Abstract An experiment was carried out to investigate the possibility of the establishment of an osteoblasts bank which could supply osteoblasts in repairing bone defect. Osteoblasts were isolated from thetibial periosteum of eight New-Zealand rabbits and cultured in votro. A bone defect, 1.5cm in length was made in both radii of each of the 8 rabbits. The cultivated osteoblasts, gelfoam as a carrier were randomly implanted into the defects of the radii of rabbits. Accordingly, the contralateral radial defects wereimplanted with gelfoam absorbed with the Hanks solution as control. The healing of bone defects was evaluated by roentgenographic examination at 2, 4, 8 and 12 weeks after operation, respectively. It was shown that the implanted cells had osteogenetic capability and could be possible to promote healing of the bone defects. It was suggested that further study needed to be carried out in this field.
Objective To explore an effective method to culture and purify canine bladder transitional epithelial cells.Methods Bladder tissue was obtained from healthy puppy under sterile conditions. Bladder mucosa was removed from the remaining tissue with fine scissor and minced into small pieces, and then were dissociated into single cell suspensions with 0.125% trypsin. The bladder epithelial cells were cultured in defined keratinocyte serum free medium. The cells were passaged and purified by 0.05% trypsin and 0.02% EDTA. Morphological characterization were studied under inverted phase contrast microscope and transmission electron microscope. Expression of cell specific marker protein was assessed by immunohistochemistry. Results Canine bladder transitional epithelial cells could be efficiently cultivated and expanded in serum-free medium without fibroblast contamination. The cells could be passaged 4-6 times without a distinguished decrease in cell proliferation. The cells were characterized by well-developed micro filament and desmosome junction under transmission electron microscope. Immunohistochemical staining with broadly reacting anticytokeratin antibodies (AE1/AE3) confirmed the epithelial phenotype of the cells.Different generations of cells showed diploid cells. Conclusion A large number of bladder transitional epithelial cells can be obtained from small bladder tissue with our digestion method. The cultured bladder epithelial cells can be proliferated to sufficient quantities for further reconstructive purposes.
Objective To investigate the influenceof insulin-like growth factor-I (IGF-I) on biological characteristics of articular chondrocytes cultured in vitro of rabbits. Methods Monolayer articular chondrocytes of 4week old rabbits were cultured in medium with IGF-I, at the concentrations of 3, 10, 30, 100, and300ng/ml. The DNA content in cells and glucuronic acid content in matrix were detected on the 2nd, 4th, 6th days after culture. Results The DNA content in cells and the glucuronic acid content in matrix in articular chondrocytes cultured in medium with IGF-I at concentrations of 3-300ng/ml were all significantly higher than those in control group (P＜0.01), which reached the peak at the concentrations of 30-100mg/ml on the 4th day. Conclusion IGF-I could obviously promote theproliferation of articular chondrocytes in vitro, and there exist time-dependent and dose-dependent effect.
Objective To establish a model of the human marrow mesenchymal stem cells (hMSCs) cultured under the hypoxic condition in adults and to investigate the biological features of MSCs under hypoxia.Methods The bone marrow was obtained by aspiration at the posterior superior iliac spine in 3 healthy adult subjects. hMSCs were isolated by the gradient centrifugation and were cultured in the DMEM-LG that contained 20％ fetal bovine serum. The serial subcultivation was performed 10-14 days later. The second passage of the hMSCs were taken, and they were divided into the following 4 groups according to the oxygen concentrations and the medium types: the normoxic group（20％O2, DMEM-LG, Group A）, the hypoxic group（1％O2, DMEM-LG,Group B）, the normoxic osteoblast induction group（20％O2, conditioned medium, Group C）, and the hypoxic osteoblast induction group（1％O2, conditioned medium, Group D）. The biological features of the cultured hMSCs under hypoxia were assessed bythe cell count, the MTT method, the colony forming unit-fibroblast, the real-time RT-PCR, and the alkaline phosphatase (ALP) activity, and the alizarinred staining. Results The hMSCs cultured in the Group B and Group D had a significantly higher proliferation rate than those in the Group A (Plt;0.01), and the culture effect was not influenced by the medium type. The hMSCs in the Group B had a significantly higher level of the colony-forming unit capability than the hMSCs cultured in the Group A(Plt;0.01). After the induction, hMSCs in the Group B had a decreasednumber of the osteoblasts than hMSCs in the Group C. The hMSCs in the Group D had a gradually-increasedactivity of ALP, which was significantly lower than that in the Group C(Plt;0.01). The RT-PCR examination revealed that ALP,osteocalcin, and mRNA expressions of collagen type Ⅰ and osteonectin in the Group Csignificantly increased （P＜0.01). By comparisonamong the 3 groups, after the 4-week culture the obvious calcium salt deposit and the red-stained calcium nodus could be observed.ConclusionHypoxia can promote the proliferation rate of hMSCs, enhance the colonyforming ability and inhibit the differentiation of the osteoblasts.
Objective To research the methods and techniques of SD rat bone marrow stromal cells （MSCs）culture in vitro and to provide a large number of MSCs for cell therapy. Methods Bone marrow from the femur and tibia of the early age SD rats was taken to culture to the passage 1-4 （P1-P4）, its growth was observed and P3 cells were evaluated by HE staining and immunohistochemisty. Results The growing speed of P1-P3 was faster than that of P0, cells fusion was 85%-90% after 3-4 d and the cells were arranged in groups liked whirlpool shape or parallel; （3.4-3.6）×104/cm2 cells were gained and the total cell number of P1-P3 was 4.08×106, 2.44×107 and 2.85×108 respectively, the rate of trypan blue rejecting stained was 95%-97%. P4’s growing speed was slower than before, 1.42×109 in 3.0×104/cm2 cells were gained, and the rate of rejecting stained was 95%. P4-cell output was amplified nearly 2 000-fold higher than P0-cell. P3 immunohistochemical analysis indicated CD105+ cells 61.9%， CD44+ 45.4%， CD29+ 16.8%， CD45+ 8.2%， CD31+ 13.7%， CD34+ 8.3% and CD11b+ 1.5%, respectively. Conclusion The culture of whole bone marrow is suitable for a large number of MSCs provision in vitro, and can meet the needs of the cell therapy research.
In thiis study,we show thai carbachol stimulates the accumulation of inositol phosphates(InsPs)in human rellnal pigment epithelium (RPE)cells and atropine blocks the carbachol-induced effect ,suggesting the existence of musearinie acelyleholine receptors in human RPE cells. In contrast,noradrenaline,serotonin, cpidermal growth factor (EGF),isoproterenol,and NECA (5'-[N-ethyl]-carboxamido-adenosine)do not influence the basal levels of InsPs.Moreover,isoprmerenol and NECA do not affect the carhaehol elevated levels of InsPs.EGF,howcvcr,does potentiate the carhaehol stimulated elevation of InsPs in a dose-dependent manner ,suggesting an interaction between EGF and musearinie receptors in cultured human RPE cells. （Chin J Ocul Fundus Dis,1994,10:220-222）
OBJECTIVE: To construct a tissue engineering skin containing melanocytes by employing tissue engineering method. METHODS: The keratinocytes, dermal fibroblasts and melanocytes were isolated and purified. Then the cells were used to construct a tissue engineering skin containing melanocytes. The location of melanocytes in the tissue engineering skin were detected by Dopa staining, transmission electron microscope (TEM) and S-100 immunohistochemical staining. RESULTS: Melanocytes can be detected in the basal layer of the constructed tissue engineering skin. The results of TEM showed that the melanocytes were in good conditions. CONCLUSION: The artificial skin containing melanocytes was successfully constructed in vitro and can be used to repair the full-thickness skin defects.
Objective To investigate the impact of photodynamic therapy (PDT) with verteporfin on the expression of pigment epithelial derivative factor (PEDF) mRNA and vascular endothelial growth factor (VEGF) mRNA in adult retinal pigment epithelial (RPE) cells in vitro. Methods The changes of cellular viability before and after PDT were assessed by methyl thiazolyl tetrazolum (MTT) colorimetric assay. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was conducted to detect the expression of PEDF and VEGF mRNA in RPE cells before and after PDT. Results PDT caused the death of RPE cells. The cellular mortality was positively correlated with the power of photocoagulation and the concentration of verteporfin. Conclusion PDT could downregulate the expression of PEDF and VEGF mRNA in adult RPE cells in vitro, which may relate to the cure or relapse of subfoveal choroidal neovascular membrane after PDT. (Chin J Ocul Fundus Dis, 2006, 22: 256-260)
Objective To investigate the expression of Human leucocyte antigen(HLA)-DP, -DQ, -DR and CD40 in human retinal pigment epithelial (RPE) cells, to determine their molecule expression in immune response process, and their abilities to stimulate T lymphocyte activation. Methods Human RPE cells were cultured with or without (IFN respectively. Expression of HLA-DP, -DQ, -DR and CD40 was measured by immunohistochemical staining. Meanwhile, peripheral blood mononuclear cells (PBMC) were cocultured with RPE cells in vitro, and then the expression of activated lymphocytes CD69 was measured by fluorescence activated cell sorter(FACS). Results Expression of HLA-DP, -DQ, -DR and CD40 antigen were enhanced by gamma;-interferon inducement. Increasing amount of CD69 positive lymphocytes were found in the co-culture system of RPE cells and PBMC. Conclusion T-lymphocytes in the peripheral blood were activated by human RPE cells which is antigen presenting cells with immunological characteristics potential.
Objective To investigate the experimental condition and mechanism of differentiation of human umbilical cord blood derived mesenchymal stem cells（hUCB-MSC）into neuron-like cells induced by recombined human epidermal growth factor （rhEGF） and taurine in vitro.Methods hUCB-MSC were primary cultured in Dulbeccoprime;s modified Eagle's medium/F12 （DMEM/F-12）which supplemented with 105U/L penicillin G, 100 mg/L streptomycin sulfate, 10% fetal bovine serum (FBS),5% autologous plasma,4 mmol Lglutamine, 30 ng/ml rhEGF.The DMEM/F-12 medium was replaced by taurine medium after 3 passages.The expression of surface antigen CD90,CD29,CD34,CD44 and CD45 were detected by flow cytometry；the expression of neuron specific enolase,rhodopsin and nestin were investigated by immunocytochemistry. The statistical method was chi square test.Results Morphologically similar to bonemarrow MSC,hUCB-MSC became attached cells after the first 5 to 7 days in culture，and reached 80% to 90% confluent after 3 to 4 weeks. Growth accelerated after passage. hUCB-MSC were positive for CD29,CD44 and CD90 but negative for CD34 and CD45. After taurine induction, 2515/3120 cells expressed NSE, 1168/3175 cells expressed rhodopsin and 903/3050 cells expressed nestin while only 234/2965 cells expressed NSE in the control group(P＜0.01).Conclusion rhEGF and taurine can induce hUCB-MSC differentiating into neuronlike or rhodopsin positive cells.