Objective To express human HT036 protein in Escherichia coli(E.coli.)and identify it.Methods The cDNA sequence obtained by PCR was cloned into the prokaryotic expression plasmid pET30a(+).The target protein was expressed in E.coli..induced by IPTG and analyzed by Western blotting.Results The interest gene was identified by restriction endonucleases digestion and DNA sequencing.The protein was highly expressed in E.coli..Conclusion We successfully expressed the HT036 protein.

Objective: To explore the relationship of discrimination perception and social anxiety of the left-behind children, focusing on the moderating effect of trait gratitude. Methods: Totally 588 left-behind children were investigated with discrimination perception questionnaire, gratitude questionnaire and Liebowitz social anxiety questionnaire. Data were analyzed by independent t test, correlation analysis and regression analysis. Results: There was a significant difference in group discrimination between the only child (2.11±0.98) and the non-only child (2.28±0.93) (t=-2.11, P<0.05). Correlation analysis showed that trait gratitude (4.93±0.89) was negatively correlated with individual discrimination (2.53±1.03) and group discrimination (2.22±0.95) (r=-0.55, r=-0.56, both P< 0.05), furtherly negatively correlated with fear of negation (0.85±0.47) and social avoidance (0.81±0.53) dimensions of social anxiety (r=-0.47, r=-0.48, both P<0.05). Additionally, discrimination perception was positively correlated with social anxiety (r=0.61, P<0.05). Regression analysis showed that discrimination perception could significantly predict social anxiety (β=0.44, P<0.01), and trait gratitude could significantly moderate this effect (β=-0.75, P<0.01). Conclusion Discrimination perception can positively predict social anxiety, and trait gratitude can moderate this effect.

Objective To screen the proteins interacting with FOXP3 in yeast two-hybrid system. Methods The "bait plasmid" pGBKT7 (named as pGBKT7-FOXP3) was constructed successfully. Using FOXP3 as bait, a human liver cDNA library was screened and the proteins interacting with FOXP3 were searched. The false positive clones were discarded by one to one yeast two-hybrid system, and the positive clones were sequenced and analyzed by bioinformatic methods. Results The bait plasmid pGBKT7-FOXP3 was constructed successfully and there was no self-activation or toxicity in AH109. Three proteins had been found in our system to be able to interact with FOXP3. They were tumor protein D52, splicing factor 3b subunit 1 and one hypothetical protein. Conclusion FOXP3 interacts with tumor protein D52, splicing factor 3b subunit 1 and one hypothetical protein, all of which may interfere in cell metabolism and function of T cell.

Objective To construct a bait vector containing human Foxp3 gene in yeast two-hybrid system in order to screen the cDNA library of T lymphocyte. Methods RT-PCR was used to amplify the Foxp3 gene fragment from the peripheral blood mononuclear cells (PBMC) with the primers designed in accordance with the sequence in GenBank. The product was inserted into pMD18-T vector. After verified with restriction endonuclease digestion of EcoRⅠ and SalⅠ, the vector was inserted into the “bait plasmid” pGBKT7 (named as pGBKT7-Foxp3). After confirmation with restricted endonuclease digestion and sequence analysis, the plasmid was transformed into the yeast cell AH109, and its toxicity and transcriptional activation was tested by both the phenotype assay and the color assay. Results The amplified product of 1 203 bp was inserted into PMD18-T vector and proven correctly by double restriction enzyme digestion. Sequence analysis revealed that the fragment was correctly inserted into pGBKT7 with a right reading frame and its expression in yeast was verified. Conclusion The bait plasmid pGBKT7-Foxp3 constructed expresses correctly, and can not activate the transcription of reporter gene alone in yeast two-hybrid system

Objective To explore the interaction between HT036(hypothetical protein HT036)and P311 by co-immunoprecipitation.Methods HA-tagged fusion protein(HA-HT036)expression vector was constructed,identified and transfected into human embryo kidney 293(HEK293)cells alone or with Myc-tagged fusion protein(Myc-P311)expression vector pCMV-Myc-p311.The interaction between P311 and HT036 was detected by co-immunoprecipitation.Results Double restriction enzyme digestion showed that pCMV-HA-HT036 was constructed correctly.When Myc-P311 was immunoprecipitated by anti-Myc antibody,HA-HT036 was identified by Western blotting with anti-HA antibody from immunoprecipitated complex.Conclusion The recombinant vector pCMV-HA-HT036 was constructed successfully.The interaction between HT036 and P311 could be identified by co-immunoprecipitation after co-expression of pCMV-HA-HT036 and pCMV-Myc-p311.The result provides an important basis for further study of the intracellular signal transduction of P311.

To explore the role of dentritic epidermal T lymphocytes ( DETCs) in immune rejection of skin allograft in mice and its related mechanism. Methods (1) Full-thickness skin was harvested from back of one male wild type (WT) C57BL/6 mouse. Epithelial cells were isolated for detection of the expression of DETCs and their phenotype with flow cytometer. Another male WT C57BL/6 mouse was used to harvest full-thickness skin from the back. Epidermis was isolated for observation of the morphological characteristics of DETCs with immunofluorescence technology. (2) Four male green fluorescence protein (GFP)-marked C57BL/6 mice, 7 female WT C57BL/6 mice (group WT), and 7 female ybT lymphocytes 8 gene knock-out (GK) C57BL/6 mice (group GK) were used. Full-thickness skin in the size of 1.4 cm x 1.4 cm on the back of mice in groups WT and GK were excised, and the wounds were transplanted with full-thickness skin in the size of 1.2 cm x 1.2 cm obtained from male GFP-marked C57BL/6 mice. The survival time of skin grafts was affirmed with small animal in vivo imager and naked eyes and recorded. (3) Two male WT C57BL/6 mice were used to isolate epithelial cells. Cells were inoculated into 48-well plate and divided into activation group (A) and control group (C) according to the random number table, with 4 wells in each group. Cells in group A were treated with 10 pL concanavalin A in the concentration of 2 microg/mL for 24 hours, while those in group C with PBS in the same volume as that in group A. The expression of interferon y in DETCs was detected with flow cytometer. (4) Four male GFP-marked C57BL/6 mice were used as donors. Fourteen female WT C57BL/6 mice were used as receptors and divided into interferon gamma neutralizing group (IN) and control group (C) according to the random number table, with 7 mice in each group. The skin transplantation model of C57BL/6 male to C57BL/6 female was established as in part (2). Before surgery and 72 hours after, mice in group IN were intraperitoneally injected with 200 pL interferon y neutralizing antibody in the concentration of 1 mg/mL, and those in group C with normal saline in the same volume as that in group IN. The survival time of skin grafts was observed and recorded using the methods in part (2), and the result of group IN was compared with that of group GK in part (2). The survival curve of skin grafts was processed with Log-rank ( Mantel-Cox) test. Results (1) The positive expression rate of DETCs in epithelial cells of skin in mouse was 7.27%, and they were all CD3 cells. DETCs were found to be scattered in the epidermis of skin in mouse with dendritic morphology. (2) The survival time of skin grafts of mice in group GK was 22-35 d, obviously longer than that in group WT (12-16 d, y2 = 14. 10 , P < 0.001). (3) Expression of interferon gamma was detected in 22. 70% DETCs in group A, which was obviously higher than that in group C (0.51%). (4) The survival time of skin grafts of mice in group IN was 19-24 d, which was obviously longer than that in group C (12-16 d, chi 2 = 13.60, P < 0.001) but close to that in group GK as in part (2) (chi2 = 0.06, P = 0.810). Conclusions DETCs are involved in promotion of immune rejection of skin allograft probably by secretinf interferon gamma.
Allografts ; Animals ; Epidermis ; Female ; Graft Survival ; immunology ; physiology ; Interferon-gamma ; immunology ; metabolism ; Lymphocytes ; Male ; Mice ; Mice, Inbred C57BL ; Skin ; Skin Transplantation ; T-Lymphocytes ; immunology

Objective To explore the relationship of discrimination perception and social anxiety of the left-behind children,focusing on the moderating effect of trait gratitude. Methods Totally 588 left-be-hind children were investigated with discrimination perception questionnaire, gratitude questionnaire and Liebowitz social anxiety questionnaire. Data were analyzed by independent t test,correlation analysis and re-gression analysis. Results There was a significant difference in group discrimination between the only child (2. 11±0. 98) and the non-only child (2. 28±0. 93) (t=-2. 11,P<0. 05). Correlation analysis showed that trait gratitude (4. 93± 0. 89) was negatively correlated with individual discrimination ( 2. 53 ± 1. 03) and group discrimination (2. 22±0. 95) (r=-0. 55,r=-0. 56,both P< 0. 05),furtherly negatively correlated with fear of negation (0. 85±0. 47) and social avoidance (0. 81±0. 53) dimensions of social anxiety ( r=-0. 47,r=-0. 48,both P<0. 05). Additionally,discrimination perception was positively correlated with so-cial anxiety (r=0. 61,P<0. 05). Regression analysis showed that discrimination perception could significant-ly predict social anxiety (β=0. 44,P<0. 01),and trait gratitude could significantly moderate this effect (β=-0. 75,P<0. 01). Conclusion Discrimination perception can positively predict social anxiety,and trait gratitude can moderate this effect.

OBJECTIVETo observe the effects of artificial dermis combined with basic fibroblast growth factor (bFGF) on the treatment of cicatrix and deep skin wounds.

METHODSThe clinical data of 72 patients with wounds repaired with artificial dermis, hospitalized in our unit from October 2010 to April 2015, conforming to the study criteria, were retrospectively analyzed. The types of wounds were wounds after resection of cicatrices, deep burn wounds without exposure of tendon or bone, and wounds with exposure of small area of tendon or bone, in a total number of 102. Wounds were divided into artificial dermis group (A, n=60) and artificial dermis+ bFGF group (B, n=42) according to whether or not artificial dermis combined with bFGF. In group A, after release and resection of cicatrices or thorough debridement of deep skin wounds, artificial dermis was directly grafted to wounds in the first stage operation. After complete vascularization of artificial dermis, wounds were repaired with autologous split-thickness skin grafts in the second stage operation. In group B, all the procedures were exactly the same as those in group A except that artificial dermis had been soaked in bFGF for 30 min before grafting. Operation area, complete vascularization time of artificial dermis, survival of skin grafts, and the follow-up condition of wounds in the two groups were recorded. Data were processed with t test and Fisher's exact test.

RESULTS(1) Operation areas of wounds after resection of cicatrices, deep burn wounds without exposure of tendon or bone, and wounds with exposure of small area of tendon or bone in the two groups were about the same (with t values from -1.853 to -0.200, P values above 0.05). Complete vascularization time of artificial dermis in wounds after resection of cicatrices, deep burn wounds without exposure of tendon or bone, and wounds with exposure of small area of tendon or bone in group B were respectively (15.6 ± 2.9), (14.7 ± 2.7), and (20.3 ± 4.4) d, and they were shorter by an average time of 2.7, 4.0, 7.4 d, respectively, as compared with those in corresponding types of wounds in group A [respectively (18.3 ± 4.7), (18.7 ± 4.2), and (27.7 ± 8.8) d, with t values from -2.779 to -2.383, P values below 0.05]. (2) The ratio of skin grafts with excellent survival in the three types of wounds in group B were higher than those in corresponding types of wounds in group A, but there were no statistically significant differences (with P values above 0.05). (3) Patients were followed up for 1 to 48 months, and there were no obvious cicatrices in skin graft sites and the donor sites during the following time.

CONCLUSIONSArtificial dermis combined with bFGF can effectively shorten the vascularization time of artificial dermis in wounds after resection of cicatrices and deep skin wounds.

Burns ; therapy ; Cicatrix ; therapy ; Debridement ; Dermis ; injuries ; Fibroblast Growth Factor 2 ; therapeutic use ; Humans ; Retrospective Studies ; Skin Transplantation ; Skin, Artificial ; Soft Tissue Injuries ; therapy ; Transplantation, Autologous ; Wound Healing

Objective:To study the effects of reactive oxygen species (ROS)-responsive antibacterial microneedles (MNs) on the full-thickness skin defect wounds with bacterial colonization in diabetic mice.Methods:Experimental research methods were adopted. The ROS-responsive crosslinker N1-(4-boronobenzyl)-N3-(4-boronophenyl)-N1, N1, N3, N3-tetramethylpropane-1,3-diaminium (TSPBA) was first synthesized, and then the polyvinyl alcohol (PVA)-TSPBA MNs, PVA-ε-polylysine (ε-PL)-TSPBA MNs, PVA-TSPBA-sodium hyaluronate (SH) MNs, and PVA-ε-PL-TSPBA-SH MNs were prepared by mixing corresponding ingredients, respectively. The PVA-TSPBA MNs were placed in pure phosphate buffer solution (PBS) and PBS containing hydrogen peroxide, respectively. The degradation of MNs immersed for 0 (immediately), 3, 7, and 10 days was observed to indicate their ROS responsiveness. The standard strains of Staphylococcus aureus ( S. aureus) and Escherichia coli (E. coli) cultured in Luria-Bertani medium containing hydrogen peroxide were divided according to the random number table (the same grouping method below) into blank control group (without any treatment, the same below) and 0 g/L ε-PL group, 1.0 g/L ε-PL group, 5.0 g/L ε-PL group, and 10.0 g/L ε-PL group with which PVA-ε-PL-TSPBA MNs containing the corresponding concentration of ε-PL were co-cultured, respectively. Bacterial growth was observed after 24 h of culture, and the relative survival rate of bacteria was calculated ( n=3). The mouse fibroblast cell line 3T3 cells at logarithmic growth stage (the same growth stage below) were divided into blank control group and 0 g/L ε-PL group, 1.0 g /L ε-PL group, 5.0 g /L ε-PL group, and 10.0 g /L ε-PL group in which cells were cultured in medium with the extract from PVA-ε-PL-TSPBA MNs containing the corresponding concentration of ε-PL, respectively. Cell growth was observed after 24 h of culture by optical microscopy, and the relative survival rate of cells was detected and calculated by cell counting kit 8 (CCK-8) assay to indicate the cytotoxicity ( n=6). Both PVA-TSPBA MNs and PVA-TSPBA-SH MNs were taken, the morphology of the two kinds of MNs was observed by optical microscopy, and the mechanical properties of the two kinds of MNs were tested by microcomputer controlled electronic universal testing machine (denoted as critical force, n=6). Six male BALB/c mice aged 6-8 weeks (the same gender and age below) were divided into PVA-TSPBA group and PVA-TSPBA-SH group, with 3 mice in each group. After pressing the skin on the back of mice vertically with the corresponding MNs for 1 minute, the skin condition was observed at 0, 10, and 20 min after pressing. Another batch of 3T3 cells were divided into blank control group, 0 g/L ε-PL group and simple 5.0 g/L ε-PL group which were cultured with the extract of PVA-ε-PL-TSPBA MNs containing the corresponding concentration of ε-PL, and 5.0 g/L ε-PL+SH group which were cultured with the extract of PVA-ε-PL-TSPBA-SH MNs with 5.0 g/L ε-PL. The CCK-8 assay was performed to detect and calculate the relative survival rate of cells cultured for 24, 48, and 72 h to indicate the cell proliferation activity ( n=6). Eighteen BALB/c mice were induced into diabetic mice model by high-sugar and high-fat diet combined with streptozotocin injection and then divided into sterile dressing group, 0 g/L ε-PL+SH group, and 5.0 g/L ε-PL+SH group, with 6 mice in each group. A full-thickness skin defect wound was made on the back of each mouse, and S. aureus solution was added to make a full-thickness skin defect wound with bacterial colonization model for diabetic mouse. The wounds of mice in 0 g/L ε-PL+SH group and 5.0 g/L ε-PL+SH group were covered with PVA-ε-PL-TSPBA-SH MNs with the corresponding concentration of ε-PL, and the wounds of mice in the 3 groups were all covered with sterile surgical dressings. The wound healing was observed on post injury day (PID) 0, 3, 7, and 12, and the wound healing rate on PID 3, 7, and 12 was calculated. On PID 12, the skin tissue of the wound and the wound margin were stained with hematoxylin and eosin to observe the growth of new epithelium and the infiltration of inflammatory cells. Data were statistically analyzed with one-way analysis of variance, analysis of variance for repeated measurement, Mann-Whitney U test, and Bonferroni test. Results:With the extension of the immersion time, the PVA-TSPBA MNs in PBS containing hydrogen peroxide gradually dissolved and completely degraded after 10 days of immersion. The PVA-TSPBA MNs in pure PBS only swelled but did not dissolve. After 24 h of culture, there was no growth of S. aureus in 5.0 g/L ε-PL group or 10.0 g/L ε-PL group, and there was no growth of E. coli in 10.0 g/L ε-PL group. The relative survival rate of S. aureus was significantly lower in 1.0 g/L ε-PL group, 5.0 g/L ε-PL group, and 10.0 g/L ε-PL group than in blank control group ( P<0.05 or P<0.01). The relative survival rate of E. coli was significantly lower in 5.0 g/L ε-PL group and 10.0 g/L ε-PL group than in blank control group ( P<0.01). After 24 h of culture, the cells in blank control group, 0 g/L ε-PL group, 1.0 g/L ε-PL group, 5.0 g/L ε-PL group, and 10.0 g/L ε-PL group all grew well, and the relative survival rate of cells was similar among the groups ( P>0.05). The needle bodies of PVA-TSPBA MNs and PVA-TSPBA-SH MNs were both quadrangular pyramid-shaped and neatly arranged, and the needle bodies of PVA-TSPBA-SH MNs was more three-dimensional and more angular. The critical force of PVA-TSPBA-SH MNs was significantly higher than that of PVA-TSPBA MNs ( Z=3.317, P<0.01). The MNs in PVA-TSPBA+SH group penetrated the skin of mice at 0 min after pressing, and the pinholes partially disappeared after 10 min and completely disappeared after 20 min, while the MNs in PVA-TSPBA group failed to penetrate the skin of mice. After 24, 48, and 72 h of culture, the proliferation activity of the cells in 5.0 g/L ε-PL+SH group was significantly higher than that of blank control group ( P<0.05 or P<0.01). In sterile dressing group, the wounds of mice healed slowly and exuded more. The wound healing speed of mice in 0 g/L ε-PL+SH group was similar to that of sterile dressing group in the early stage but was faster than that of sterile dressing group in the later stage, with moderate exudation. The wound healing of mice in 5.0 g/L ε-PL+SH group was faster than that in the other two groups, with less exudation. The wound healing rates of mice in 5.0 g/L ε-PL+SH group were (40.6±4.2)%, (64.3±4.1)%, and (95.8±2.4)% on PID 3, 7, and 12, which were significantly higher than (20.4±2.7)%, (38.9±2.2)%, and (59.1±6.2)% in sterile dressing group and (21.6±2.6)%, (44.0±1.7)%, and (82.2±5.3)% in 0 g/L ε-PL+SH group ( P<0.01). The wound healing rates of mice in 0 g/L ε-PL+SH group on PID 7 and 12 were significantly higher than those in sterile dressing group ( P<0.05 or P<0.01). On PID 12, the wounds of mice in 5.0 g/L ε-PL+SH group were almost completely epithelialized with less inflammatory cell infiltration, the wounds of mice in 0 g/L ε-PL+SH group were partially epithelialized with a large number of inflammatory cell infiltration, and no obvious epithelialization but a large number of inflammatory cell infiltration was found in the wounds of mice in sterile dressing group. Conclusions:The composite MNs prepared by TSPBA, PVA, ε-PL, and SH can successfully penetrate mouse skin and slowly respond to ROS in the wound to resolve and release antibacterial substances, inhibit bacterial colonization, and promote the repair of full-thickness skin defect wounds with bacterial colonization in diabetic mice.

OBJECTIVETo explore the interaction between P311 and transforming growth factor beta 1 (TGF-β1) in murine fibroblasts and its effect on the function of fibroblasts.

METHODSSkin fibroblasts obtained from five neonatal P311 wild-type C57BL/6 mice and P311 gene knock-out C57BL/6 mice were cultured. The second passage of fibroblasts were used in the following experiments. All experiments were repeated for 3 times. (1) The fibroblasts of P311 wild-type mice were divided into blank control group and P311 over-expression group according to the random number table (the same grouping method below), with 36 wells in each group. Fibroblasts in blank control group were transfected with 10 μL control vector, and fibroblasts in P311 over-expression group were transfected with equal efficiency P311 expression adenovirus vector. After being cultured for 48 hours, the mRNA expression level of P311, and the mRNA and protein expression levels of TGF-β1, α-smooth muscle actin (α-SMA), and collagen type I of fibroblasts in both groups were determined with real-time fluorescent quantitative RT-PCR and Western blotting (the same detection methods below), respectively. (2) After cultured reaching the cell density of 80%-90%, the mRNA and protein expression levels of TGF-β1, α-SMA, and collagen type I of the fibroblasts of P311 wild-type mice and P311 gene knock-out mice, with 4 flasks in each type of fibroblasts, were determined. (3) The fibroblasts of P311 wild-type mice were divided into blank control group and 5, 10, 15, 20, and 25 ng/mL TGF-β1 groups after being starved treatment with DMEM medium containing 1% FBS for 3 hours, with 2 flasks in each group. Fibroblasts in blank control group were routinely cultured, while fibroblasts in the latter five groups were treated with 5, 10, 15, 20, and 25 ng/mL TGF-β1, respectively. After being cultured for 48 hours, the mRNA expression levels of P311 in fibroblasts of the six groups were determined. Another fibroblasts of P311 wild-type mice were divided into blank control group and 10 ng/mL TGF-β1 group, with 6 wells in each group, and the protein expression levels of P311 in both groups were determined by immunofluorescence staining. (4) The fibroblasts of P311 wild-type mice were divided into blank control group and 10 ng/mL TGF-β1 group after being starved treatment as above, with 2 flasks in each group, and fibroblasts in blank control group were routinely cultured, while fibroblasts in the latter group were treated with 10 ng/mL TGF-β1. After being cultured for 48 hours, the mRNA and protein expression levels of α-SMA and collagen type Ⅰ were determined. The fibroblasts of P311 gene knock-out mice were grouped and treated as above, and the mRNA and protein expression levels of α-SMA and collagen type I were determined. Data were processed with one-way analysis of variance and t test.

RESULTS(1) The mRNA expression level of P311 of fibroblasts in P311 over-expression group was increased nearly 300 000-fold compared with that in blank control group (t=9.942, P<0.001). The mRNA expression levels of TGF-β1, α-SMA, and collagen type I of fibroblasts in P311 over-expression group, and the protein expression levels of pro-TGF-β1, activated TGF-β1, α-SMA, and collagen type I of fibroblasts in P311 over-expression group were significantly higher than those in blank control group (with t values from 8.192 to 49.090, P values below 0.01). (2) The mRNA expression levels of TGF-β1, α-SMA, and collagen type I in fibroblasts of P311gene knock-out mice were significantly lower than those in fibroblasts of P311 wild-type mice (with t values from 8.157 to 22.270, P values below 0.01). The protein expression levels of pro-TGF-β1, α-SMA, and collagen type I in fibroblasts of P311 gene knock-out mice were significantly lower than those in fibroblasts of P311 wild-type mice (with t values from 2.995 to 12.600, P<0.05 or P<0.01), and the protein expression levels of active TGF-β1 were similar in two types of fibroblasts (t=1.070, P>0.05). (3) The mRNA expression levels of P311 of fibroblasts in blank control group and 5, 10, 15, 20 and 25 ng/mL TGF-β1 groups were 1.28 ± 0.44, 3.61 ± 0.91, 6.64 ± 0.92, 6.58 ± 1.04, 1.79 ± 0.31, 0.16 ± 0.06, respectively. Compared to the mRNA expression level of P311 of fibroblasts in the blank control group, the mRNA expression levels of P311 of fibroblasts in 5 and 20 ng/mL TGF-β1 groups were similar (with t values respectively 2.302 and 0.955, P values above 0.05), while they were significantly higher in 10 and 15 ng/mL TGF-β1 groups (with t values respectively 5.630 and 4.710, P values below 0.001), and they were significantly lower in 25 ng/mL TGF-β1 group (t=2.509, P<0.01). The protein expression level of P311 of fibroblasts in 10 ng/mL group was higher than that in blank control group. (4) The mRNA and protein expression levels of α-SMA and collagen type I of fibroblasts of P311 wild-type mice in 10 ng/mL TGF-β1 group were significantly higher than those in blank control group (with t values from 3.523 to 14.290, P<0.05 or P<0.01). The mRNA and protein expression levels of α-SMA and collagen type I of fibroblasts of P311 gene knock-out mice in 10 ng/mL TGF-β1 group were significantly higher than those in blank control group (with t values from 4.895 to 14.870, P<0.05 or P<0.01).

CONCLUSIONSThe interaction between P311 and TGF-β1 in murine fibroblasts exists and it may enhance the differentiation of fibroblasts in combination.

Actins ; metabolism ; Animals ; Cell Differentiation ; Cells, Cultured ; Collagen Type I ; metabolism ; Fibroblasts ; cytology ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Nerve Tissue Proteins ; genetics ; metabolism ; RNA, Messenger ; Transfection ; Transforming Growth Factor beta1 ; metabolism