杆状病毒介导的GFP转染与退变的兔椎间盘髓核细胞

目的:检测杆状病毒转染正常与退变的椎间盘髓核细胞的效率以及杆状病毒介导的GFP基因(Ac/CMV/GFP)在正常与退变细胞中的表达水平。方法:取新西兰大白兔的髓核细胞进行离体培养,第1组:健康幼兔;第2组:椎间盘退变模型兔;第3组:椎间盘发生自然退变的成年兔。以200MOI杆状病毒介导的绿色荧光蛋白(GFP)转染离体培养的细胞,于第48h采用流式细胞仪检测转染效率,同时采用HPIAS2000图像分析软件半定量分析外源性GFP基因的表达水平。结果:3组细胞被转染的百分率分别为84.4±3.4,82.9±5.7,81.8±5.5(P>0.05)。外源性基因表达荧光蛋白的光密度值分别为0.0054±0.0029,0.0052±0.0032,0.0056±0.0031(P>0.05)。结论:杆状病毒转染椎间盘髓核细胞的效率与椎间盘退变程度无关,杆状病毒介导的外源性基因能在正常与退变的髓核细胞中高水平的表达。

Baculovirus-mediated GFP Gene Transference to Rabbit Degenerative Nucleus Pulposus Cells

Objective: To determine the efficacy of baculovirus-mediated marked GFP gene (Ac/CMV/GFP) to rabbit normal and nucleus pulposus cells and to assess the level of GFP expression in vitro. Methods: Rabbit nucleus pulposus cells were isolated and cultured from Lumber intervertebral discs of young health rabbits, degeneration-model rabbits, and adult rabbits. Nucleus pulposus cells were treated with 200 MOI Ac/CMV/GFP for 48 hours. The percentage of GFP-positive cells was analyzed by flow cytometry and the level of GFP expression was semi-quantified using HPIAS 2000 imagine analysis system. Results: The percentage of GFP-positive cells of three experimental groups were 84.4±3.4, 82.9±5.7 and 81.8±5.5, respectively. The fluorescence intensity value of the three experimental groups was 0.005 4±0.002 9, 0.005 2±0.003 2 and 0.005 6±0.003 1, respectively. Conclusion: In vitro transducibility of rabbit intervertebral disc cells by Ac/CMV/GFP is relatively insensitive to disc degeneration grade. Exogenous marked genes can be expressed in degeneration and nondegeneration cells in high level.