OBJECTIVE: To analysis the biological characteristics of human fibroblasts transfected by human telomerase reverse transcriptase (hTERT) eucaryotic expression plasmid pGRN145. METHODS: Fibroblasts from children’s foreskin were isolated and cultured in vitro, and the fibroblasts were transfected by pGRN145 with Lipofec-tAMINE PLUS Reagent. After strict screening of hygromycin B, the positive clones were subcultured. The telomerase activity was detected by RT-PCR and TRAP-PCR technique. The cell generation cycle and apoptosis rate were detected by flow cytometry to investigate the proliferative characteristics after transfection, and the chromosome karyotype of transformed cells was analyzed. The collagen secreted by transformed cells was detected by immunohistochemical staining. RESULTS: The morphological properties of fibroblasts did not change obviously after transfection. There were telomerase activity in transfected fibroblasts, while it could not be detected in pre-transfection fibroblasts. The cell generation cycle had no obvious changes between pre-transfection and post-transfection. However, the apoptosis rate of transfected fibroblasts were decreased compared with that of pre-transfection. The fibroblasts transfected by pGRN145 maintained the normal diploid karyotype, as well as the cells could normally secret type I and III collagen. CONCLUSION: The human fibroblasts transfected by pGRN145 has telomerase activity with prolonged life span of culture, which preliminarily proves the availability of establishing standard seeding cell lines of tissue engineering by hTERT plasmid transfection techniques.
Objective To study the effects of dermal template on the biological behaviors of fibroblasts during wound healing. Methods A total of 120 rats were made fullthickness wound modes on the dorsum and divided into 4 groups,in group 1, the wounds were allowed to heal by contraction（ConT）；in group2, the wounds covered with fullthickness skin grafts( FTSG); in group 3, the wounds were with split thickness skin grafts (STSG); and ingroup 4, the wounds were covered by dermal regeneration template with overlying thin splitthickness autograft (ADMT).The specimens were obtained at one week, two weeks, four weeks, six weeks,and twelve weeks respectively. The expressions of α smooth muscle actin(αSMA，characteristic of MFB）,fibronectin(FN),integrin α2,β1 and transforming growth factor β1(TGF-β1) were examined by immunohistochemical analysis. Results Positive expression of α-SMA、FN、integrin α2β1 and TGF-β1 in ADMT groups was significantly lower than that in STSG group and ConT group, but higher than that in FTSG group（P＜0.05）. Conclusion Dermal regeneration template can inhibit the transformation of FB to MFB and restrain the expressionof FN,integrin α2,β1,and TGF-β1 in fibroblasts which might reduce thepossibility of hypertrophyic scaring, and improve wound healing.
Abstract To observe the effect of fibroblast growth factor (FGF) on wound healing, 50 mice were divided into 5 groups. On the back of every mouse, 2 wounds were made by operative cuts, one for experiment and the other for control. The wounds of the experimental group were covered with 0.5ml FGF solution (contented FGF 300 μg/ml, heparin 100 μg/ml), whereas the wounds of the control group were covered with 0.5ml 0.9% NaCl solution. All of the wounds were dressed by sterilized gauze, and received the same treatment once a day. After 1,3,5,7,10 days, the mice in every group were sacrificed and the tissues of the wounds were collected and prepared for microscopic examination. The results showed that the capillaries and fibroblasts in the experimental group were markedly increased and reached the peak 2～3 days earlier than those in the control group. It was suggested that FGF promoted the formation of granulation tissue and the wound healing.
Objective To construct a mammalian expression vector ofbasic fibroblast growth factor (bFGF) and to investigate the expression of bFGFin vitro and in vivo. Methods A mammalian expression vector pcDNA3.1/myc-His(-)C-bFGF was constructed with gene cloning technique. The mammalian expression system was prepared and purified. The expression of bFGF cDNAin cultured transfected cells was examined by RT-PCR and cell immunohistochemistry. The recombinant plasmids, pcDNA3.1/myc-His(-)C-bFGF and pCD2-VEGF121, were transferred into rabbit cervical muscle by direct injection of plasmid following electric pulses in vivo. The transferred gene expression and the biological effect were measured by use of histochemistry and immunohistochemistry analysis. Results The eukaryon expression system pcDNA3.1/myc-His(-)C-bFGF could express the target protein bFGF in vitro. The recombinant plasmids, pcDNA3.1/myc-His(-)C-bFGF and pCD2-VEGF121 were transferred into muscles flap in vivo successfully. The active proteins bFGF and VEGF121were expressed at high levels. Blood vessels increased significantly in the muscles, and blood circulation was improved by local angiogenesis. Conclusion Theeukaryon expression vector of bFGF is constructed and can be expressed successfully in vitro and in vivo. That is a primary preparation for the research on tissue transplantation and tissue engineering with bFGF gene therapy.
OBJECTIVE: To explore the effect of Fas/Apo-1 and Bcl-2 gene expression on mechanism of scar formation. METHODS: Immunohistochemical method was applied to defect the expression of Fas and Bcl-2 protein in fibroblasts from 10 cases with normal skin, 10 cases with hypertrophic scar and 10 cases with keloid. RESULTS: The positive expression rate of Bcl-2 protein in keloid was 83.2%, significantly higher than that in hypertrophic scar (38.6%), (P lt; 0.01), and the positive expression rate in hypertrophic scar and keloid was higher than that in normal skin (6.78%), (P lt; 0.01). But the positive expression rate of Fas/Apo-1 protein was 78.4% in normal skin 80.4% in hypertrophic scar, 84.4% in keloid respectively, which showed no significant difference among them (P gt; 0.05). CONCLUSION: Bcl-2 gene but Fas gene may take part in the formation of pathologic scar.
Objective To investigate the effect of vanadate on prol iferation and collagen type I production of rat medial collateral l igament (MCL)fibroblasts. Methods A total of 12 adult male SD rats were included, weighing 350-375 g. MCL was cut into small pieces (1 mm × 1 mm × 1 mm) in aseptic conditions, and then placed and cultured in culture chambers. Fibroblasts were passaged with 0.25% trypsin. The vanadate (0, 1.0, 2.5, 5.0 ng/mL) was added in the 3rd passage MCL fibroblasts, respectively, and the samples were divided into 4 groups (0, 1.0, 2.5, 5.0 ng/mL groups). MTT was used to measure the cell prol iferation. The production of collagen type I was measured by RT-PCR and ELISA. Twelve samples in each group were measured. Results In fibroblast prol iferation, the absorbency values of the 1.0, 2.5, 5.0 ng/mL groups were significantly different from that of the 0 ng/mL group (P lt; 0.05). The absorbency values of the 0, 1.0, 2.5, and 5.0 ng/mL groups were 0.213 ± 0.016, 0.327 ± 0.023, 0.449 ± 0.137, and 0.561 ± 0.028, respectively. In collagen secretion, vanadate in 1.0, 2.5, 5.0 ng/mL groups could significantly induce the production of collagen type I (P lt; 0.05) ina dose-dependent manner. The expressions of collagen type I of 0-5 ng/mL groups were 0.47 ± 0.02, 0.51 ± 0.03, 0.60 ± 0.01, and 0.72 ± 0.02, respectively. There was significant difference between the 1.0, 2.5, 5.0 ng/mL groups and 0 ng mL group (P lt; 0.05). RT-PCR displayed a dramatic increase of band density. The ratio of band density in the 0-5 ng mLgroups was 1.37 ± 0.76, 1.97 ± 0.53, 2.41 ± 0.94, and 2.73 ± 0.82, respectively. The gene expression of collagen type I in the 1.0, 2.5 and 5.0 ng/mL groups was higher than that in the 0 ng/mL group, and there was significant difference (P lt;0.05). There were statistical significant differences among 1.0, 2.5 and 5.0 ng/mL groups in each index mentioned above.Conclusion Vanadate can effectively induce the prol iferation of fibroblasts and the production of collagen type I in vitro, which may provide a new approach to the treatment of MCL injury.
ObjectiveTo investigate relationship between ultrastructural changes and expression of basic fibroblast growth factor of diabetic retinopathy in rats.MethodsDiabetes was induced in rats with a single injection of streptozotocin (STZ) and divided into normal control group and 1- , 3- and 5- month diabetes group. The paraffin slide was observed by in-situ hybridization and immunohistochemistry, and retinal ultrastructure was examined by transmission electron microscopy.ResultsNo change of retinal ultrastructure was found in the control group. Different degrees of ultrastructure lesion were found in 1-month diabetic rats with fragmental increase of thickness of basement membrane, swelling of endothelial cells and obvions fingerlike processes in the capillary cavity, disconcentration of heterochromatin both in endothelium and pericyte, and swelling and degeneration of mitochondrion. The edema of endothelial cells of 3-month diabetic rats was more serious than that of 1month ones, and the capillary cavity was nearly occluded. In 5-month diabetic rats, the basement membrane was unevenly thickened, or obviously split. The positive rate of in-situ hybridization in 3-month diabetic rats was 77.8% while the positive rate of immunohistochemical stain was 55.6%, which increased to 88.9% in 5-month diabetic rats.ConclusionsThe occurrence of the ultrastructural changes in STZ rats with diabetic retinopathy is earlier than that of the expression of bFGF.(Chin J Ocul Fundus Dis, 2003,19:348-351)
OBJECTIVE To study the early protective effects of basic fibroblast growth factor(bFGF) on the experimental acute spinal cord injury. METHODS Thirty-four SD rats were randomly divided into three groups, and were subjected to contusion of thoracolumbar spinal cord. A thin plastic tube was placed in subarachnoid space below the injury level for perfusion. The bFGF-treated rats were received 20 microliters bFGF(containing bFGF 100 U) at once, 30 min, 1, 2, 3, 4, 6, 12, 24 and 48 hours after injury, and an equal volume of normal saline was given to the control group at the same time. The injured spinal cord was detected by morphological observation and biochemical index after injury. RESULTS The degree of ionic disorder in bFGF-treated rats was significantly ameliorated and the contents of H2O were also markedly decreased. The morphological finding showed that the damages of gray and white matter in bFGF-treated rats were slighter than those of saline-treated rats. CONCLUSION bFGF has some protective effects on the secondary lesion of early spinal cord injury in rats.
ObjectiveTo investigate the effects of adipose-derived stem cell released exosomes (ADSC-Exos) on wound healing in diabetic mice.MethodsThe ADSCs were isolated from the adipose tissue donated by the patients and cultured by enzymatic digestion. The supernatant of the 3rd generation ADSCs was used to extract Exos (ADSC-Exos). The morphology of ADSC-Exos was observed by transmission electron microscopy. The membrane-labeled proteins (Alix and CD63) were detected by Western blot, and the particle size distribution was detected by nanoparticle tracking analyzer. The fibroblasts were isolated from the skin tissue donated by the patients and cultured by enzymatic digestion. The 5th generation fibroblasts were cultured with PKH26-labeled ADSC-Exos, and observed by confocal fluorescence microscopy. The effects of ADSC-Exos on proliferation and migration of fibroblasts were observed with cell counting kit 8 (CCK-8) and scratch method. Twenty-four 8-week-old Balb/c male mice were used to prepare a diabetic model. A full-thickness skin defect of 8 mm in diameter was prepared on the back. And 0.2 mL of ADSC-Exos and PBS were injected into the dermis of the experimental group (n=12) and the control group (n=12), respectively. On the 1st, 4th, 7th, 11th, 16th, and 21st days, the wound healing was observed and the wound healing rate was calculated. On the 7th, 14th, and 21st days, the histology (HE and Masson) and CD31 immunohistochemical staining were performed to observe the wound structure, collagen fibers, and neovascularization.ResultsADSC-Exos were the membranous vesicles with clear edges and uniform size; the particle size was 40-200 nm with an average of 102.1 nm; the membrane-labeled proteins (Alix and CD63) were positive. The composite culture observation showed that ADSC-Exos could enter the fibroblasts and promote the proliferation and migration of fibroblasts. Animal experiments showed that the wound healing of the experimental group was significantly faster than that of the control group, and the wound healing rate was significantly different at each time point (P<0.05). Compared with the control group, the wound healing of the experimental group was better. There were more microvessels in the early healing stage, and more deposited collagen fibers in the late healing stage. There were significant differences in the length of wound on the 7th, 14th, and 21st days, the number of microvessels on the 7th and 14th days, and the rate of deposited collagen fibers on the 14th and 21st days between the two groups (P<0.05).ConclusionADSC-Exos can promote the wound healing in diabetic mice by promoting angiogenesis and proliferation and migration of fibroblasts and collagen synthesis.
This study is aimed to investigate the effects of mechanical stretch on the expression of transforming growth factor-β1 (TGF-β1) and fibroblast growth factor-2 (FGF-2), and the signaling pathway in human bronchial epithelioid (16HBE) cells under mechanical stretch. Using loading device with flexible substrate (FX-4000T) to stretch 16HBE cells, we found that the stretching elongation was 15%, at frequency of 1 Hz, stretching for 0.5 h, 1 h, 1.5 h and 2 h. Choosing the higher expression of TGF-β1, FGF-2 and Ca2+ group to carry out intervention experiments, we used the cells pretreated with canonical transient receptor potential 1 (TRPC1) channel antagonist SKF96365, protein kinase C (PKC) inhibitor HA-100, and thereafter mechanical stretch to interpose. Compared with those in the blank control group, TGF-β1 and FGF-2' protein and mRNA, intracellular Ca2+ fluorescence intensity were higher, and the differences were statistically significant (P < 0.05) at the 4 time points, 0.5 h, 1 h, 1.5 h and 2 h. At 0.5 h, the increasing rate was the highest. TGF-β1 protein and mRNA, FGF-2 protein and mRNA, intracellular Ca2+ luorescence intensity in the stretch+SKF96365 and stretch+HA-100 intervented group were decreased, the differences were statistically significant than those in 0.5 h stretch group (P < 0.05) without intervention. The expression of TGF-β1, FGF-2 was up-regulated in 16HBE cells under mechanical stretch, PKC, TRPC1, and Ca2+ may participate in the signal path.