NADPH氧化酶参与人晶状体上皮细胞增殖及其表达

【摘要】目的观察烟酰胺二磷酸腺苷（NADPH）氧化酶的抑制剂能否抑制人晶状体上皮细胞增殖，探讨NADPH氧化酶亚单位NOX的同源异构体存人晶状体上皮细胞mRNA的表达情况：设计实验性研究。研究对象人晶状体上皮细胞永生系（SRA01／04）。方法实验分为4组，SRA0I／04中加入表皮生长因子（EGF）（EGF组），加入NADPH氧化酶抑制剂二亚苯基碘（DPI）（DPI组），先后加入DPI和EGF（DPI＋EGF组），不加入上述物质为对照组。利用酶标仪和活细胞计数CCK一8试剂盒检测各组人晶状体上皮细胞OD450值。用RT—PCR方法检测NOX的同源异构体在SRA0I／04中的表达，将RT—PCR产物测序，利用NCBIBI。AST软件对测序结果与cDNA进行序列对比。主要指标人晶状体上皮细胞的OD450值，NOX的同源异构体表达情况及其与人其他组织的序列对比：结果加入CCK-83．5小时后，EGF组比对照组OD450值升高了12．0％（P=0．000）；DPI组比对照组OD450值下降了25．5％（P=0．000）；DPI＋EGF组OD450值比EGF组降低了26．1％（P=O．000）。RT—PCR结果表明，NOX蛋白的5种同源异构体包括NOXl、NOX2（gp91phox）、NOX3、NOX4、NOX5]的mRNA在SRA01／04中均有表达。人晶状体上皮细胞中的NOXl—5的序列与人其他组织的相似性分别为100％、100％、99．7％、100％、100％。结论NADPH氧化酶可能促进人晶状体上皮细胞的增殖；NOX同源异构体NOXl、NOX2、NOX3、NOX4、NOX5的mRNA在人晶状体上皮细胞SRA0I／04中均有表达，其中NOXl明显弱于其他四种NOX。

NADPH oxidase participation in cell proliferation of human lens epithelial cells and its expression

Objective To study whether the inhibitor of nicotinamide adenine dinucleotide phosphate （NADPH） oxidase can inhibit the proliferation of human lens epithelial cells and to investigate which isoforms of NOX, one of the subunits of NADPH oxidase, are expressed in human lens epithelial cells at mRNA level. Design Experimental study. Participants Human lens epithelial ceils （SRA 01/04）. Methods The cells were divided into four groups： EGF group was added with EGF in SRA 01/04, DPI group was added with the inhibitor of NADPH oxidase-diphenylene iodonium （DPI）, DPI ＋EGF group was added with EGF after DPI and control group was added with nothing. Using Microplate reader anA Cell Counting Kit-8 to determine OD450 value of SRA 01/04 of each group. The expressinn of NOX family, including NOX1, NOX2 or gp91phox, NOX3, NOX4 and NOX5, was detected with RT-PCR. The RT-PCR products were sequenced to confirm their identities by NCBI BLAST. Main Outcome Measures OD450 of SRA 01/04, expression of NOX and their sequence alignments with other human tissues. Results After added with CCK-8 for 3.5 h, the OD450 value of EGF group increased 12.0% compared with control group （P=0.000）. The OD450 value of DP1 group decreased 25.5% compared with control group （P=0.000）. The OD450 value of DPI＋EGF group decreased 26.1% compared with EGF group （P=0.000）. RT-PCR using primers speeifie for mRNAs of the five isoforms of the NOX proteins documented that mRNA encoding NOX1 through NOX5 were constitutively present in SRA 01/04 cells. The similarity of sequences of NOX1-NOX5 in human lens epithelial cells with other human tissues was 100%, 100%, 99.7%, 100% and 100%, respectively. Conclusions NADPH oxidase complex could promote cell proliferation in human lens epithelial ceils. SRA 01/04 cells constitutively produced mRNA encoding five isoforms of NOX proteins, NOXI was much weaker than the other four NOXs. （ Opbthalmol CHN, 2009, 18：107-113）