ABSTRACT
Background Scarring of surgical area,the most important factor,leads to the failure of glaucoma filtering surgeries.Therefore,more and more attentions are paid to the causes and process of scar formation.Objective This study was to compare the differences of proliferation and migrating abilities of fibroblasts between filtering bleb scar tissue and normal Tenon capsular tissue,and to investigate the inhibitory effects of miRNA-200a (miR-200a) on biological behavior of conjunctival fibroblasts.Methods Normal Tenon capsular tissue and filtering bleb scar tissue were collected during the strabismus surgery and glaucoma filtering surgery,respectively for the primarily culture of fibroblasts.The proliferation (absorbency,A) of the cells was assayed by cell counting kit-8 (CCK8) method;the relative migrating distance of the cells was measured by cell scratch test;and the relative expressions of transforming growth factor-β1 (TGF-β1)mRNA and miR-200a mRNA in the cells were detected by realtime fluorescence quantitative PCR.TGF-β1 mimic of 0,1,2 and 5 ng/ml was added in the medium of human normal Tenon capsular-derived fibroblasts (HTFs),and 0.00,0.25,0.50,1.00 μg/ml TGF-β1 inhibitor was added in the medium of human scarring-derived fibroblasts (HSFs) for 24 hours,respectively,and CCK8 was used to evaluate the proliferation of the cells.The relative migrating distance as well as the relative expressions of miR-200a mRNA were analyzed in the 2 ng/ml TGF-β1 mimic-or 1.00 μg/ml TGF-β1 inhibitor-treated cells.Results The primary conjunctival presented the spindle and star-like in shape with large body and oval nuclei.The cells showed the positive response for keratin and vimentin antibodies.The A values were 1.476±0.110 in the HSFs and 0.958±0.074 in the HTFs,with a significant difference between them (t =24.900,P=0.016).The relative expressions of TGF-β1 mRNA were significantly higher in the HSFs than those in the HTFs,and the relative expressions of miR-200a were evidently lower in the HSFs than those in the HTFs,showing significant differences between them (t =6.358,P =0.024;t=7.394,P =0.018).Compared with the 2 ng/ml TGF-β1 mimic-treated HTFs,the relative migrating distance increased,while the expression level of miR-200a mRNA was significantly reduced in the 2 ng/ml TGF-β1 mimictreated HSFs (all at P＜0.05);Compared with the 1.00 μg/ml TGF-β1 inhibitor-treated HTFs,the relative migrating distance decreased,but the expression level of miR-200a mRNA was significantly elevated in the 1.00 μg/ml TGF-β1 inhibitor-treated HSFs (all at P＜0.05).Conclusions The proliferation and migrating abilities are stronger in the HSFs than those in the HTFs,which probably is regulated by the expression of miR-200a in the cells.The miR-200a plays a negative feedback for the effect of TGF-β1 promoting proliferation and migration of fibroblasts.
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Objective To analyze type IV collagen a5 chain mRNA and to study the relationship between genotype and phenotype in X-linked Alport syndrome. Methods Total RNA was isolated from the cultured skin fibroblasts of 21 unrelated Chinese X-linked Alport syndrome patients (17 males and 4 females),then ?5 chain (Ⅳ) mRNA was analyzed by using reverse-transcription-polymerase reaction (RT-PCR) and direct sequencing. Meanwhile,by using PCR and direct sequencing,detection of COL4A5 gene mutations at genomic DNA level was carried out. Results Abnormal ?5 chain IV cDNA was detected in 21 X-linked Alport syndrome patients,and seventeen mutations detected in this study were novel mutations. In 15/21 of patients,identical COL4A5 mutations were detected both at mRNA level and genomic DNA level,and in 6/21 of patients with splicing-site mutations,changes in transcript structure differed from changes in genomic DNA level. 16/21 of patients belonged to X-linked juvenile kindreds,and 2/21 of patients to adult kindreds. Conclusion Different type of mutations in COL4A5 can lead to the severe form of X-linked Alport syndrome,and mRNA-based procedures can both directly detect mutations in the coding sequences,as well as changes in transcript level or structure,and can identify some abnormalities that would otherwise have been missed by DNA-based procedures.
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Object To investigate the effects of Panax notoginseng saponins (PNS) on myocardial Gs? mRNA expression of seriously scalded rat. Methods A 30% skin full thickness scald model was produced by immersing rat in 95 ℃ water for 10 s. The effects of PNS on myocardial Gs? mRNA level were observed with dot blotting hybridization and in situ hybridization technique; effects on cAMP and adenyl cyclase (AC) activities were determined with radioimmunoassay. Results Myocardial Gs? mRNA, AC activity and cAMP content were reduced significantly 3 h after scalding. PNS (100, 200 mg/kg) could markedly increase the level of myocardial Gs? mRNA expression. The elevated quantity was correlated markedly with PNS dosage (r = 0.95, P