摘要
目的探讨加减驻景方含药血清对AKT基因转染后人视网膜色素上皮细胞(ARPE-19)细胞表达VEGF的影响。方法制备SD大鼠加减驻景方含药血清及空白血清,建立AKT基因转染ARPE-19细胞模型,体外培养,分为5个组,正常组为ARPE-19细胞常规培养,转染模型组(模型细胞,常规条件培养),含药血清组(模型细胞,给予含药血清干预培养)、空白血清组(模型细胞,给予空白血清干预培养)和阳性对照组(模型细胞,给予康柏西普干预培养)。设立不同时间和不同浓度,通过CCK-8法检测各组血清及阳性组药物对细胞毒性、增殖的影响;通过ELISA检测各组细胞上清液中VEGF的表达。结果蛋白质印迹法(Western blot)显示,转染模型组与正常组比较,AKT蛋白的表达量明显增加,差异有统计学意义(P <0.05)。CCK-8示,细胞培养24、48h后,AKT基因转染可促进ARPE-19细胞增殖(P<0. 05),培养72h后,对细胞增殖影响不大(P> 0.05)。10、20μg/ml康柏西普及5%、10%的含药血清对模型细胞的增殖有抑制作用(P <0. 05),各浓度空白血清组对模型细胞组的增殖影响不大(P>0. 05)。酶联免疫吸附试验(ELISA)检测显示:转染模型组细胞上清液中VEGF表达量高于正常组(P<0. 05),康柏西普组与含药血清组均显著低于模型组(P <0.05),其中康柏西普组低于含药血清组,差异有统计学意义(P<0. 05)。空白血清组与模型组比较,差异无统计学意义(P>0. 05)。结论 AKT基因转染后可诱导ARPE-19细胞的增殖,促进VEGF的表达。加减驻景方含药血清对AKT基因转染后ARPE-19细胞的增殖及VEGF的表达有抑制作用。加减驻景方可能通过抑制VEGF的过度表达发挥干预CNV生成的作用。
Objective To investigate the effect of addition and subtraction of sera containing medicinal herbs on the expression of VEGF in human retinal pigment epithelial cells(ARPE-19) cells after AKT gene transfection. Methods SD rats serum containing the prescription of Jiajianzhujing and the content of distilled water in serum were prepared. AKT gene transfected ARPE-19 cell model was established and Western blot method was used to verify the successful construction. The effects of the prescription and distilled water in serum at different concentration(2. 5%, 5%, 10%) and Conbercept(5, 10, 20 g/ml) cell vitality was observed by cell counting kit(CCK-8) assay. The effect of serum and Conbercept on the expression of VEGF in model cells was assessed by ELISA. Results Result of Western blot assay confirmed that : compared with the normal group, the expression of AKT protein increased significantly in the model group(P < 0. 05). Results of CCK-8 measure confirmed that After be cultured for 24 h and 48 h, AKT gene transfection can increase the value of ARPE-19 cells(P <0. 05), but after cultured for 72 h, there was little difference(P>0. 05). While the 10,20 μg/ml of Conbercept group and 5% and 10% contained serum group,were lower than model cells group(P <0. 05). After cultured for 72 h,there was little or no difference in proliferation between model cell and control group(P > 0. 05). Results of the expression of VEGF in the supernatant of cells detected by ELISA shown that the model group was higher than that of normal cells group( P < 0. 05), serum and Conbercept group were significantly lower than the model group( P < 0. 05), no statistically significant difference between the blank serum group and model group cell difference(P > 0. 05). Conclusion The AKT gene transfection can induce proliferation of ARPE-19 cells and promote the expression of VEGF. The modified Zhu Jing Fang medicated serum has inhibitory effect on the proliferation and VEGF expression of ARPE-19 cells transfected with AKT gene. The effect of intervention on the generation of CNV may be played by suppressing the overexpression of VEGF.
作者
褚文丽
陈水龄
亢泽峰
张丛青
李维义
刘健
Cha Wenli;Chen Shuiling;Kang Zefeng(Eye Hospital,Chinese Academy of Chinese Medical Sciences,Beijing 100040,China)
出处
《医学研究杂志》
2018年第12期55-59,共5页
Journal of Medical Research
基金
国家自然科学基金资助项目(面上项目)(81574032)
