Objective To determine whether fibroblasts can be used to promote endochondral bone formation in vivo by transfer of human bone morphogenetic protein-2(hBMP-2) into fibroblasts. Methods pcDNA3-hBMP-2 was constructed by use of gene clone and recombined technique.NIH3T3 fibroblasts were transfected with pcDNA3hBMP-2. The positive cell clones were selected with G418. In NIH3T3 fibroblaststransferred with pcDNA3-hBMP-2, the expression of hBMP-2 was determined by in situ hybridization and immunohistochemical analysis; alkaline phosphatase activity was measured. hBMP-2producing fibroblasts were implanted into nude mouse muscle to observe endochondral bone formation in vivo. Results pcDNA3-hBMP-2 was successfully constructed. In NIH3T3 fibroblasts transfected with -pcDNA3-hBMP-2,the BMP-2 expression was stable; alkaline phophatase activity was much higher than that in nontransfectedNIH3T3 cells. Endochondral bone formation invivo was observed at the site of implantation 4 weeks later.Conclusion Fibroblasts transfected by hBMP-2 gene can be used to promote endochondral bone formation in vivo.
This experiment was designed to observe the proliferative effects of platelet derived wound healing factor (PDWHF) of different concentrations on fibroblasts from rat wounds and on epithelial from human wounds. Cultured fibroblasts from rat wound and epithelia from human wound were randomly divided into three groups. (1) In blank control, the cells were treated with basic medium (BM, contains 1640/0.5% FCS); (2) the positive control, the cells were treated with 1640/10% FCS and (3) in the PDWHF group, the cells were treated respectively with BM/1% PDWHF, BM/3% PDWHF, BM/5% PDWHF, BM/7% PDWHF, BM/10% PDWHF, BM/12% PDWHF, respectively. The Cells were collected after 48 hours culturing with BM or PDWHF, and the cell proliferation was measured by MTT method according to the OD values. The result showed that the PDWHF could remarkably enhance the proliferation of fibroblasts and epithelial cells when its concentration was between 1% and 7%, which was obviously higher than that of the blank control (P lt; 0.01). When the concentration of PDWHF reached 10%, its proliferative effect was not remarkable when compared with the blank control, When the concentration of PDWHF reached 12%, it showed inhibitory effect on fibroblasts and manifested no obvious inhibitory effect on epithelial cells. It was concluded that the PDWHF was a combination of a variety of growth factors. In a certain range of concentration, the PDWHF might effectively promote the proliferation of fibroblasts and epithelial cells. Howerve, when its concentration reached to relatively higher level, its effect was not remarkable any more, or even showed inhibitory effect on cell proliferation.
Objective To construct human brain-derived neurotrophic factor retroviral vector-pLXSN (hBDNFpLXSN), and to evaluate the bioactivity of hBDNF. Methods The genome mRNA was extracted from embryonic brain tissue of a 5-month-old infant, the hBDNF gene sequence was obtained with RT-PCR technology, and hBDNF-pLXSN constructed in vitro was used to infect the fibroblasts (NIH/3T3). The expression of hBDNF was identfied by the immunohistochemistry method, and the NIH/3T3 and BDNF biological activities were determined by culture of the PC12 cells and dorsal root gangl ia. Results The hBDNF-pLXSN was constructed successfully by sequencing analyses. The infected NIH/3T3 showed positive expression of hBDNF. The infected NIH/3T3 could product hBDNF. Bioactivity of the products could support the PC12cell survival and neurite growth in the primary cultures of dorsal root gangl ia neurons of mice. Conclusion hBDNF-pLXSNvirus has the abil ity to infect NIH/3T3 and make it expressed and secreted hBDNF with the biological activity. It can be used to treat facial paralysis as a gene therapy.
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 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 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 observe the effects of keratinocytes on proliferation and collagen secretion of fibroblasts. Methods The conditioned medium,collected from cultured keratinocytes, was added to the cultured fibroblasts as the tested groups(12.5％, 25% and 50％ groups) and DMEM as control group. The MTT, hydroxyproline coloricmetric method and flow cytometer were employed to measure the fibroblast proliferation, the collagen secretion andthe change of the cell cycle.Results In fibroblast proliferation, the absorbency(A) value of tested groups was significantly different from that of the control group (P＜0.01). A value increased as increasing concentration, there was statistically significant difference betweetheconcentrations of 25%,50% and the concentration of 12.5%(P＜0.01), but no statistically significant difference between the concentrations of 25% and 50%(P＞0.01). In collagen secretion, there was no statistically significant difference between the tested groups and the control group(P＞0.01), and between the tested groups(P＞0.01). In cell cycle, 50% of conditioned medium could make the fibroblast pass the limit of G1/S and S/G2 period, the cell rates of S,G2-M period increased. Conclusion The conditioned medium from keratinocytes can increase fibroblasts proliferation, have little effect on general collagen secretion.
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 evaluated the role of wt-P53 protein in telomerase regulation in keloid fibroblasts(KFBs). Methods The fibroblasts were derived from humankeloid tissue which was proved by pathological diagnosis. KFBs were divided into 2 groups, the transfection group and the untransfection group. wt-p53 gene was transfected into the fibroblasts by adenovirus vectors in the transfection group. The KFBs untransfected with wt-p53 gene served as control (untransfection group). After 48 hours of transfection, the expression of wt-P53 protein was analyzed by both Western blotting and immunofluorescence method, respectively. The telomerase activity was evaluated by TRAP-ELISA after 1-7 days of transfection. Results All the KFBs from 2 groups expressed wt-P53 protein. But the expression level of wt-P53 protein in the transfection group was significantly higher than that in the untransfection group.At the same time of high expression of wt-P53 protein, the telomeraseactivity of KFBs in transfection group was significantly lower than that in theuntransfection group(P＜0.05). Conclusion High level expression of wt-P53 protein can transiently inhibit the telomerase activity of KFBs.
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.