Objective To investigate the effects of simvastatin on the collagen synthesis of rat pulmonary arterial smooth muscle cells ( PASMCs ) induced by hypoxia. Methods Under hypoxic condition, rat PASMCs were cultured with different concentrations of simvastatin. Collagen synthesis of PASMCs with or without simvastatin were measured by 3H-proline incorporation assay. The mRNA expression of TGF-β1 and the contents of super oxide dismrtase ( SOD) ,malondialdehyde ( MDA) in mediumwere also measured. Results The incorporation data of 3H-TdR in the hypoxia group was significantly increased as compared with that in the control group ( P lt;0. 01) , and simvastatin significantly reduced the incorporation data of 3H-TdR induced by hypoxia. The expression of TGF-β1 mRNA in the hypoxia group was significantly increased as compared with that in the control group ( P lt; 0. 01 ) , and simvastatin could significantly inhibited hypoxia-induced expression of TGF-β1 mRNA in a dose-dependent manner. Compared with the hypoxia group, the expression of TGF-β1 mRNA decreased by 55% in simvastatin( 10 - 6mol /L) group ( P lt; 0. 01) , and by 70% ( P lt; 0. 01) in simvastatin ( 10 - 5mol /L) group. Compared with the control group, the activity of SOD was reduced and the contents of MDA were increased significantly in the hypoxia group. Simvastatin can increase the activity of SOD and reduced the content of MDA in a dose-dependent manner. Conclusions Simvastatin can decreases collagen synthesis of PASMCs. This effect might be explained that simvastatin can reduce lipid peroxide and expression of TGF-β1 mRNA.
Objective Astragalus polysaccharide (APS) has promoting angiogenesis function. To explore the effects of APS collagen sponge on enhancing angiogenesis and collagen synthesis so as to provide evidence for the future tissue engineering appl ication as a kind of angiogenic scaffold. Methods APS collagen sponges were prepared by covalent binding with collagen polypeptides by using of crossl inking agents at the ratio of 1 ∶ 1 (W/W). Twenty 10-week-old SpragueDawley rats (10 males and 10 females, and weighing 200-250 g) were selected. Longitudinal incision was made at both sides of the back to form subcutaneous pockets. APS collagen sponges of 5 mm × 5 mm × 5 mm at size were implanted into the left pockets as the experimental group, collagen sponges without APS of the same size into the right pockets as the control group. The general conditions were observed after operation. At 3, 7, 14，and 21 days, 5 rats were sacrificed and the samples were harvested to count the number of microvessels, to measure the contents of the hydroxyprol ine (Hyp), and to detect the mRNA expressions of angiopoetin 1 (Ang1), matrix metalloproteinases 9 (MMP-9), and tissue inhibitors of metalloproteinases 1 (TIMP-1). Results All rats were al ive during experiment period. The number of microvessels increased gradually, and reached the peak at 14 days in 2 groups; the expermental group was significantly higher than the control group (P lt; 0.05). The contents of Hyp increased gradually in 2 groups, and the experimental group was significantly higher than the control group (P lt; 0.05). The mRNA expressions of Ang1 and MMP-9 in the experimental group were significantly higher than those in the control group at 3, 7, and 14 days (P lt; 0.05); the mRNA expression of TIMP-1 in the experimental group was significantly lower than that in the control group at 3 days and was significantly higher at 14 and 21 days (P lt; 0.05). Conclusion The APS collagen sponges can improve angiogenesis and collagen synthesis in wound heal ing by regulating the expressions of Ang1, MMP-9, and TIMP-1.
Objective To investigate the effects of NGF on the prol iferation, mitotic cycle, collagen synthesis and migration of human dermal fibroblasts (HDFs), and to explore the function of NGF on the wound heal ing. Methods The 3rd generation of HDFs were incubated with various concentrations of NGF (0, 25, 50, 100, 200 and 400 ng/mL), the cell prol iferation was measured with MTT assay. After treated with NGF at 0, 100 ng/mL, the cell cycle of HDFs was determined by flow cytometry (FCM). Hydroxyprol ine and real-time fluorescence quantitative PCR (FQ-PCR) were used to measure collagen synthesis at protein level and mRNA level respectively. The in vitro cell scratch wound model was set up to observe the effect of NGF (0, 50, 100 and 200 ng/mL) on the migration of HDFs after 24 hours of culture. Results Absorbance value of HDFs for different concentrations of NGF (0, 25, 50, 100, 200, and 400 ng/ mL) showed that NGF did not influence the prol iferation of HDFs (P gt; 0.05). When HDFs were treated with NGF at 0 and 100 ng/mL, the result of FCM analysis showed that percentage of HDFs in G0/G1, S, G2/M phases were not changed (P gt; 0.05). Compared with control group, the expression of Col I and Col III were not significantly different, measured by both hydroxyprol ine and FQ-PCR (P gt; 0.05). The rates of HDFs’ migration at various concentrations of NGF (0, 50, 100, 200 ng/ mL) were 52.12% ± 6.50%, 80.67% ± 8.51%, 66.33% ± 3.58%, and 61.19% ± 0.97%, respectively, indicating that NGF could significantly enhanced the migration of HDFs at 50 and 100 ng/mL (P lt; 0.05). Conclusion NGF does not influence prol iferation, mitotic cycle and collagen synthesis of HDFs, but significantly enhanced migration in an in vitro model of wounded fibroblasts.
Objective To investigate the influence of enamel matrix proteins (EMPs) on the attachment, prol iferation and pre-mRNA of type I collagen synthesis of cultured human dermal fibroblast cells. Methods Human dermal fibroblast cells were obtained from human acrobystia and cultured in DMEM medium with 10% FBS. The 3rd to 6th passage cells were used. Ninety-six-well plates and 6-well plates were pre-coated with different concentrations of EMPs (50, 100, 150 and200 μg/ mL). ① The cell attachment experiment: 0.2 mL cells suspension at the concentration of 1 × 106/mL was added to the pre-coated 96-well plates as the experimental groups (groups A, B, C and D based on different concentrations of EMPs). At 1.5, 3.0, and 4.5 hours after inoculation, the attached cells were measured by MTT method. ② The cell prol iferation experiment: 0.2 mL cells suspension at the concentration of 5 × 104/mL was added to the pre-coated 96-well plates as the experimental groups (groups A1, B1, C1 and D1 based on the different concentrations of EMPs). At 2, 4, 6 and 8 days after inoculation, the cells were measured by MTT method. ③ The synthesis experiment of pre-mRNA: 2 mL cells at the concentration of 1 × 106/mL was added to the pre-coated 6-well plates as the experimental groups (groups A2, B2, C2 and D2 based on different concentrations of EMPs). At 5 days after inoculation, the synthesis of pre-mRNA was measured by RT-PCR method. Human dermal fibroblast cells were added to the un-coated plates as the control groups. Results ① The cell attachment experiment: There were significant differences in attachment cells between the control group, group A and the groups B, C and D (P lt; 0.05). There were no significant difference between group A and control group (P lt; 0.05). ② The cell prol iferation experiment: At 2 days, there were no significant differences in absorbance between the control group and the experimental groups (P gt; 0.05); at 4 days and 6 days, the absorbance of groups B1 (0.598 ± 0.020 and 0.639 ± 0.016 ), C1 (0.582 ± 0.017 and 0.641 ± 0.020) and D1 (0.574 ± 0.021and 0.635 ± 0.021) was significantly higher than that of the control group (0.548 ± 0.021 and 0.605 ± 0.019, P lt; 0.05); at 8 days, the absorbance of group B1 (0.629 ± 0.012) and group C1 (0.631 ± 0.014) was significantly higher than that of the control group (0.606 ± 0.031, P lt; 0.05). ③ The synthesis experiment of pre-mRNA: The synthesis of type I collage pre-mRNA of groups B2, C2 and D2 was significantly higher than that of the control group. Conclusion EMPs stimulate human dermal fibroblast cell attachment, prol iferation and synthesis of type I collage pre-mRNA, and its maximal effect can be achieved at the concentration of 100 μg /mL.
Objective To investigate the influence of lipopolysaccharide(LPS) on the proliferation and collagen synthesis of normal human skin fibroblasts so as to elucidate its relation with skin wound healing. Methods Fibroblasts wereisolated and cultured in vitro, and then exposed to different doses of LPS(0.005, 0.010, 0.050, 0.100, 0.500, and 1.000 μg/ml) from E.coli055∶B5 respectively. Then the absorbance (A) value of fibroblasts was determined with the colorirneteric thiazolylblue (MTT) assay, and the cell number was counted under inverted phase contrast microscope from the 1st day to the 9th day after LPS administration, and collagen synthesis of fibroblasts in culture medium was measured with the method of pepsin digestion after incorporation of 3Hproline into stable, single-layered, confluent fibroblasts at 7 days after LPS administration. Results Compared with control group, A value increased with the increasing concentration of LPS (0.005 μg/ml 0.500 μg/ml) and LPS of 0.100 μg/mlgroup had the best effect. The difference was remarkable from the 5th day to the 9th day(P＜0.05). A value decreased when challenged with the LPS of 1.000 μg/ml and the difference was remarkable from the 3rd day to the 9th day(P＜0.05). Cell number increased with theadministration of LPS of different concentrations (0.005 μg/ml 0.500 μg/ml) and LPS of 0.100 μg/mlgroup had the best effect. The difference was remarkable from the 1st day to the 6th day(P＜0.05). Cell number decreased remarkably when challenged with LPS of 1.000 μg/ml and the difference was remarkable from the 2nd day to the 9th day(P＜0.05). Collagen synthesis increased when challenged with LPS of different concentrations (0.005 μg/ml 0.500 μg/ml) and the 0.100 μg/ml group had the best effect. However, when the dose of LPS reached 1.000 μg/ml, it inhibited collagensynthesis. Conclusion LPS could promote the proliferation andcollagen synthesis of fibroblasts within a certain range of low doses, but over-high dose ofLPS might inhibit the proliferation and collagen synthesis of fibroblasts, suggesting that LPS of certain concentrations might contribute to wound healing, while excessive LPS has negative effect on wound healing.
ObjectiveTo investigate the effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), a hypoxia-inducible factor-1α (HIF-1α) inhibitor, on hypoxia induced rat pulmonary arterial adventitial fibroblasts (AFs) proliferation and collagen synthesis, and explore the molecular mechanism.MethodsUnder hypoxic condition, rat AFs were cultured in DMEM medium supplemented with 10% fetal bovine serum in vitro. The cells were divided into five groups, ie. a normoxia group, a hypoxia group and three hypoxia+YC-1 groups (treated with YC-1 at concentration of 0.01, 0.05 and 0.1 mmol/L, respectively). The cells proliferation was determined by MTT method. Collagen synthesis of AFs was measured by 3H-proline incorporation assay. The expression of HIF-1α in AFs in different conditions was measured by Western blot, and the mRNA expression of transforming growth factor-β1 (TGF-β1) was measured by reverse-transcription polymerase chain reaction.ResultsThe proliferation rate and the incorporation data of 3H-proline in the hypoxia group were significantly increased as compared with those in the control group (both P<0.01). YC-1 significantly reduced the proliferation rate and incorporation data of3H-proline induced by hypoxia in a dose-dependent manner. YC-1 could also down-regulate the expressions of HIF-1α and TGF-β1 mRNA significantly (both P<0.01). Compared with the hypoxia group, the expressions of HIF-1α and TGF-β1 mRNA decreased respectively by 65% and 61% in the hypoxia+YC-1 (0.1 mmol/L) group (bothP<0.01).ConclusionsYC-1 can inhibit hypoxia-induced AFs proliferation and collagen synthesis in a dose-dependent manner. The mechanism may relate to YC-1’s inhibitory effect on expressions of HIF-1α and TGF-β1 mRNA.