ABSTRACT
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.
ABSTRACT
Background Researches demonstrated that ciliary neurotrophic factor (CNTF) can enhance survival and promote differentiation of neutron.Meanwhile,CNTF also is thought to play an important role in the development of visual pathway.But,less studies are reported in the relationship of CNTF and form deprivation amblyopia.Objective This study was to investigate the expressions of CNTF in visual cortex area 17 in form deprivation amblyopia model.Methods Twelve 4-week-old cats were randomized into normal group and form deprivation amblyopia group.Monocular form deprivation amblyopic models were established in 6 cats by eyelids suture method.Pattern visual evoked potential(P-VEP) was recorded to evaluate the amblyopic models 16 weeks later following the eyelids suturing.Then,bilateral visual cortex tissue was incised at a vertical in sagittal axis fashion to prepare the section.Nissl staining was used to detect the morphologies of neurons.Expression of CNTF in Ⅰ-Ⅵ layers of visual cortex area 17 was located and quantified by immunochemistry.The positive cell number and gray scale for CNTF were calculated and compared between two groups.The use of the animals complied with Regulations for the Administration of Affairs Coucerning Experimental Animals by State Science and Technology Commission.Results Compared with the normal group,P-VEP amplitude was significantly reduced (6.11 ±1.56 μV vs.11.42±t.92 μV) and latency was significantly prolonged(75.77±9.83 ms vs.58.56±7.17 ms) in the form deprivation amblyopia group (t=5.272,3.464,P＜0.05).Nissl staining showed that the number of neurons in the form deprivation amblyopia group was less than that in the normal group.In the form deprivation amblyopia group,neurons became shrinkage and turned round,cytoplasmic processes get shortened,and the nucleus became small.The number of Nissl bodies was decreased.lmmunochemistry showed the positive neutrons for CNTF in Ⅰ-Ⅵ layers of visual cortex area 17 in hoth normal cats and model cats with the more positive cells in Ⅱ-Ⅳ layers.Compared with the normal group,the positive cell number for CNTF was significantly reduced in Ⅱ-Ⅳ layers of visual cortex area 17 in the form deprivation amblyopia group (Ⅱ layer:95.93±8.24 vs.116.25±6.52;I layer:102.65±7.45 vs.125.23±8.21;Ⅳ layer:l10.65±6.85 vs.139.54±4.26) (t=4.737,4.989,8.773,P＜0.05).In addition,the gray scale of CNTF positive cells was significantly lower in Ⅱ-Ⅳ layers of visual cortex area 17 in the form deprivation amblyopia group than that the normal group (Ⅱ layer:106.98 ± 8.86 vs.138.65 ± 6.38 ; Ⅲ layer:109.56 ± 8.69 vs.149.59 ±8.55;Ⅳ layer:l16.65 ±9.52 vs.155.76±9.87) (t=7.105,8.043,6.986,P＜0.05).Both CNTF positive cell number and gray scale in Ⅰ,Ⅴ,Ⅵ layers of visual cortex area 17 had no significant differences between two groups (P＞0.05).Conclusions Form deprivation in critical period of a new born animal may lead to distributing abnormality of CNTF in visual cortex,which maybe play a role in the development of form deprived amblyopia.