Y overabundant collagen synthesis, analysis of collagen of type I and III gene transcription and protein expression levels was completed using Real-Time PCR and Western blot after 72 h of TLP treatment. As shown in Figure 2, the expression of Col I/III in the high TLP expression group was significantly elevated above levels observed in control groups (P,0.05), up-regulated more than 1.5 times and 2 times respectively. Furthermore, with cytokine TGF-b1 stimulation, the amount of the synthesized collagen in the high TLP expression group was also markedly increased than the control groups (P,0.05). Protein level analysisFigure 2. The mRNA levels changes of TGF-b1, Col I, and Col III after TLP treatment. The groups were designed as Cell-Lv-TLP (HSFs transfected with recombinant lentivirus), Cell-Lv (HSFs transfected with control Tubastatin-A manufacturer lentivirus ), Cell (HSFs without any treatment), Cell-Lv-TLP+TGFb1 (HSFs transfected with recombinant lentivirus and stimulated by the cytokine TGF-b1), Cell-Lv+TGF-b1 (HSFs transfected with control lentivirus and stimulated by TGF-b1), and Cell+TGF-b1 (HSFs stimulated by TGF-b1 ). After RNA isolation and Sermorelin manufacturer reverse transcription, TGF-b1 mRNA was quantified by quantitative PCR, data represents mean6SD, n = 5. * means P,0.05 vs. the control groups with or without TGF-b1. doi:10.1371/journal.pone.0055899.gEffects of TLP on Synthesis of CollagensFigure 3. Detection of TLP gene expression and its influence on the synthesis of Col I/III protein. (A) Immunoblot analysis of lysate samples for TLP, TGF-b1, Col I, and Col III. (B, C, D, E) Determination of grey value of TLP, TGF-b1, Col I, and Col III revealed in A. Experiments were repeated 3 times, and data are expressed mean6SD, N = 3. * means P,0.05 between the two groups. doi:10.1371/journal.pone.0055899.galso showed the similar tendency towards to Figure 2 but except collagen III. As shown in Figure 3E, although there was no statistical significance existing between groups, among all the panel experiments under same conditions we obtained the consistent results that the Col III expression were about enhanced by 10?15 after TLP treatment.While the variation were not so apparent under TGF-b1 stimulation. Notably, the amount of the expressive TGF-b1 appeared constant (Figure 3C).hypertrophic scars were also markedly higher than those observed in normal skin samples both mRNA and protein levels(shown in Figure 5).MTT AssayTo determine the effect of TLP on the cell viability, MTT assays were performed. The results showed that before 12 h after TLP treatment, there was no obvious statistical difference among all groups. However, when at 24 h cell viability was increased by up to 40 (Figure 6). Upon treatment with TGF-b1, variation within cell viability became more apparent among the experimental groups. Thus, TLP may act cooperatively with TGF-b1 to increase cell proliferative viability. Moreover, compared to control cells, the lentivirus transfected cells showed no sign of cytotoxicity and weak multiplication capacity, that is to say, the gene delivery vector was safe and showed no conspicuous effect on cells.Alteration of the Expression of p-Smad2 and p-Smad3 Affected by TLPThe intrinsic mechanism of alteration in collagen expression triggered by TLP is further revealed by examining the expression levels of Smad2/P-Smad2 and Smad3/P-Smad3 (shown in Figure 4). Under conditions of high TLP expression, the level of p-Smad3 decreased approximately by 25 (P,0.05). Conversely, t.Y overabundant collagen synthesis, analysis of collagen of type I and III gene transcription and protein expression levels was completed using Real-Time PCR and Western blot after 72 h of TLP treatment. As shown in Figure 2, the expression of Col I/III in the high TLP expression group was significantly elevated above levels observed in control groups (P,0.05), up-regulated more than 1.5 times and 2 times respectively. Furthermore, with cytokine TGF-b1 stimulation, the amount of the synthesized collagen in the high TLP expression group was also markedly increased than the control groups (P,0.05). Protein level analysisFigure 2. The mRNA levels changes of TGF-b1, Col I, and Col III after TLP treatment. The groups were designed as Cell-Lv-TLP (HSFs transfected with recombinant lentivirus), Cell-Lv (HSFs transfected with control lentivirus ), Cell (HSFs without any treatment), Cell-Lv-TLP+TGFb1 (HSFs transfected with recombinant lentivirus and stimulated by the cytokine TGF-b1), Cell-Lv+TGF-b1 (HSFs transfected with control lentivirus and stimulated by TGF-b1), and Cell+TGF-b1 (HSFs stimulated by TGF-b1 ). After RNA isolation and reverse transcription, TGF-b1 mRNA was quantified by quantitative PCR, data represents mean6SD, n = 5. * means P,0.05 vs. the control groups with or without TGF-b1. doi:10.1371/journal.pone.0055899.gEffects of TLP on Synthesis of CollagensFigure 3. Detection of TLP gene expression and its influence on the synthesis of Col I/III protein. (A) Immunoblot analysis of lysate samples for TLP, TGF-b1, Col I, and Col III. (B, C, D, E) Determination of grey value of TLP, TGF-b1, Col I, and Col III revealed in A. Experiments were repeated 3 times, and data are expressed mean6SD, N = 3. * means P,0.05 between the two groups. doi:10.1371/journal.pone.0055899.galso showed the similar tendency towards to Figure 2 but except collagen III. As shown in Figure 3E, although there was no statistical significance existing between groups, among all the panel experiments under same conditions we obtained the consistent results that the Col III expression were about enhanced by 10?15 after TLP treatment.While the variation were not so apparent under TGF-b1 stimulation. Notably, the amount of the expressive TGF-b1 appeared constant (Figure 3C).hypertrophic scars were also markedly higher than those observed in normal skin samples both mRNA and protein levels(shown in Figure 5).MTT AssayTo determine the effect of TLP on the cell viability, MTT assays were performed. The results showed that before 12 h after TLP treatment, there was no obvious statistical difference among all groups. However, when at 24 h cell viability was increased by up to 40 (Figure 6). Upon treatment with TGF-b1, variation within cell viability became more apparent among the experimental groups. Thus, TLP may act cooperatively with TGF-b1 to increase cell proliferative viability. Moreover, compared to control cells, the lentivirus transfected cells showed no sign of cytotoxicity and weak multiplication capacity, that is to say, the gene delivery vector was safe and showed no conspicuous effect on cells.Alteration of the Expression of p-Smad2 and p-Smad3 Affected by TLPThe intrinsic mechanism of alteration in collagen expression triggered by TLP is further revealed by examining the expression levels of Smad2/P-Smad2 and Smad3/P-Smad3 (shown in Figure 4). Under conditions of high TLP expression, the level of p-Smad3 decreased approximately by 25 (P,0.05). Conversely, t.
