组织蛋白酶D绿色荧光蛋白复合质粒转染慢性光损伤成纤维细胞的研究

目的 探讨组织蛋白酶D绿色荧光蛋白复合质粒（GFP-CatD）技术在成纤维细胞慢性光损伤研究中的作用。 方法 长波紫外线（UVA）25 J/cm2每天照射人皮肤成纤维细胞1次，共21 d，构建慢性光损伤成纤维细胞模型。构建GFP-CatD质粒，将其转染入慢性光损伤成纤维细胞，荧光显微镜观察绿色荧光标记的组织蛋白酶D表达，Western印迹检测组织蛋白酶D表达，流式细胞仪检测转染GFP-CatD后的慢性光损伤成纤维细胞凋亡率，噻唑蓝（MTT）法检测细胞增殖率。 结果 慢性光损伤成纤维细胞转染GFP-CatD质粒96 h，可见绿色荧光蛋白组织蛋白酶D在慢性光损伤细胞胞质内表达。Western印迹结果显示，慢性光损伤成纤维细胞转染GFP-CatD质粒后，组织蛋白酶 D蛋白表达为未转染细胞的1.28倍。细胞凋亡检测结果发现，转染后的光老化成纤维细胞凋亡细胞率为4.29% ± 1.30%，与无转染的空白对照组凋亡细胞率3.03% ± 1.70%比较，差异无统计学意义（P > 0.05）。MTT细胞增殖检测结果发现，转染后的光老化成纤维细胞的细胞增殖率为45.20% ± 4.70%，无转染质粒的光老化成纤维细胞为43.60% ± 3.90%（P > 0.05）。 结论 GFP-CatD复合质粒转染慢性光损伤成纤维细胞可示踪到组织蛋白酶 D荧光蛋白，且不影响慢性光损伤成纤维细胞原有的正常生物活性和周期。

Performance of transfection with a complex plasmid encoding green fluorescent protein tagged cathepsin D in researches on chronic photodamaged fibroblasts

Zheng Yue, Chen Haiyan, Xu Qingfang, Ye Congxiu, Liu Huixian, Yi Jinling, Lai Wei. Department of Dermatovenereology, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, China Corresponding author: Lai Wei, Email: drlaiwei@163.com 【Abstract】 Objective To evaluate the performance of transfection with a complex plasmid encoding green fluorescent protein tagged CatD （GFP-CatD） in researches on chronic photodamaged fibroblasts. Methods Human dermal fibroblasts （HSFs） were irradiated with ultraviolet A （UVA） at 25 J/cm2 once a day for 21 consecutive days to establish a chronic photodamaged cell model. A plasmid encoding GFP-CatD was constructed and transfected into some chronic photodamaged fibroblasts （experimental group）. The photodamaged HSFs receiving no treatment served as the blank control group, and those transfected with the negative plasmid encoding GFP only as the negative control group. After additional culture, fluorescence microscopy and Western-blot analysis were performed to observe and measure the expression of GFP-CatD in HSFs respectively, flow cytometry and methyl thiazolyl tetrazolium （MTT） assay to evaluate the apoptosis and proliferation of chronic photodamaged fibroblasts respectively. Results Fluorescence microscopy showed the expression of GFP-CatD in cytoplasm of chronic photodamaged fibroblasts at 96 hours after transfection with the GFP-CatD-encoding plasmid. Western-blot analysis revealed that the expression of CatD in the experimental group was 1.28 times that in the blank control group. There were no significant differences in the apoptosis rate （4.29% ± 1.30% vs. 3.03% ± 1.70%, P > 0.05） or proliferative rate （45.20% ± 4.70% vs. 43.60 ± 3.90%, P > 0.05） between the experimental group and blank control group. Conclusion CatD could be traced in chronic photodamaged fibroblasts with no changes in biological activity or cell cycle after transfection with the GFP-CatD-encoding complex plasmid.