RNA干扰MMP-2基因对人晶状体上皮细胞增殖及移行能力的影响

目的　研究ＲＮＡ干扰基质金属蛋白酶-２（ｍａｔｒｉｘｍｅｔａｌｌｏｐｒｏｔｅｉｎａｓｅ-２，ＭＭＰ-２）表达对体外培养的人晶状体上皮细胞（ｈｕｍａｎｌｅｎｓｅｐｉｔｈｅｌｉａｌｃｅｌｌｓ，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（ｓｈｏｒｔｈａｉｒｐｉｎＲＮＡ，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ｈＬＥＣｓＳＲＡ０１／０４，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Ｗｅｓｔｅｒｎｂｌｏｔ检测转染后２４ｈＭＭＰ-２ｍＲＮＡ及ＭＭＰ-２蛋白的表达水平。ＭＴＴ比色法测定细胞转染后２４ｈ、４８ｈ以及７２ｈ时ｈＬＥＣｓ的增殖能力。细胞划痕实验方法测定转染后２４ｈ、４８ｈ时ｈＬＥＣｓ的移行愈合率。结果　转染２４ｈ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２４ｈ与４８ｈ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-２ｓｈＲＮＡ可以有效降低ｈＬＥＣｓＳＲＡ０１／０４ＭＭＰ-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。

Effects of RNA interference targeting MMP-2 0n proliferation and migration of human lens epithelial cells

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 sigruficant 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.