OBJECTIVE This paper aims to explore the new method of continuous delivery of epidermal growth factor to wounds by transfected fibroblasts to promote wound repair. METHODS It was constructed a novel chimeric expression plasmid in which the biologically active portion of the human epidermal growth factor (EGF) gene was fused in-frame to the human granulocyte colony-stimulating factor signal sequence. RESULTS Clonally selected human fibroblasts transfected with this construct could secrete biologically active EGF. After the transplantation of irradiated gene-transfected fibroblasts suspended in fibrin glue to murine full-thickness wounds, EGF could be demonstrated for at least seven days in the wounds, slowly decreasing from initially 470 ng/L to 140 ng/L in 7 days. No EGF was found in the wound at 14 days. CONCLUSION A single application of irradiated EGF gene transfected fibroblasts to wounds can continuously deliver the transgene in vivo and can be used to administer drugs to the wound bed during the crucial first seven days of wound-healing.
Objective To investigate the possible mechanism of the fibroblasts inducing the vascularization of dermal substitute. Methods Fibroblasts were seeded on the surface of acellular dermal matrix and cultivated in vitro to construct the living dermal substitute. The release of interleukin 8 (IL 8) and transfonming growth factor β 1(TGF β 1) in culture supernatants were assayed by enzyme linked immunosorbent assay, the mRNA expression of acid fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) were detected by RT-PCR. Then, the living substtute was sutured to fullth ickness excised wound on BALBouml;C m ice, and the fate of fibroblast w as observed by using in situ hybridizat ion. Results Fibroblasts cultured on acellular dermalmat rix p ro liferated and reached a single2layer confluence. Fibroblasts could secret IL 28 (192. 3±15. 9) pgouml;m l and TGF-B1 (1. 105±0. 051) pgouml;m l. There w as the mRNA exparession of aFGF and bFGF. Fibroblasts still survived and proliferated 3 weeks after graft ing. Conclusion Pept ides secreted by fibroblasts and its survival after graft ing may be relat ive to the vascularizat ion of the dermal subst itute.
OBJECTIVE :To investigale effect of subretinal fluld(SRF)on proliferalion of the cellular elements of PVR.
METHOD:The effect of SRF of 28 patients with rhegmatogenous retinal detachment proliferation of the cultured human retinal pigment epithelial cells(RPE),retinal glial cells (RG),and fibroblast (FB)was observed and detected by the methods of cell-counting and 3H-TdR in DNA synthesis.
RESULTS:The range of proliferatinn-stimulating activity was 52.5%~233.3%,
36.4% ~ 177.8%，55.4% ~277.8% above the baseline in 1:10 dilution of these 3 kinds ,of cellular elements,and there was no significant difference among them.
CONCLUSION ;The stimulating effect of SRF on the cellular proliferation was thougt to be due to the actions from certain growth factors.
（Chin J Ocul Fundus Dis,1996,12: 233-235）
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 Influence of irradiation and phenytoin sodium on modulatory activities of wound fluid on proliferation of fibroblasts and collagen synthesis was studied. METHODS The male Wistar rats were used in this study. The rats were divided into irradiated and non-irradiated groups, and in each of them it was subdivided into phenytoin group and control. A 7 cm long incisional wound was made on the back of each rat, in which a polyvinyl alcohol sponge (PVAS) with a size of 1.0 cm x 0.4 cm was implanted into the wound and the wound was sutured up. The PVAS was prepared by rinsing in running water over night and then was boiled for 30 minutes. Before implantation, the sponge was immersed in phenytoin sodium solution (10 mg/l ml) or normal saline (as control). From each wound the wound fluid and fibroblasts were collected. The methods of incorporation of 3H were adopted to assess the proliferation of fibroblasts and synthesis of collagen. RESULTS It was shown that proliferation of fibroblasts and collagen synthesis were stimulated by wound fluid remarkably on 5 to 8 days after wounding, and that 6 Gy to total-body irradiation wound decrease this effect. It was also noted that topical phenytoin sodium increased the modulatory activity of wound fluid irrespective of being irradiated or not. CONCLUSION It could be drawn that, after total-body irradiation, stimulation of hyperplasia of fibroblasts and collagen synthesis by wound fluid was markedly lowered indicating the total-body irradiation resulted in changes of local conditions of the wound which was unbenefitted to repair of tissue cells, while phenytoin sodium could enhance the stimulating action of wound fluid on proliferation of fibroblasts and synthesis of collagen which was beneficial to wound healing.
OBJECTIVE: To fabricate artificial human skin with the tissue engineering methods. METHODS: The artificial epidermis and dermis were fabricated based on the successful achievements of culturing human keratinocytes(Kc) and fibroblasts (Fb) as well as fabrication of collagen lattice. It included: 1. Culture of epidermal keratinocytes and dermal fibroblasts: Kc isolated from adult foreskin by digestion of trypsin-dispase. Followed by comparison from aspects of proliferation, differentiation of the Kc, overgrowth of Fb and cost-benefits. 2. Fabrication of extracellular matrix sponge: collagen was extracted from skin by limited pepsin digestion, purified with primary and step salt fraction, and identified by SDS-PAGE. The matrix lattice was fabricated by freeze-dryer and cross-linked with glutaraldehyde, in which the collagen appeared white, fibrous, connected and formed pores with average dimension of 180 to 260 microns. 3. Fabrication artificial human skin: The artificial skin was fabricated by plating subcultured Kc and Fb separately into the lattice with certain cell density, cultured for one week or so under culture medium, then changed to air-liquid interface, and cultured for intervals. RESULTS: The artificial skin was composed of dermis and epidermis under light microscope. Epidermis of the skin consisted of Kc at various proliferation and differentiation stages, which proliferated and differentiated into basal cell layer, prickle cell layer, granular layer, and cornified layer. Conifilament not only increased in number, but also gathered into bundles. Keratohyalin granules at different development stages increased and became typical. The kinetic process of biochemistry of the skin was coincide with the changes on morphology. CONCLUSION: Tissue engineered skin equivalent has potential prospects in application of repairing skin defect with advantages of safe, effective and practical alternatives.
Objective To investigate an effect of compressive stress on proliferation and apoptosis of human hyperplastic scar fibroblasts（HSFb） in vitro. Methods HSFb were obtained from a 20 year old female patient who developed a hyperplastic scar 3 months after operation for a largearea burn. HSFb were isolated, and were cultured in vitro with the simplified airpressure controlled cellculture instrument, and then they were randomly divided into the following 8 groups: the control group (no stress) and the 7 continuous compressive stress groups, which respectively underwent the 5, 10, 15, 25, 50, 100 and 150mmHg(1mmHg=0.133 kPa) pressure treatment for 4d ays. The absorbance (A) of the cell and the inhibition ratio (IR) of the cell proliferation were determined by the MTT assay, the cell growth cycle was determined by the flow cytometer, and the cell apoptosis was observed by the AnnexinV binding with PI labeling method. Results In the 5, 10, 15, 25, 50, 100 and 150mmHg pressure groups and the control group, the A values of the cells were 0.228±0.004, 0.226±0.003, 0.213±0.005, 0.180±0.005, 0.172±0.007, 0.165±0.004, 0.164±0.004 and 0.230±0.005, respectively; the IRs of the cell proliferation were 0.8%，2.0%，7.3%，21.7%，252%， 28.2% and 0, respectively;the ratios of the cells in G1 were 71.80％±0.44％, 72.32％±0.40％, 74.56％±1.01％, 82.82％±2.76％, 86.77％±2.06％, 88.23％±1.27％, 89.11％±1.74％ and 71.6％±0.49％,respectively; the cell apoptosis ratios were 4.22％±0.49％, 5.12％±0.74％ , 8.58％±0.79%, 19.28％±1.40%, 25.60％±1.21%, 3580％±2.39%, 36.18％±2.38% and 4.00％±0.36％, respectively. In the 5 and 10mmHggroups there were no statistically significant differences in all the above parameters when compared with those in the control group (P＞0.05); however, in the 15, 25,50, 100 and 150mmHg groups there were statistically significant differences in the above parameters when compared with those in the control group (P＜0.05). Furthermore, in the 10, 15, 25 and 50 mmHg groups, there were statistically significant differences in the Avalue of the cells and the ratios of the cells in G 1 when compared with each other (P＜0.01). By contrast, there were no statistically significant differences in the 50, 100 and 150 mmHg groups when compared witheach other (P＞0.05). In the 10, 15, 25, 50 and 100mmHg groups there werestatistically significant differences in the cell apoptosis ratio when comparedwith each other (P＜0.01). In the 100 and 150 mmHg groups there were no such statistically significant differences when compared with each other (P＞0.05).Conclusion A continuous compressive stress when given properly can have a combined effect of the proliferation inhibition and the apoptosis promotion on HSFb in vitro, and this kind of combined effects can becomeone of the important mechanisms for the pressure therapy in treating hyperplastic scar.
Objective To evaluate the effects of cryopreserved cultured allogenic dermal fibroblasts on angiogenesis and fibroplasia while artificial dermis grafting by spraying the cells on the graft bed.Methods Full thickness skin defect was made on the back of Wistar rat, fibroblasts mixed into fibrin glue (fibroblast group) and same amount fibrin glue (control group) were sprayed separately between the wound bed and artificial dermis in cell density of 1.0×105 cells/cm2 before the artificial dermis was grafted. On day 5 after grafting, the graft and surrounding tissue were examined histologically for angiogenesis and fibroplasia in the dermis and wound bed with hematoxylin eosin stain, VEGF antibody stain, Masson’s trichrome stain and India ink stain. Evans blue perfusion methodwas also used for detecting the angiogenesis quantitatively.Results In the fibroblast group, the angiogenesis of graft bed was significantly accelerated onday 5 after grafting; the numbers of the newly formed capillaries were 9.64±2.36/HP in the fibroblast group and 3.88±1.62/HP in the control group (P＜0.05). And on day 10 after grafting the angiogenesis was accelerated not only in graft bed but also in the artificial dermis when compared with control group, the newly formed capillaries network was clearly observed in the artificial dermis. Otherwise, the synthesis of collagen was increased in the dermis on day 10 after grafting in the fibroblast group when compared with control group. The immunoreactivity of VEGF antibody in the fibroblast group also showed a ber expression than that in control group on day 5 after grafting, the numbers of positive cells were 46.04±8.90/HP in the fibroblast group and 30.08±7.76/HP in the control group(P＜0.05).Conclusion Transplantation of cryopreserved dermal fibroblasts while artificial dermis grafting can accelerate the angiogenesis and fibroplasia in the artificial dermis and graft bed, thereby accelerate the formation of dermallike tissue in the artificial dermis.
Objective To compare the efficiency of epidermis cell culture between big graft method and small strip method. Methods The big graft method was to cut the skin tissue reticularly from dermis layer while the epidermis were not cut off. After it was digested fully in trypsin, theepidermis was separated from skin and was used to culture epidermal cells. The small strip method was routine. The time to cut the skin and to separate the epidermis was recorded, and the number and quality of cells were compared between two methods. Results It took 8-10 minutes to cut an area of 5 cm2 skin into small strips and 1-2 minutes into big grafts. It took 10-15 minutes to separate the epidermis from the same area skin by small strip method and 2 minutes by big graft method. The cells showed better vigor and its number was more by big grafts than by small strips.The chance of fibroblast contamination was reduced obviously. Conclusion The big graft method is simpler than the small strip method and can culture more epidermis cells with less chance of fibroblast contamination.
Objective To establish a method of constructing skin-equivalents (SE) by the hair follicle stem cells (HFSC) and the fibroblasts. Methods The K19 immunostainning was employed to localize the HFSC in the human scalp from the cosmetic surgery. The isolated HFSC through the enzyme digestion were seeded on the dermal equivalent (DE) formed by polymerization of the fibroblasts and collagen. After being cultured between the air-liquid interface for 14 days, SE were harvested and used for an evaluation. Results HFSC were located mainly in the outer root sheath in the hair follicle. Based on DE, the growing HFSC could build a fullydeveloped and multilayered epidermis with the basal membrane formedb etween the epidermis and the dermis. The fibroblasts were active and spread evenly in the collagen matrix. Conclusion The hair follicle stem cells located in the outer root sheath can be successfully used to construct skin-equivalents in vitro and have a promising clinical use in the treatment.
