| Objective: to observe effect of secreted protein,acidic and rich in cysteine on expression of collagen type I of the cultured human fibroblasts of hypertrophic scar by real-time fluorescence quantitative RT-PCR.Methods:①Hypertrophic scars of Upper and lower limbs were derived from 2 cases of Department of Plastic Surgery, First Affiliated Hospital of Anhui Medical University. Hypertrophic scars of abdomen and back were derived from 2 cases of Department of burns, First Affiliated Hospital of Anhui Medical University. Fibroblasts were derived from four Hypertrophic scars. The III-V generation exponential phase of growth cell were used for experiment.②Different concentrations of recombinant human SPARC (final concentration of 2.5,5,10 pmol ? L-1) on the 3-5 generation of hypertrophic scar fibroblasts, respectively intervention 3h. using Trizol reagent extracted total RNA from fibroblasts, nucleic acid detector test Optical density and RNA quality was identified by 1% agarose gel electrophoresis, the cDNA, obtained by Reverse transcription reaction , -20℃to save. Diluted cDNA into the 5 gradient (104,103,102,101,100), respectively target genes and restricted reference materials collagen type I geneβ-actin of SYBR Green I Real-time quantitative PCR reaction, standard curves prepared two sets of genes and to detect extended increase efficiency.③The product of PCR was analyzed by the melting curve, and making 1% agarose gel electrophoresis to determine product specificity. collagen type I mRNA relative expression using Comparative Delta-delta Ct method for analysis.④comparing between the numbers by SPSS 11.0 statistical software for analysis of variance and t test pairwise comparison was be used.Results: Four Hypertrophic scar specimens in primary culture were survived well and spread for the 3-5 generation of experiments, the role of 3h with different concentrations of SPARC after the termination of culture, entered the final analysis.①The total RNA extracted for 1% agarose gel electrophoresis to determine product-specific, results showed that the total RNA extracted from the quality of pure, no significant degradation, can be the next step RT-PCR reaction.②The reverse transcriptase product was 95℃10 s denaturation, then 95℃5 s, 60℃30 s repeated 50 cycles for amplification, using Rotor Gene3000 system Comparative Delta-delta Ct method analysis, the results compared with the blank control group, SPARC2.5pmol·L-1, 5pmol·L-1, SPARC10.0pmol·L-1 experimental group, collagen type I mRNA expression increased 1.5,3.4,3.9 times.③comparing between the number of each group were be used by the SPSS 11.0 statistical software for analysis of variance and pairwise comparison t test (P<0.05), showed statistically significant difference.④PCR product for the melting curve analysis showed that collagen type I andβ-actin gene PCR product of the melting curve peaks were 85℃and 89℃, melting temperature uniformity, as a single sharp peak, indicating no primer-dimers and non-specific amplification, experiment results are reliable.Conclusion: Recombinant human SPARC stimulation in all experimental groups composed of a relatively blank control fibroblast collagen type I mRNA expression was significantly increased, suggesting that SPARC in hypertrophic scars may have to promote hypertrophic scar fibroblasts secretion of collagen type I's role in hypertrophic scar formation may have an important role in the process for the study of hypertrophic scar formation mechanism and treatment of hypertrophic scars to provide new ideas.
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