OBJECTIVETo explore transient expression of the eukaryotic expression plasmid carrying human platelet-derived growth factor-B (hPDGF-B) in gingival fibroblasts of Beagle dog.

METHODSPlasmid carrying hPDGF-B (EX-A0380-M03) was amplified and identified, and then transfected into gingival fibroblasts of Beagle dog. Reverse transcription polymerase chain reaction (RT-PCR), immunocytochemistry, enzyme-linked immunosorbent assay (ELISA) and Western bolt were choose to detect the expression of hPDGF-B.

RESULTSTarget gene carried by EX-A0380-M03 was hPDGF-B. Green fluorescence protein (GFP) expressed by transfected gingival fibroblasts was observed under inverted phase contrast fluorescence microscope (IPCFM) (after 24 hours) and the transfection efficiency was 18%-38% (after 48 hours). Serials other methods (RT-PCR, immunocytochemistry, and ELISA) mentioned above also convinced that cells expressed hPDGF-B, and the protein that was a kind of fusion protein composed of PDGF-BB and GFP was identified by Western blot.

CONCLUSIONEukaryotic expression plasmid carrying hPDGF-B was transfected into gingival fibroblasts successfully, and a kind of fusion protein was expressed.

Animals ; Dogs ; Fibroblasts ; Gingiva ; Plasmids ; Platelet-Derived Growth Factor ; Proto-Oncogene Proteins c-sis ; Transfection

OBJECTIVETo construct a recombinant human platelet-derived growth factor-B (PDGF-B) adenoviral vector and to transfect it into human periodontal ligament stem cells (PDLSC).

METHODSThe recombinant plasmid pAd-PDGF-B was constructed by homologous recombination and confirmed by restriction endonucleases digestion. Recombinant adenovirus was packaged in HEK293 cells. PDLSC were transfected with recombinant adenovirus and PDGF-B expression was confirmed. Expression of collagen type I gene was determined by quantitative analysis of the products of RT-PCR. The cell proliferation was determined with MTT colorimetric assay.

RESULTSThe recombinant plasmid pAd-PDGF-B was confirmed by restriction endonucleases digestion. EGFP expression was observed on the third day after transfecting, and the expression of PDGF-B was detected. Immunohistochemical methods revealed that PDGF-B was expressed in PDLSC. Levels of expression of collagen type I gene were increased significantly by transfer of the exogenous PDGF-B gene to PDLSC. At the same time, findings indicated that Ad-PDGF-B stimulated PDLSC proliferation. MTT assay indicated the absorbance of PDLSC by stimulating with Ad-PDGF-B was (0.68 +/- 0.02), P < 0.01.

CONCLUSIONSUsing the AdEasy system, the human PDGF-B recombinant adenovirus can be rapidly obtained. These results indicate that recombinant adenoviruses encoding PDGF-B transgenes could modulate proliferative activity of PDLSC, enhance the high expression of collagen type I and lay the foundation for periodontal tissue regeneration and dental implant gene therapy.

Adenoviridae ; genetics ; Cells, Cultured ; Genetic Vectors ; Humans ; Periodontal Ligament ; cytology ; Proto-Oncogene Proteins c-sis ; genetics ; Stem Cells ; Transfection

Objective: To investigate the clinicopathological and cytogenetic features of cryptic COL1A1-PDGFB fusion dermatofibrosarcoma protuberans (CC-DFSP). Methods: Three cases of CC-DFSP diagnosed in West China Hospital, Sichuan University, Chengdu, China from January 2021 to September 2021 were studied. Immunohistochemistry for CD34 and other markers, fluorescence in situ hybridization (FISH) for PDGFB, COL1A1-PDGFB and COL1A1, next-generation sequencing (NGS), reverse-transcriptase polymerase chain reaction (RT-PCR) and Sanger sequencing were performed. Results: There were three cases of CC-DFSP, including two females and one male. The patients were 29, 44 and 32 years old, respectively. The sites were abdominal wall, caruncle and scapula. Microscopically, they were poorly circumscribed. The spindle cells of the tumors infiltrated into the whole dermis or subcutaneous tissues, typically arranging in a storiform pattern. Immunohistochemically, the neoplastic cells exhibited diffuse CD34 expression, but were negative for S-100, SMA, and Myogenin. Loss of H3K27me3 was not observed in the tumor cells. The Ki-67 index was 10%-15%. The 3 cases were all negative for PDGFB rearrangement and COL1A1-PDGFB fusion, whereas showing unbalanced rearrangement for COL1A1. Case 1 showed a COL1A1 (exon 31)-PDGFB (exon 2) fusion using NGS, which was further validated through RT-PCR and Sanger sequencing. All patients underwent extended surgical resection. Except for case 3 with recurrence 2 years after surgical resection, the other 2 cases showed no recurrence or metastasis during the follow-up. Conclusions: FISH has shown its validity for detecting PDGFB rearrangement and COL1A1-PDGFB fusion and widely applied in clinical detection. However, for cases with negative routine FISH screening that were highly suspicious for DFSPs, supplementary NGS or at least COL1A1 break-apart FISH screening could be helpful to identify cryptic COL1A1-PDGFB fusions or other variant fusions.
Female ; Humans ; Male ; Collagen Type I, alpha 1 Chain ; Dermatofibrosarcoma/pathology* ; In Situ Hybridization, Fluorescence ; Oncogene Proteins, Fusion/genetics* ; Proto-Oncogene Proteins c-sis/genetics* ; Skin Neoplasms/pathology* ; Adult

PURPOSE: Platelet transplantation is a novel therapeutic strategy for acceleration of wound healing. When applying platelets, efficacy of adding thrombin to stimulate growth factor release from platelets has already been proven. However, no quantitative data of the thrombin treatment has been reported. The purpose of this study is to determine the optimal thrombin concentration to maximize growth factor release of platelets. In particular, this study was designed to quantify levels of platelet derived growth factor(PDGF)-BB, which is a major growth factor containing in the platelets, in vitro. METHODS: Fresh platelets were obtained from a blood bank. They were suspended in DMEM/F-12 and incubated with thrombin of various concentrations. The concentrations of thrombin tested were 0, 6.25, 12.5, 25, 50, 100, 200, and 400IU/mL. After 30 minutes, 1, 3, 5, and 7 days, the levels of PDGF-BB were measured using enzyme linked immunosorbent assay. Platelets from four donors were included in this study. Each sample was tested in triplicate and the mean value was used as a data for each sample. RESULTS: The addition of thrombin increased the level of PDGF-BB. Increases in storage time of platelets resulted in decreased levels of PDGF-BB. Higher levels of PDGF were detected in consort with increased thrombin concentrations. However, there was no significant difference between samples of 200 and 400IU/mL concentrations. CONCLUSION: The results indicate that adding thrombin accelerates the release of growth factors from platelets and the optimal thrombin concentration to maximize this function is 200IU/mL.
Acceleration ; Blood Banks ; Blood Platelets ; Enzyme-Linked Immunosorbent Assay ; Humans ; Intercellular Signaling Peptides and Proteins ; Proto-Oncogene Proteins c-sis ; Thrombin ; Tissue Donors ; Transplants ; Wound Healing

OBJECTIVETo acquire sufficient PDGF-BB protein and provide the basis for the further studies of its role on the fracture healing and trauma reconstruction and its clinical applications.

METHODSConstructed the prokaryotic expression vector pQE-PDGF-B with the gene rearrangement technique, and the monomeric form of recombinant PDGF-B expressed in E. coli M15.

RESULTSPDGF-B mature peptide gene was inserted into prokaryotic expression vector pQE30, which was confirmed by PCR, enzyme digestion and sequencing identification; the expressed products of pQE-PDGF-B in E. coli showed a single protein on SDS-PAGE, and their expression level was about 15% of the total bacterial protein. The molecular weight of the purified PDGF-B protein was about 15 KDs on SDS-PAGE.

CONCLUSIONSThe construction of recombinant plasmid and preparation of the monomeric protein of PDGF-B provides a solid foundation for further studying the function of PDGF-BB and producing biologically PDGF-BB protein.

Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; In Vitro Techniques ; Platelet-Derived Growth Factor ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-sis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection

Animals ; Artifacts ; Bicuspid ; Blood Platelets ; Dogs ; Intercellular Signaling Peptides and Proteins ; Mandible ; Molar ; Nitrogen Mustard Compounds ; Osseointegration ; Osteogenesis ; Proto-Oncogene Proteins c-sis ; Titanium ; Transplants

OBJECTIVETo investigate the synergistic effect of transforming growth factor-beta1 (TGF-beta1) and platelet-derived growth factor-BB (PDGF-BB) on the expression of integrin beta3, in periodontal membrane of rat orthodontic tooth.

METHODSAn orthodontic tooth movement model was established. Up to 32 experimental rats were randomly divided into four groups according to a random number table. The four groups were injected with 1% PBS, TGF-beta1 (5 ng), PDGF-BB (10 ng), and combined TGF-beta1 (5 ng) and PDGF-BB (10 ng) in the buccal submucosal, respectively. The volume injected in each group was 0.1 mL. The animals were then sacrificed on the 10th day. The left maxillary first molar and periodontal tissue were taken. Different expressions of integrin beta3 were detected in periodontal tissues through immunohistochemistry. Mean optical density (OD) values of the positive fields were examined. The data obtained were analyzed through ANOVA. The data followed normal distribution, and were compared via t-test.

RESULTSCompared with the control groups, the expression of integrin beta3 was higher in the experimental groupin tension sides (P < 0.01). Significant differences in tension sides between the single-injection groups and the combined group were observed (P < 0.01). Compared with the control groups, the expression of integrin beta3 was higher in the experimental group in compression sides (P < 0.05). In addition, there was no significant differences in compression sides between the single-injection groups and the combined group (P > 0.05).

CONCLUSIONIn terms of local regulatory factors, TGF-beta1 combined with PDGF-BB enhance the expression of integrin beta3 in the periodontal membrane and accelerate periodontal remodeling. The synergistic effect of the two growth factors is better than the single growth factor.

Animals ; Integrin beta3 ; Molar ; Periodontal Ligament ; Platelet-Derived Growth Factor ; Proto-Oncogene Proteins c-sis ; Rats ; Tooth Movement Techniques ; Transforming Growth Factor beta1

Alcohols ; Animals ; Disease Models, Animal ; Female ; Kidney Diseases ; chemically induced ; metabolism ; pathology ; Liver Diseases, Alcoholic ; metabolism ; pathology ; Proto-Oncogene Proteins c-sis ; biosynthesis ; Rats ; Rats, Wistar

OBJECTIVETo study the effect of PDGF on dermal blood vessel reconstruction by transplanted tissue-engineering skin containing PDGF-B gene to rats.

METHODSThe recombined eukaryotic expression vector, pcDNA3.1-hPDGF-B, was constructed and transfected into fibroblasts mediated by LipofectAMINE. Keratinocytes + acellular dermal matrix (group A), keratinocytes + acellular dermal matrix + fibroblasts (group B), keratinocytes + acellular dermal matrix + fibroblasts with PDGF gene (group C) were recombined respectively, then transplanted them to rat dorsum and evaluated the reconstruction of blood vessels in the dermis after 2, 4, 6 week postoperation.

RESULTSIn 2-4 weeks after skin grafting the vascularization rate in group C was higher than that of group B and group A. The vascularization rates in all groups had no significant differences in six weeks (P > 0.05).

CONCLUSIONPDGF-B gene plays an important role in reconstruction of blood vessels in the dermis at early tissue-engineering skin grafting, which ensures the take of grafted tissue-engineering skin.

Acellular Dermis ; Animals ; Male ; Neovascularization, Physiologic ; Proto-Oncogene Proteins c-sis ; genetics ; Rats ; Rats, Sprague-Dawley ; Skin ; blood supply ; Skin Transplantation ; Skin, Artificial ; Swine ; Tissue Engineering ; methods ; Transfection

OBJECTIVETo observe the effects of the grafting of a platelet-derived growth factor gene-modified artificial composite skin on rat wounds with full thickness defect.

METHODSPlatelet derived growth factor-B (PDGF-B) eukaryotic expression plasmid was constructed, and the fibroblasts were transfected with it by liposome mediation. Artificial composite skins 1 and 2 were constructed respectively. The skin1 was composed of keratinocyte, porcine acellular dermal matrix and PDGF-B gene-transfected fibroblasts while the skin 2 contained keratinocyte, porcine acellular dermal matrix and fibroblasts. The two kinds of composite skin were grafted onto wounds on the rat back to form composite skin group 1 (C1) and 2 (C2), respectively, with 18 rats in each group. Eight rats with wounds without treatment served as control (C) group. The survival rate of the composite skin was observed at 2 post-operative weeks (POWs). The rat wounds were examined grossly on 2, 4 and 6 POWs for the calculation of wound contraction rate. Wound tissue samples were harvested for histological examination.

RESULTS(1) Up to 2 POWs, 14 grafts in C1 group survived completely, 3 with partial survival and 1 failure. In C2 group, 10 skin grafts survived completely, 4 with partial survival and 4 failures. (2) A scab was formed in the wound at 2 POW in C group. The surface of the grafted skin in C1 group was smooth, elastic, and showed good anti-friction properly, and it was better in quality compared with that in other two groups at 6 POW. (3) The wound contraction rate of the grafts in C group of rats was higher than that in C1 and C2 groups at 2, 4 and 6 POWs, while that in C1 was lower than that in C2 group. (4) Capillary formation was more intense in the grafted skins in C1 group at 2 POWs, and the epithelia differentiated well into 7 to 10 layers of epithelial cells with compact and orderly arrangement and evenly distributed fibrous tissue at 6 POWs.

CONCLUSIONRepair of the wound with artificial composite skin containing PDGF-B gene could improve the quality of wound healing.

Animals ; Cells, Cultured ; Female ; Humans ; Proto-Oncogene Proteins c-sis ; genetics ; Rats ; Rats, Sprague-Dawley ; Skin Transplantation ; methods ; Skin, Artificial ; Swine ; Tissue Engineering ; Transfection ; Wound Healing