ObjectiveTo study the effects of recombinant human growth hormone (rhGH) on proliferation of human rectal cancer cell in vitro. MethodsThe experiment was divided into control group,rhGH group,Oxaliplatin (LOHP) group and rhGH+LOHP group. The double proliferation time of cells,cell inhibition rate,cell cycle, proliferation index (PI) and DNA inhibition rate of human rectal cancer line,HR8348,were studied by cell culture, MTT assay and flow cytometry on different concentration of rhGH. ResultsIn vitro the markedly accelerated effects of rhGH on multiplication of HR8348 cell line were not found: there was no statistical significance as compared rhGH group with control group or compared rhGH+LOHP group and LOHP group （Pgt;0.05）. The double proliferation time of cells was markedly lengthened, cell inhibition rate and the cells arrested in G0-G1 phase were obviously increased, meanwhile, the cells in S phase （P＜0.05） and G2-M phase and PI were markedly decreased and DNA inhibition rate was obviously risen as compared rhGH+LOHP group with control group or rhGH+LOHP group and rhGH group （P＜0.01）.ConclusionIn vitro rhGH does not accelerate the multiplication of human rectal cancer cells.
Objective To culture astrocytes of human optic nerve and establish the cell lines for further study of healing process after optic nerve trauma. Methods Astrocytes of infantile optic nerve were cultured by tissue inoculation or tissue digestion with 0.25 % trypsin and 0.06% EDTA. The second and fourth passage cells were stained with HE and anti-GFAP, S-100 protein, vimentin, and CD34 antibodies. Results The trypsinized astrocytes of infantile optic nerver eached confluence in 7 days. The cultured cells were in polygonal shape with processes and the cytoplasm was abundant. These cells were positive in GFAP, S-100 protein and vimentin staining, and negative in CD34 staining. Conclusions Astrocytes of human optic nerve can be successfully cultured by trypsinization rather than tissue inoculation. (Chin J Ocul Fundus Dis, 2001,17:144-146)
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 observe the effects of silks on attachment, shape and function of chondrocytes cultured in vitro. METHODS: The silks from silk worm cocoons were digested by trypsin and coated with polylactic acid to from three dimensional scaffolds for rabbit rib chondrocyte culture. The growth and shape of chondrocytes were observed with phase contrast microscopy, scanning electron microscopy. RESULTS: The chondrocytes were adhered to silks slowly after chondrocytes were seeded into silk scaffolds and cells fixed on silks well 1 or 2 days later. Cells began to proliferate after 3 days and multiplicative growth was observed on the 6th day. Microholes of silk scaffolds were filled with chondrocytes 2 weeks later. Scanning electron microscopy showed that there was a lot of extracellular matrix surrounding cells. CONCLUSION: Silks are ideal for attachment, growth and function maintenance of chondrocytes, and silks can be used as scaffolds for chondrocytes in three dimensional culture.
Objective To investigate the effect of hypoxia on the exp ression and function of integrin receptor αvβ3 of bovine retinal vascular endotheliocytes. Methods Bovine retinal vascular endotheliocy tes in the culture dishes coated by vitronectin was put into the normal and hypoxemic condition, respectively. Enzyme linked immunosorbent assay and cell adhesion analysis were used to detect the expression and function of integrin receptor αvβ3 in bovine retinal vascular endotheliocytes, respectively. Results Under the condition of hypoxia, the expression of αvβ3 increased gradually, and reached the peak at the 48th hour. The expression of αvβ3 at the 60th and 72nd hour in hypoxia group was higher than that in the normal group. Bovine retinal vascular endotheliocytes absorbed more Vn of extra-cellular matrixes (ECM) after cultured under hypoxemic condition for 24 hours.Conclusion Hypoxia may up-regulate the expression of αvβ3, which promote the adsorbability of endotheliocytes.(Chin J Ocul Fundus Dis，2004，20：360-363)
In order to study the biological properties of fibroblasts isolated from different tissues. The fibroblasts from normal skin, hypertrophic scar and keloid were cultured, respectively, in vitro, and their morphologies and growth kinetics were compared. The results revealed that although fibroblasts in keloid were irregularly arranged, crisscross and overlapping with loss of polarization, there was no significant difference in the 3 groups so far the cellular morphology of fibroblast itself, cellular growth curve, cellular mitotic index, cloning efficiency and DNA content provided those cultures were in the same cellular density and culture conditions. It was concluded that fibroblasts isolated from culture of normal skin, hypertrophic scar and keloid in vitro showed no significant difference in morphology and growth kinetics, on the contrary, their biological behaviors were quite similar.
Objective To investigate the effect of dissociation of the human retinal pigment epithelium (RPE) cells by ficoll hypaque gradient centrifugation.Methods The primary human RPE cells and subcultured human RPE cells were dissociated with ficoll gradient centrifugation solution (d=1.077 g/ml)and the same divid ed cells as the control were dissociated with routine normal culture medium cent rifugation. The Trypan blue (0.4%) rejection staining was used, and the mouse anti-human monoclonal anti-body and fluorescein isothiocyanate (FITC) labeled rabbit anti-mouse IgG were utilized for indirect immunoreactivity for the test of human cytokeratin (CK) in active RPE cells cytoplasm. Flow cytometry assay was used to analyzed the percentages of CK positive staining RPE cells. simultaneously, the cells configuration, growth condition, the rate of clone formation, and the purifying result were observed under the fluorescent and confocal microscope.Results The survival rate and positive rate of CK of RPE cells in experimental group were higher than those in the control（P＜0.001）, but the number of the cells was reduced. The cells in the experimental group were integrated round with smooth border, symmetrical staining, homogeneous configuration and higher rate of clone formation （P＜0.001）. Conclusions RPE cells disas sociated with ficoll gradient centrifugation have the better dissociation effects. (Chin J Ocul Fundus Dis，2003，19：333-404)
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 establ ish the methods to get high activity, high purity, and adequate Schwann cells (SCs), and to provide sufficient seed cells for the peripheral nerve repair. Methods Six 5-day-old, male or female, Sprague Dawley rats were selected and the sciatic nerve (control group) and dorsal root gangl ion (DRG) (ex perimental group) were harvested.Then the sciatic nerves and DRG were digested by co-enzyme and dispersed by medium containing serum to isolate SCs. Freshlyisolated SCs from rats were cultured, purified and subcultured. The 1st generation of SCs were chosen to draw the growth curve of SCs by the counting method and to detect the prol iferation of SCs by MTT assay at 8 days of culture, the purity of SCs by immunocytochemistry of anti-S-100 and the brain-derived neurotrophic factor (BDNF) concentration by ELISA. Results A total of 36-43 DRGs could be obtained in each rat. The number of obtained single SC in experimental group [(7.5 ± 0.6）× 106] was significantly higher than that in control group [(3.5 ± 0.4）× 106 ] (t=13.175, P=0.000). SCs reached logarithm prol iferation phase at 3 days. With time, the cell number and the prol iferation absorbance (A) value of 2 groups all showed upward trend. The number and A value of experimental group were significantly higher than those of control group (P lt; 0.05). The SCs purity of experimental group (92.08% ± 3.45%) was significantly higher than that of control group (77.50% ± 3.57%) (t=6.689, P=0.001).The concentrations of BDNF at 3 days and 5 days in experimental group were significantly higher than those of control group (P lt; 0.05). Conclusion The sufficient amount, high purity, and viabil ity of SCs from DRGs can meet the needs of studies on peripheral nerve repairment.
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.