RESUMO
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.
RESUMO
Background Human paired box gene 6 (PAX6)encodes a transcriptional regulator.It is essential for eye and brain morphogenesis.Mutation of PAX6 gene isresponsible for many congenital ocular malformations,such as aniridia.Aniridia is a autosomal dominant inheritance mode.Objective In this study,PAX6 gene mutation was analyzed in three Chinese families with aniridia through polymerase chain reaction (PCR) and sequencing.Methods The blood specimens were collected from 5 suffers and normal individuals of 3 aniridia families to extract DNA.The sequences of extron 4-13 were designed based on PAX6 gene.The primer was amplified by PCR and sequenced and compared with the known PAX6 gene sequence.This study complied with Declaration of Helsinki and approved by ethic committee of Sichuan University.Written informed consent was obtained from each individual before any medial examination.ResultsThere were 5 suffers in the 3 families.A heterozygous mutation (c.718 C＞T) in PAX6 gene was identified in 2 patients of family A.This mutation caused an amino acid substitution of arginine to termination codon at position 240 ( p.Arg240X) of PAX6 protein.No similar change in the normal families.No any the alteration of PAX6 gene was detected in family B whatever suffers and normal individuals.In family C,a deletion mutation of c.331 delG ( p.Val111 SerfsX13 ) in PAX6 gene was found.The deletion of one base caused frame shift mutation of PAX6 protein,and no such mutation was seen in other families.Conclusions Mutation of PAX6 gene appeares to be causative mutations of the disease in family A and C.