CAJ | 학술논문

背景后发性白内障是现代白内障囊外摘出术后导致视力下降的常见并发症.基质金属蛋白酶(MMPs)是参与降解除多糖以外全部细胞外基质(ECM)的重要蛋白酶,探讨MMP-3对后发性白内障的基因治疗一直是研究热点,而纤连蛋白(FN)是MMP-3的主要降解底物,能间接反映MMP-3的表达情况.目的构建pEGFP-N1-MMP-3真核重组质粒并转染晶状体上皮细胞(LECs),观察MMP-3在LECs中的表达,探讨后发性白内障基因治疗的可能性.方法取6只新鲜猪晶状体,将囊膜用组织块法进行原代培养,获得LECs.用E.coli DH5α MMP-3和E.coli DH5α pEGFP-N1质粒构建MMP-3的真核表达重组质粒pEGFP-N1-MMP-3,通过双酶切,DNA测序分析鉴定人MMP-3片段的准确性.将构建的pEGFP-N1-MMP-3转染至猪LECs中,通过绿色荧光蛋白(GFP)间接观察MMP-3在猪LECs中的表达,用Western blot法检测pEGFP-N1-MMP-3真核重组质粒转染后LECs表达产物FN的相对表达量,间接评估MMP-3的表达量. 结果双酶切鉴定结果符合质粒pEGFP-N1及目的片段MMP-3大小.软件分析测序结果表明,重组质粒pEGFP-N1-MMP-3中的MMP-3基因与GenBank中的人MMP基因序列相似性达99.6％,表示目的片段已正确插入pEGFP-N1载体.原代培养猪LECs,将重组质粒pEGFP-N1-MMP-3转染入猪LECs,荧光显微镜下可见明显的GFP绿色荧光.Western blot检测表明,FN在pEGFP-N1 -MMP-3真核重组质粒转染组的表达量为0.666±0.008,空质粒转染组FN的表达量为0.326±0.071,差异有统计学意义(P=0.000). 结论成功构建了pEGFP-N1-MMP-3质粒,并能够在猪LECs中表达,为进一步研究该蛋白在LECs中的表达与功能奠定了基础.
배경 후발성백내장시현대백내장낭외적출술후도치시력하강적상견병발증.기질금속단백매(MMPs)시삼여강해제다당이외전부세포외기질(ECM)적중요단백매,탐토MMP-3대후발성백내장적기인치료일직시연구열점,이섬련단백(FN)시MMP-3적주요강해저물,능간접반영MMP-3적표체정황.목적 구건pEGFP-N1-MMP-3진핵중조질립병전염정상체상피세포(LECs),관찰MMP-3재LECs중적표체,탐토후발성백내장기인치료적가능성.방법 취6지신선저정상체,장낭막용조직괴법진행원대배양,획득LECs.용E.coli DH5α MMP-3화E.coli DH5α pEGFP-N1질립구건MMP-3적진핵표체중조질립pEGFP-N1-MMP-3,통과쌍매절,DNA측서분석감정인MMP-3편단적준학성.장구건적pEGFP-N1-MMP-3전염지저LECs중,통과록색형광단백(GFP)간접관찰MMP-3재저LECs중적표체,용Western blot법검측pEGFP-N1-MMP-3진핵중조질립전염후LECs표체산물FN적상대표체량,간접평고MMP-3적표체량. 결과 쌍매절감정결과부합질립pEGFP-N1급목적편단MMP-3대소.연건분석측서결과표명,중조질립pEGFP-N1-MMP-3중적MMP-3기인여GenBank중적인MMP기인서렬상사성체99.6％,표시목적편단이정학삽입pEGFP-N1재체.원대배양저LECs,장중조질립pEGFP-N1-MMP-3전염입저LECs,형광현미경하가견명현적GFP록색형광.Western blot검측표명,FN재pEGFP-N1 -MMP-3진핵중조질립전염조적표체량위0.666±0.008,공질립전염조FN적표체량위0.326±0.071,차이유통계학의의(P=0.000). 결론 성공구건료pEGFP-N1-MMP-3질립,병능구재저LECs중표체,위진일보연구해단백재LECs중적표체여공능전정료기출.
Background Posterior capsular opacification(PCO) is common complication after extrecapsular extract of cataract.Matrix metalloproteinases-3 (MMP-3) can degrade all the extracellular matrix except polyose.The gene therapy of PCO upon MMP-3 is the researching hot topic.Fibronectin ( FN ) is a degrade gelatin,so its expression can reflect the effect of MMP-3 on LECs indirectly. Objective The aim of this study was to construct MMP-3 eukaryotic recombination plasmid and transfect to lens epithelium cells(LECs) for the observation of MMP3 expression,and to explore the feasibility of gene therapy for after cataract. Methods Six fresh lenses were obtained from pigs.LECs were cultured using explant method.The eukaryotic expression vector pEGFP-N1-MMP-3 was reconstructed with MMP-3 and pEGFP-N1 plasmids.The accuracy of MMP-3 gene fragment was confirmed by double enzyme digestion and DNA sequencing analysis.After transfecting pEGFP-N1-MMP-3 into LECs of pig,the expression of MMP-3 protein in the cells was indirectly observed by green fluorescent protein.The expression of FN in LECs was detected using Western blot. Results The result of double enzyme digestion was consistent with the base number of pEGFP-N1 plasmids and target fragment.By enlacing the result of DNA sequencing analysis with software,the resemblance of the DNA sequence of MMP-3 from recombination plasmid pEGFP-N1-MMP-3 and that of homo MMP-3 was 99.6％,indicating that the target fragment was inserted to pEGFP-N1 plasmids successfully.Green fluorescence for GFP was seen in the LECs in pEGFP-N1-MMP-3 transfected group,but absent response for GFP was in empty vector group.Western blot revealed that the relative expression level of FN in LECs was 0.666±0.008 in pEGFP-N1-MMP-3 trasfected group and 0.326 ±0.071 in empty vector group,with a significant difference between these two groups(P=0.000). Conclusions Eukaryotic recombination plasmid pEGFP-N1-MMP-3 is successfully constructed,and MMP-3 can be expressed in LECs after transfected.These results lay a foundation for the further research of MMP-3 gene therapy for PCO.