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 explore the expression of alpha-smooth muscle actin (alpha-SMA) induced by transforming growth factor beta 1 (TGF-beta 1). METHODS: Five samples of hypertrophic scars and three samples of normal mature scars were collected as the experimental and control groups respectively. The fibroblasts were isolated from scars, and cultured in 2-dimension or 3-dimension culture system. The immunohistochemical staining method of LSAB were used to investigate the expression of alpha-SMA in fibroblasts in the different concentration of TGF-beta 1. RESULTS: The expression of alpha-SMA in 3-dimension culture system were markedly lower than those in 2-dimension culture system with respect to the fibroblasts in the experimental group. The expression of alpha-SMA in fibroblasts were different in response to various TGF-beta 1 concentration, it was more effective at the concentration of 5 ng/ml. The expression of alpha-SMA in the fibroblasts from hypertrophic scars seemed to be more sensitive to TGF-beta 1 compared to that of the normal mature scars. CONCLUSION: There are concentration-dependent in the expression of alpha-SMA induced by TGF-beta 1 in scar fibroblasts in vitro. The biological characteristics of the fibroblasts from hypertrophic scars and normal mature scars and their sensitivity to the inducement of TGF-beta 1 were different. The inducement of TGF-beta 1 may be depressed by extracellular matrix components and that may decrease the expression of alpha-SMA.
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）
ObjectiveTo investigate the antibacterial pretreatment protocol for primary fibroblast cell culture from transbronchial biopsies in patients with benign tracheal stenosis (BTS). MethodsFifteen specimens of intratracheal hyperplastic granulation tissue were obtained from 14 BTS patients by transbronchial biopsies. The tissues were divided into 3 groups according to different antibacterial pretreatment with 5 specimens in each group. An usual concentration of antibiotics pretreatment group (group 1) was pretreated by washing with PBS contained 1%-2% penicillin/streptomycin. A high concentration of antibiotics pretreatment group (group 2) was pretreated by washing with PBS contained 6% penicillin/streptomycin. An alchohol and high concentration of antibiotics pretreatment group (group 3) was pretreated by washing with 75% alcohol 3-4 seconds firstly,then by washing with 6% penicillin/streptomycin. After different pretreatment,all tissues were cultured with tissue culture method in the same condition. ResultsThe primary fibroblasts were successfully cultured from the tissue pretreated by method 2 and 3,but not cultured from the tissue pretreated by method 1 with large amount of bacteria. There were significant differences in the furthest radius of cell proliferation between different culture time points in three groups (P<0.01). The differences in the furthest radius of cell proliferation between three groups were not different at 24,48 or 72 h (P>0.05),but were significant between three groups at 96 h (P<0.05). ConclusionAn pretreatment protocol with high concentration of antibiotics or 75% alcohol is feasible in human primary fibroblasts culture from small specimens obtained by transbronchial biopsy.
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.
OBJECTIVE: To study the relationship between intracellular actin and scar contracture. METHODS: Fibroblasts from 10 cases of hypertrophic scar and 5 cases of keloid were cultured in vitro. Total actin, filamentous actin(F actin), globular actin (G actin) and the ratio of F to G actin(F/G) were measured by densitometry after differential extraction and separation by polyacrylamide gel electrophoresis in the presence of sodium sulfate. RESULTS: Total actin, F actin, G actin and F/G in hypertrophic scar fibroblasts were 2.38 ng/10(4) cells, 0.98 ng/10(4) cells, 1.42 ng/10(4) cells and 0.68 respectively, while in keloid fibroblasts were 1.68 ng/10(4) cells. 0.46 ng/10(4) cells, 1.26 ng/10(4) cells, and 0.36 respectively. There was significant differences between two tissues fibroblasts in the items of total actin, F actin, G actin, and F/G (P lt; 0.01), while no significant difference in G actin (P gt; 0.05). CONCLUSION: Total intracellular actin, F actin, and F/G may play an important role in the scar contracture. The hypertrophic scar and keloid can be distinguished by the contents of total intracellular actin, F actin and F/G.