摘要
目的分别建立人N-Myc下游受调节基因2(N-Myc downstream regulated gene2,NDRG2)表达上调、下调的培养人晶状体上皮细胞稳定转染细胞株,并观察NDRG2对晶状体上皮细胞增殖能力的影响。方法分别用质粒PLenti6-NDRG2、PLenti6-cherry、PLKO-NDRG2shRNA、PLKO-scramble以及PAX2和PMD2G构建慢病毒载体,感染培养人晶状体上皮细胞系(SRA01/04),通过药物筛选获得NDRG2表达上调、下调的晶状体上皮细胞株。采用Real-time PCR、Western blot等方法检测NDRG2的表达。通过MTT、EdU等方法检测所获得细胞株的生长曲线及增殖能力。结果 NDRG2 mRNA在过表达对照组和过表达组细胞中的相对表达量分别为1.04±0.38、6.57±0.97,两组间差异有统计学意义(P<0.05);NDRG2mRNA在干涉对照组和干涉组细胞中的表达量分别为1.05±0.31、0.49±0.14,两组间差异有统计学意义(P<0.05)。NDRG2过表达和干涉慢病毒载体感染后,SRA01/04细胞中NDRG2 mRNA的表达分别出现了明显的上调和下调。Western blot检测结果显示:与各自对照组相比,在NDRG2过表达组和干涉组细胞中,NDRG2蛋白的表达量分别出现了明显的升高和降低。MTT检测结果显示:与其对照组相比,NDRG2过表达组细胞生长曲线表现出降低趋势,干涉组细胞生长曲线表现出升高趋势,在培养后5d检测时差异有统计学意义(P<0.05)。EdU检测结果显示:过表达对照组Cherry-SRA01/04细胞、过表达组NDRG2-SRA01/04细胞EdU阳性表达率分别为(41.57±2.53)%、(33.53±3.03)%,两组间差异有统计学意义(P<0.05),提示NDRG2表达上调后DNA的合成受到抑制。干涉对照组Scramble-SRA01/04细胞和干涉组shNDRG2-SRA01/04细胞EdU阳性表达率分别为(27.70±1.05)%、(46.43±4.01)%,差异有统计学意义(P<0.05),提示NDRG2表达下调后DNA合成速度加快。结论通过病毒载体感染联合药物筛选,分别建立了NDRG2表达上调和下调的培养人晶状体上皮细胞稳定转染细胞株,且NDRG2基因对晶状体上皮细胞的增殖能力具有负向调节作用。
Objective To establish human lens epithelial cell strains cultured artificially with upward or downward expression of N-Myc downstream regulated gene 2 （NDRG2）, and investigate the effects of NDRG2 on cellular multiplication capacity. Methods Cultured human lens epithelial cell strains （SRA01/04） were infected by lentiviral vectors constructed using plasmids of PLentiS-NDRG2, PLentiS-cherry, PLKO-NDRG2 shRNA, PLKO-scramble, PAX2, and PMD2G, and human lens epithelial cell strains with upward or downward expression of NDRG2 obtained through drug screening. The expression of NDRG2 was detected by Real-time PCR and Western blot. The growth curve and multiplication capacity were analyzed by MTT and EdU. Results The relative expression level of NDRG2 mRNA in overexpression control group and overexpression group was 1.04 ± 0.38 and 5.57 ± 0.97, respectively, and the difference between the two groups was statistically significant （ P 〈 0.05 ） ; Expression of NDRG mRNA in interference control group and interference group was 1.05 ± 0.31 and 0.49 ± 0.14, respectively, the difference between which suggested a statistical significance （P 〈 0.05 ）. With the overexpression and infection with interference lentiviral vector, the expression of NDRG2 mRNA in SRA01/04 cells was significantly up and down,respectivly. Western blot test results indicated that compared with the two control groups, the expression of NDRG protein in overexpression group and interference group obviously rised and falled, respectively. MTT test results showed that compared with the control group, the cell growth curve of NDRG2 overexpression group showed a decreasing trend;Interference group suggested an increasing trend. The difference tested on the fifth day of cultivation had a statistical significance （P 〈 0.05 ）. EdU test results demonstrated that the positive expression rate of EdU in Cherry-SRA01/04 cells of overexpression control group and overexpression group was （41. 57 ± 2. 53）% and （33.53 ± 3.03 ） % , respectively. The statistically significant （ P 〈 0.05 ） difference between these two groups indicated that with the increased expression of NDRG, DNA synthesis was inhibited. The positive expression rate of EdU in Cherry-SRA01/04 cells of interference group（46.43±4.01 ）% was significantly higher than that of interference control group （27.70 ± 1.05 ） % （P 〈 0.05 ）, and the difference between them had a statistical significance （P 〈 0.05 ） ,indicating DNA synthesis increased after the decreasing of NDRG2 expression. Conelusion Human lens epithelial cell strains cultured artificially with upward or downward expression of NDRG2 can be obtained through lentiviral vector infection and drug screening. NDRG2 has a negative regulation effect on the multiplication capacity of human lens epithelial cells.
出处
《眼科新进展》
CAS
北大核心
2013年第5期415-418,422,共5页
Recent Advances in Ophthalmology
基金
国家自然科学基金资助(编号:30801275)
德国洪堡基金会仪器设备捐赠基金资助(编号:V-8151/02085)~~
关键词
NDRG2
晶状体上皮细胞
细胞增殖能力
N-myc downstream regulated gene 2
lens epithelial cells
multiplication capacity
