摘要
目的研究RNA干扰基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)表达对体外培养的人晶状体上皮细胞(human lens epithelial cells,hLECs)增殖、移行的影响。探讨RNA干扰技术抑制LECs增殖、移行的可行性,以探索防治后发性白内障的新方法。方法设计构建含有靶向MMP-2的短发夹RNA(short hairpin RNA,shRNA)质粒载体和不含靶向MMP-2的shRNA阴性对照质粒载体,体外转染hLECs SRA01/04,分别标记为MMP-2沉默组和阴性对照组;以等体积培养液代替转染质粒培养作为空白对照组。实时荧光定量PCR和Western blot检测转染后24 h MMP-2 mRNA及MMP-2蛋白的表达水平。MTT比色法测定细胞转染后24 h、48 h以及72 h时hLECs的增殖能力。细胞划痕实验方法测定转染后24 h、48 h时hLECs的移行愈合率。结果转染24 h后MMP-2沉默组mRNA及其蛋白的相对表达水平分别为0.202±0.075、80.856±2.165,与空白对照组1.041±0.163和184.419±3.584比较分别下降了80.6%和55.1%,差异均有统计学意义(均为P<0.05),而阴性对照组与空白对照组相比差异无统计学意义(P>0.05);MTT比色法检测结果显示,各时间点的细胞增殖能力MMP-2沉默组与阴性对照组比较,差异无统计学意义(P>0.05),而2组较空白对照组均有所下降,差异均有统计学意义(均为P<0.05);细胞划痕实验示转染24 h与48 h后MMP-2沉默组的移行愈合率分别为(20.36±4.14)%和(23.19±5.62)%,较空白对照组(41.26±4.57)%和(67.61±8.80)%以及阴性对照组的(36.28±2.28)%和(74.48±9.21)%均明显降低,差异均有统计学意义(均为P<0.05),阴性对照组与空白对照组相比差异无统计学意义(P>0.05)。结论靶向MMP-2 shRNA可以有效降低hLECs SRA01/04 MMP-2mRNA和蛋白表达,抑制细胞的移行,但尚不能认为对细胞增殖能力有影响。RNA干扰MMP-2的表达可有效抑制hLECs的移行。
Objective To investigate the effects of RNA interference targeting matrix metalloproteinase-2（ MMP-2） on proliferation and migration of human lens epithelial cells（ hLEC） in vitro, and explore the new methods of prevention and treatment of posterior capsule opacification（ PCO）. Methods Two plasmid vectors were designed and synthesized, one of them contained a short hairpin RNA（ shRNA） targeting MMP-2 inserted and another one was inserted without expression of MMP-2. Then hLEC SRA01 /04 in vitro culture were transfected by the two plasmid vectors,named MMP-2 silence group and negative control group respectively, and hLEC were cultured in DMEM of an equal volume compared with the upper two groups as blank control group.At 24 hours after transfection,MMP-2 mRNA and protein in hLEC were detected by real-time PCR and Western blot. The ability of proliferation was determined by MTT assay at 24 hours, 48 hours and 72 hours after transfection. Cell scratch assay was used at 24 hours and 48 hours after transfection to evaluate the ability of migration. Results At 24 hours after transfection, the relative quantities of MMP-2 mRNA and protein of MMP-2 silence group were 0. 202±0. 075 and 80.856±2. 165,which were decreased by 80. 6% and 55. 1% respectively compared with blank control group（ 1. 041±0. 163 and 184. 419±3. 584, all P〈0. 05）, and there was no statistical difference between negative control group and blank control group（ P〉0. 05）; MTT assay showed that there was no statistical difference in cell proliferation between MMP-2 silence group and negative control group（ P〉0. 05）, and the two groups had statistically difference compared with blank control group（ all P〈0. 05）; Cell scratch assay was quantified by ratio of migrated heal, and the ratio of MMP-2 silence group were（ 20. 36±4. 14） % and（ 23. 19±5. 62） % respectively at 24 hours and 48 hours after transfection,which were significant decreased compared with blank control group [（ 41. 26±4. 57） % and（ 67. 61±8. 80） %]and negative control group [（ 36. 28±2. 28） % and（ 74. 48±9. 21） %]（ all P〈0. 05）,however, there was no statistical difference between negative control group and blank control group（ P〉0. 05）. Conclusion MMP-2 shRNA can effectively reduce secretion of MMP-2 mRNA and protein of hLECSRA01 /04 in vitro culture, simultaneously,migration is inhibited. However, there is no enough evidence to prove that proliferation is connected to the change of MMP-2.
出处
《眼科新进展》
CAS
北大核心
2015年第3期235-239,共5页
Recent Advances in Ophthalmology
