细胞培养上清中hNGFβ表达量与对照组同期相比有显著差异,P<0.01;且hNGFβ可持续表达至15d。Ad-hNGFβ转染细胞后所表达的活性产物以hNGFβ二聚体的形式存在。流式细胞仪测得细胞增殖指数与对照组比明显升高,P<0.01。结论:Ad-hNGFβ可有效介导hNGFβ基因转染原代培养的脊髓星形胶质细胞,并且成功表达有活性的hNGFβ,增加细胞活性。

Objective： To transfer the human beta-nerve growth factor gene recombinant adenovirus vector （Ad- hNGFβ） into rat spinal cord astrocyte in vitro and to examine whether it could deliver potent peptides in the nervous system and observe the cell toxic action as well. Methods. The astrocytes from neogenesis SD rat spinal cord were mechanically dissociated, and the DMEM/F-12 medium was adopted to primary culture and passaged the cells, and then identified by GFAP （glial fibrillary acidic protein） immumofluo- rescence method. The recombinant Ad-hNGFβ was transfected into AST cells at an MOI of 25, 50 and 100 respectively, and the toxicity of Ad-hNGFβ to astrocyte was analyzed by MTT method. The expression of NGFβ were identified by ELISA in astrocyte cultured supernatant at 3, 6, 9, 12 and 15 d respectively. The capacity to stimulate the proliferation and the apoptosis ratio was determined by flow cytometry. Relative molecular weight was measured to determine the construction by Western blot. Results： The astrocytes from neogenesis SD rat spinal cord were successfully cultured, which showed positive activity to GFAP. Recombinant adenovirus vector had no obvious toxicity to astrocytes at multiplicity of infection（MOI）at 25, 50 and 100. The expression of hNGFβ were significant differet between control group and testing group（P〈0.01）; the hNGFβ expression were gradually decreased with the extension of time. The expressed hNGFβ had the capacity to stimulate the proliferation of astrocytes. The construction of expressed protein is dimeric of hNGFβ. Conclusion. The spinal cord astrocytes can be infected directly by the recombinant adenovirus containing hNGFβ gene and express biologically active protein efficiently. Recombinant adenovirus vector has no obvious cell toxic effect and it could release beta-NGF to elevate the cytoactive at a suitable MOI.