加减驻景方含药血清对人视网膜色素上皮细胞AKT转染后AKT/mTOR通路的影响  

Effect of Jiajian-Zhujing Decoction on the AKT/mTOR signaling pathway in ARPE-19 cells after AKT transfection

作　　者：褚文丽 亢泽峰[1] 陈水龄 郝雪莲 刘健[1] 李维义 陶方方 Chu Wenli;Kang Zefeng;Chen Shuiling;Hao Xuelian;Liu Jian;Li Weiyi;Tao Fangfang(The Eye Clinic,Eye Hospital,China Academy of Chinese Medical Sciences,Beijing 100040,China;The Eye Clinic of Traditional Chinese Medicine,Shenzhen Eye Hospital,Shenzhen 518040,China;Ophthalmology Department,Xiyuan Hospital,China Academy of Chinese Medical Sciences,Beijing 100091,China)

机构地区：[1]中国中医科学院眼科医院眼科门诊,北京100040 [2]广东省深圳市眼科医院中医眼科,518040 [3]中国中医科学院西苑医院眼科,北京100091

出　　处：《国际中医中药杂志》2020年第4期347-352,共6页International Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金(1574032);国家中医药管理局岐黄工程亢泽峰歧黄学者工作室项目(2019年);深圳市医疗卫生三名工程(SZSM201812090);北京市石景山区亢泽峰名医传承工作室项目(2016年)。

摘　　要：目的观察加减驻景方含药血清对人视网膜色素上皮细胞(ARPE-19)AKT转染后VEGF表达的影响,探讨其作用机制。方法制备加减驻景方含药血清及空白血清,将ARPE-19细胞按随机数字表法分为正常组、模型组、空白血清组、含药血清组、康柏西普组及联合组。除正常组外,其余各组细胞建立AKT转染细胞模型。正常组与模型组细胞常规培养,空白血清组加入10%空白血清培养,含药血清组加入10%含药血清培养,康柏西普组加入20μg/ml康柏西普干预培养,联合组加入10%含药血清和20μg/ml康柏西普干预培养。采用CCK-8法检测各组ARPE-19细胞增殖情况。采用实时定量PCR法检测各组细胞AKT、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)、VEGF mRNA水平。采用Western blot检测各组细胞AKT、mTOR、VEGF蛋白表达。结果培养24、48、72 h后,与模型组比较,含药血清组、康柏西普组和联合组细胞增殖率下降(P<0.05)。干预24、48 h,含药血清组、康柏西普组和联合组细胞AKT mRNA[24 h:(3.10±0.48)、(1.97±0.14)、(1.26±0.24)比(4.77±0.68);48 h:(3.52±0.82)、(2.62±0.77)、(1.10±0.19)比(6.12±1.21)]、mTOR mRNA[24 h:(3.02±0.26)、(2.45±0.75)、(1.13±0.15)比(4.48±0.80);48 h:(1.29±0.30)、(1.30±0.57)、(0.65±0.19)比(2.54±0.62)]、VEGF mRNA[24 h:(3.33±0.62)、(2.18±0.20)、(1.55±0.28)比(5.53±1.02);48 h:(2.35±0.54)、(1.23±0.28)、(0.93±0.25)比(3.59±0.40)]表达及AKT蛋白[24 h:(0.45±0.09)、(0.25±0.05)、(0.14±0.04)比(0.62±0.04);48 h:(0.36±0.06)、(0.23±0.04)、(0.14±0.03)比(0.54±0.08)]、mTOR蛋白[24 h:(0.35±0.05)、(0.24±0.02)、(0.18±0.02)比(0.52±0.09);48 h:(0.23±0.04)、(0.29±0.04)、(0.14±0.03)比(0.40±0.10)]、VEGF蛋白[24 h:(0.14±0.03)、(0.33±0.04)、(0.24±0.03)比(0.54±0.10);48 h:(0.24±0.03)、(0.17±0.02)、(0.11±0.02)比(0.42±0.10)]较模型组降低(P<0.05),且联合组AKT、mTOR、VEGF mRNA及蛋白表达较康柏西普组降低(P<0.05)。结论AKTObjective To study the mechanism of the effect of Jiajian-Zhujing Decoction on the expression of VEGF on ARPE-19 cells after AKT transfection.Methods To prepare the serum and blank serum of Jiajian-Zhujing Decoction and divide ARPE-19 cells into the normal group,model group,blank serum group,medicated serum group,Conbercept group and combined group.Except normal group,this research established AKT transfected cell model.Then cultured the normal group and model group with conventional method,and the blank serum group was cultured with 10%blank serum,the medicated serum group was cultured with 10%medicated serum,the Conbercept group was cultured with 20μg/ml Conbercept,the combined group was cultured with 10%medicated serum and 20μg/m Conbercept.The proliferation of ARPE-19 cells in each group was detect by the CCK-8 method.The levels of AKT,mTOR and VEGF mRNA were detected by real-time quantitative PCR.Western blot was used to detect the expression of AKT,mTOR and VEGF.Results After being cultured for 24,48 and 72 hours,compared with the model group,the cell proliferation rate in blank serum group,medicated serum group,Conbercept group and combined group significantly decreased(P<0.05).Compared with the model group,the expression of AKT mRNA(24 h:3.10±0.48,1.97±0.14,1.26±0.24 vs.4.77±0.68;48 h:3.52±0.82,2.62±0.77,1.10±0.19 vs.6.12±1.21),mTOR mRNA(24 h:3.02±0.26,2.45±0.75,1.13±0.15 vs.4.48±0.80;48 h:1.29±0.30,1.30±0.57,0.65±0.19 vs.2.54±0.62),VEGF mRNA(24 h:3.33±0.62,2.18±0.20,1.55±0.28 vs.5.53±1.02;48 h:2.35±0.54,1.23±0.28,0.93±0.25 vs.3.59±0.40),AKT protion(24 h:0.45±0.09,0.25±0.05,0.14±0.04 vs.0.62±0.04;48 h:0.36±0.06,0.23±0.04,0.14±0.03 vs.0.54±0.08),mTOR protion(24 h:0.35±0.05,0.24±0.02,0.18±0.02 vs.0.52±0.09;48 h:0.23±0.04,0.29±0.04,0.14±0.03 vs.0.40±0.10),VEGF protion(24 h:0.14±0.03,0.33±0.04,0.24±0.03 vs.0.54±0.10;48 h:0.24±0.03,0.17±0.02,0.11±0.02 vs.0.42±0.10)significantly decreased(P<0.05),and the combined group was significantly lower than that of the Co

关 键 词：中药血清药理学 视网膜色素上皮 转染 血管内皮生长因子 加减驻景方 

分 类 号：R77[医药卫生—眼科] R2[医药卫生—临床医学]