增生细胞核抗原核酶对兔视网膜色素上皮细胞增生的抑制作用

Inhibitory effects of proliferating cell nuclear antigen ribozyme on proliferation of retinal pigment epithelial cells in rabbits

目的探讨增生细胞核抗原(proliferative cell nuclear antigen,PCNA)核酶(ri-bozyme,Rz)对PCNA表达及细胞增生的影响,为进一步基因治疗增生性玻璃体视网膜病变提供实验基础。方法设计构建针对PCNA的锤头状核酶(PCNα-Rz),通过阳离子脂质体Lipofectamine2000介导转染体外培养的视网膜色素上皮细胞(经此处理的为Rz载体组),观察其在细胞中的分布,应用3H-TDR掺入实验及四甲基偶氮唑盐比色实验检测细胞增生及活性,应用免疫组织化学技术检测细胞内PCNA表达水平,并设立裸Rz组、空载体组和对照组进行对照研究。结果在脂质体介导下PCNα-Rz可成功转染培养的视网膜色素上皮细胞,较裸PCNα-Rz转染效率明显增高;Rz载体组细胞增生活性显著低于裸Rz组、空载体组及对照组,细胞生长抑制率显著提高(81.2%);Rz载体组细胞PCNA表达水平(9.2±1.3)%显著低于裸Rz组(27.5±4.3)%、空载体组(37.4±5.2)%和对照组(43.6±5.8)%,差异均有显著统计学意义(均为P<0.01)。结论PCNα-Rz能显著抑制培养的兔视网膜色素上皮细胞中PCNA的表达,降低细胞的增生活性,此项技术有望应用于人眼并可能成为防治增生性玻璃体视网膜病变的有效手段。

Objective To investigate the inhibitory effects of ribozyme（Rz）on expression and cell proliferation of proliferating cell nuclear antigen（PCNA）so as to provide foundation of a new genetic therapy for proliferative vitreoretinopathy（PVR）.Methods Hammerhead ribozyme targeting PCNA（PCNα-Rz）was synthesized and transduced into cultured rabbit retinal pigment epithelial（RPE）cells mediating by positive ion liposome Lipofectamine 2000（PCNα-Rz assisted with Lipofectamine group）.Cell proliferation and activity were measured by MTT assay and 3H-TDR incorporation method. The levels of PCNA＇were detected by immunohistochemical technique. The other three groups were set up, PCNα-Rz group, Lipofectmnine group and control group. Results The cultured rabbit RPE cells could be successfully transfected by the synthesized PCNα-Rz mediated by liposome, and significantly higher than that transfected by PCNα-Rz. The activity of cell proliferation and the expression level of PCNA in PCNα-Rz assisted with Lipofectamine group were significantly lower than those in PCNα-Rz group, Lipofectamine group and control group. The inhibition ratio of cell growth was obviously increased to 81.2%. While the expression levels of PCNA were（9. 2 ± 3.1）% in PCNα-Rz assisted with Lipofectanfine group, （27. 5 ± 4. 3 ）% in PCNα-Rz group, （ 37.4± 5.2 ） % in Lipofectamine group and （ 43.6 ± 5.8 ） % in control group （ all P 〈 0.01 ）. Conclusions PCNα-Rz can obviously inhibit the expression level of PCNA and cut down the activity of cell proliferation in the cultured rabbit RPE cells, which may be a potential way to prevent and cure PVR in human eyes.