ObjectiveTo establish human bladder cancer cell line with silenced Fibulin-5 gene and observe the effects and mechanism of Fibulin-5 gene silencing on the proliferation activity and migration of the bladder cancer cells.MethodsThe human bladder cancer cells 5637 were divided into group F5 and group NC, and the cells in group F5 were infected with Fibulin-5 RNA interference (RNAi) lentivirus while the cells in group NC were infected with negative-control virus. Then the expression of Fibulin-5 mRNA was detected by real-time quantitative polymerase chain reaction, the cell proliferation activity was detected by MTT, the migration rate was detected by wound healing method, and the expression levels of proteins in receptor tyrosine kinase (RTK) pathway were detected by PathScan RTK Signaling Antibody Array Kit.ResultsThe Fibulin-5 mRNA expression decreased significantly by Fibulin-5 RNAi lentivirus (0.067±0.013 in group F5 vs. 1.001±0.000 in group NC), and the gene silencing efficiency reached 93.3%, so the Fibulin-5 silencing cell line was established successfully. Comparing with group NC, the relative absorbance value and migration rate of cell 5637 in group F5 decreased significantly (P<0.01); in addition, the expression levels of anaplastic lymphoma kinase, Axl, p44/42 mitogen activated protein kinase, and Src protein were up-regulated in group F5 (P<0.05).ConclusionFibulin-5 may play a role in the proliferation and migration of bladder cancer cells, and may have an inhibitory effect on extracellular signal-regulated kinase and its signaling pathway proteins.
ObjectiveTo systematically review the diagnostic value of FibroScan for the staging of liver fibrosis in chronic hepatitis B. MethodsWe searched the PubMed, EMbase, Web of Knowledge, CBM, WanFang Data and CNKI databases for studies investigated the diagnostic value of FibroScan for hepatic fibrosis B from Jan. 1st, 2003 to Aug. 31st, 2013. Two reviewers independently screened literature according to the exclusion and inclusion criteria, extracted data and assessed methodological quality of included studies. Then, Stata 13.0 software was used to analyze the data. ResultsA total of 15 studies involving 2 588 patients were included. The results of meta-analysis showed that:the pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio and the AUC of SROC were 0.77 (95%CI 0.69 to 0.83), 0.84 (95%CI 0.70 to 0.87), 3.8 (95%CI 2.6 to 5.6), 0.29 (95%CI 0.22 to 0.38), 13 (95%CI 8 to 21), 0.82 (95%CI 0.82 to 0.88) for hepatic fibrosis; and were 0.81 (95%CI 0.73 to 0.87), 0.89 (95%CI 0.86 to 0.92), 7.5 (95%CI 5.3 to 10.3), 0.21 (95%CI 0.14 to 0.31), 36 (95%CI 20 to 65), 0.93 (95%CI 0.90 to 0.95) for early hepatic cirrhosis, respectively. ConclusionThe current evidence suggests that FibroScan is of good accuracy in the diagnosis of early hepatic fibrosis but not for hepatic cirrhosis in patient with chronic hepatitis B.
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.
In order to explore further the regulatory factors to the potentiality in inducing osteogenesis by fibroblasts, the fibroblasts were isolated, and purified from human skin, and were grown in incubation in the media of EGF, IL-6, TNF-alpha and BMP2 at different concentrations for two weeks, then, the markers for osteogenic features were investigated by biochemistry, histochemistry and electron microscopic observations. It was found that the combined use of TNF-alpha and BMP2 could stimulate fibroblasts to secrete alkaline phosphatase, osteocalcin and collagen, and the morphological changes of the fibroblasts were also very striking. In the extracellular matrix, the collagen fibrils, with or without periodicity, were arranged regularly or randomly oriented, and numerous minute calcium granules were interspersed among them. The fibroblasts were interwoven one on top of another in the form of multilayer structure and on the surface, there were secreting granules and piling up of calcium crystals which coalessed steadily and increased in size in forming bony nodules. It was considered that TNF-alpha and BMP2 were capable of inducing the fibroblasts to form bone.
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 investigate the changes of fibroblast growth factor (FGF) in burn wounds. METHODS: The FGF expression in the center of wound granulation, the edge of wound, the healed part of wound, the normal skin of patients, and the heal course of second degree burn wounds were detected by immunohistochemical methods. RESULTS: The expression intensity of FGF was different in the different sites of third degree burn wounds. The highest contents of FGF was in the center granulation of burn wounds, the less was in the borderline of wound and healed skin, and the least was in the healed skin. FGF expression mainly concentrated in the middle layer of wound, and almost no FGF expression in normal skin. The most FGF expression was occurred at 14 days after injury in second degree of burn wound. CONCLUSION: The changes of FGF in wounds are closely related to the wound healing, and rational use of FGF can promote wound healing.
Objective To investigate the influence of lipopolysaccharide(LPS) on the proliferation and collagen synthesis of normal human skin fibroblasts so as to elucidate its relation with skin wound healing. Methods Fibroblasts wereisolated and cultured in vitro, and then exposed to different doses of LPS(0.005, 0.010, 0.050, 0.100, 0.500, and 1.000 μg/ml) from E.coli055∶B5 respectively. Then the absorbance (A) value of fibroblasts was determined with the colorirneteric thiazolylblue (MTT) assay, and the cell number was counted under inverted phase contrast microscope from the 1st day to the 9th day after LPS administration, and collagen synthesis of fibroblasts in culture medium was measured with the method of pepsin digestion after incorporation of 3Hproline into stable, single-layered, confluent fibroblasts at 7 days after LPS administration. Results Compared with control group, A value increased with the increasing concentration of LPS (0.005 μg/ml 0.500 μg/ml) and LPS of 0.100 μg/mlgroup had the best effect. The difference was remarkable from the 5th day to the 9th day(P＜0.05). A value decreased when challenged with the LPS of 1.000 μg/ml and the difference was remarkable from the 3rd day to the 9th day(P＜0.05). Cell number increased with theadministration of LPS of different concentrations (0.005 μg/ml 0.500 μg/ml) and LPS of 0.100 μg/mlgroup had the best effect. The difference was remarkable from the 1st day to the 6th day(P＜0.05). Cell number decreased remarkably when challenged with LPS of 1.000 μg/ml and the difference was remarkable from the 2nd day to the 9th day(P＜0.05). Collagen synthesis increased when challenged with LPS of different concentrations (0.005 μg/ml 0.500 μg/ml) and the 0.100 μg/ml group had the best effect. However, when the dose of LPS reached 1.000 μg/ml, it inhibited collagensynthesis. Conclusion LPS could promote the proliferation andcollagen synthesis of fibroblasts within a certain range of low doses, but over-high dose ofLPS might inhibit the proliferation and collagen synthesis of fibroblasts, suggesting that LPS of certain concentrations might contribute to wound healing, while excessive LPS has negative effect on wound healing.
Objective To evaluate the effect of the treatment of necrosis of femoral head with the free vascularized fibula grafting. Methods From October 2000 to February 2002, 31 hips in 26 patients with ischemic necrosis of the femoral head were treated with free vascularized fibula graft. Among these patients, 21 patients (25 hips) were followed up for 6-18 months(12 months on average). According to Steinberg stage:Ⅱ period, 5 hips;Ⅲ period,8 hips; Ⅳ period, 12 hips.Results Among 25hips, their Harris Hip Score at all satges were improved during the follow-up. The symptom of pain diminished or disappeared after operation. The patient’s ability to work and live was notlimited or only slightly limited during the follow-up. Radiographic evaluation showed that most femoral heads improved (18 hips) or unchanged (6 hips) and only oneworsened.Conclusion The free vascularized fibular grafting is a valuable method for femoral head necrosis. With this method, we can prevent or delay the process of the disease.
Objective To establish an effective way to cryopreserveprecartilaginous stem cells(PSCs) of neonate rat. Methods PSCs ［fibroblast growth factor-3(FGFR-3) positive cells］ were isolated and purified by magnetic cell sorting method. PSCs were cultured and amplified to the third generation. PSCs were preserved in liquid nitrogen. The biological properties of cryopreserved PSCs were investigated by reverse transcriptase polymerase chain reaction(RT-PCR), immunohistochemistry, and immunofluorescence. Results Immunohistochemical and immunofluorescent analysis showed widespread expression of FGFR-3 in cryopreserved PSCs. FGFR-3 could be dectected by RT-PCR in cryopreserved PSCs.Cryopreserved PSCs kept high cell viability, and phenotypic and proliferation characteristics of PSCs in vivo.Conclusion Cryopreservation of PSCs can supply adequate qualified cells for repairing the defects of epiphyseal growth plate by tissue engineering technique.
Objective To investigate the phenotypic change and proliferation of fibroblasts in human inflammatory strictured bile duct wall. Methods We observed the density and ultrastructure of fibroblasts, and the histologic structure in human normal bile duct wall and inflammatory strictured bile duct wall by light and electron microscope.Results The results showed that fibroblasts were the main source of extracellular matrix production in bile duct wall. The phenotype of fibroblasts in inflammatory strictured bile duct wall changed obviously, quiescent fibroblasts were activated and transformed to myofibroblasts, with massive proliferation. Conclusion These data suggest that massive proliferation of activated fibroblasts and myofibroblasts is the main source of extracellular matrix overproduction which results in inflammatory bile duct stricture.