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.
【Abstract】ObjectiveTo establish an efficient, effective hepatocyte isolation technique in order to increase cell production and decrease the prime cost. Methods The inferior vena cava below diaphragm was dissected and ligatured, and the inferior vena cava below liver was separated. Subsequently, the liver was perfused with EGTA through the portal vein while the inferior vena cava below liver was opened, and then the liver was harvested. The liver tissue was cut into 1 mm×1 mm×1 mm and digested at 37 ℃ water bath with Ⅳ collagenase for 30-40 minutes, then the hepatocytes were purified and cultured in CO2 incubator. The production and function of hepatocytes were assessed. ResultsThe isolated hepatocytes using this technique were more than 95% among the all isolated cells. No statistic difference was found in cell production and cell function comparing with traditional technique. But this technique was simplified and more economically. ConclusionThis modified hepatocyte isolation technique is efficient and effective. It can ensure the amount of production and purity of hepatocytes.
Objective To establish a method for primary culture of iris pigment epithelial cells(IPE). MethodsEnzyme-Assisted microdissection was used to isolate and cultivate the IPE cells.An identification was made with microscopic and immunohistochemical observations.Results IPE were successfully sultured and showed on differences with RPE in primary culture and subculture.ConclusionEnzyme-Assisted microdissection is a reliable and quick method for the isolation of IPE.
Objective To investigate the molecular mechanism of apoptosis in cultured human retinal pigment epithelial (RPE) cells. Methods The growth media of confluency human RPE cells were replaced with a daunoblastinacontaining one at a dose of 180mu;g/L,and the cells were incubated for 12 hr at 37℃.After incubation with the drug,the medium was withdrawn,fresh medium was added and incubation was carried out for an additional 24 hr.Apoptosis was monitored by light microscopy,enzyme linked immunosorbent assay(ELISA)and terminal deoxynucleotidyl transferase mediated biotin-dUTP nick-end labelling(TUNEL)staining.The expression of bax and bcl-2 were evaluated by immuncoytochemical staining with anti-human bcl-2 and bax antibodies. Results After the RPE cells treated with daunoblastina,shrinkage of cytoplasm and nucleus was identified.The ratio of nucleus to cytoplasm was increased.TUNEL staining showed that many cells were positive staining.The amount of apoptotic cells was directly proportional to the drug dose.The integral optical desity values for expression of bax inereased by 22.0%(Plt;0.05), and that of bcl-2 did not change significantly(Pgt;0.05). Conclusions During human RPE cell apoptosis induced by daunoblastina,overexpression of bax or low bcl-2/bax ratio were demonstrated.The results suggest that bax and bcl-2 gene expression could play a role in regulation of RPE cell apoptosis. (Chin J Ocul Fundus Dis, 1999, 15: 153-156)
Objective:To study the effects of growth factor on the proliferation of the cultured huamn retinal glial cells. Methods:EGF(0.5～100.0ng/ml) and NGF (0.5～10.0ng/ml) were added to cultures of human retinal glial cells and the proliferation rates of the cells were measured by MTT method. Results:EGF at a dosage ranging from 0.5ng/ml to 100.0ng/ml and NGF (0.05～10.0ng/ml) stimulated the cellular proliferation effectively with their EC 50 of 17ng/ml and 0.7 ng/ml respectively. Conclusion:Both EGF and NGF NGF had an effective stimulation on human retinal glial cell proliferation.They may play a role in the formation of PVR. (Chin J Ocul Fundus Dis,1998,14:33-34)
Objective To make an experimental research of the tissue engineered rat submandibular glands (SMG) cells growing on a collagensponge scaffold under an optimal culture condition. Methods The Wistar rat (8 days old) SMG cells of the second generation were seeded onthesurface of the collagen sponge scaffold (5 mm×5 mm×2 mm) and were cultured under a physiologically optimal condition for 3 weeks. At 1, 2 and 3 weeks, the cultured cells were observed on their shapes and structures by the histological examination and the scanning electron microscopy. The cultured cells underwentthe immunohistochemistry research (the cytokratin 813,CK8.13;αsmooth muscular actin,αSMA) staining performed at 3 weeks of the culture, and the amylaseactivity analysis (the Amano method) performed at 1 day, 1, 2 and 3 weeks of the culture for an evaluation on the secretion function of the cells; the ultrastructures of the cells were also observed by the transmission electron microscopy for an identification of their origins. Results The observatio n under the scanning electron microscope showed that at 1 week after the cellseeding, the seeded cells were attached to the collagen sponge scaffold surface, with no cell process formed; at 2 weeks the cells increased, with formation of the cell process that was anchored on the collagen sponge scaffold surface; and at 3 weeks, the scaffold surfaceattached cells increased, with formation of thefiliform fibers in the surface layer of the cells. The immunohistochemistry staining showed that the cultured epithelial cells of SMG were bly positive for the specific antibody of CK8.13, and the myoepithelial cells were positive forthe specific antibody of αSMA. The transmission electron microscopy showed that in the surface layer of the cultured epithelial cells of SMG the microvilli,plasm crease, and zymogen granules were observed, with a big and ovalshaped nucleus in the cell, and mitochondria and rough endoplasmic reticulum in the cytoplasm of the cell. The amount of amylase secreted by the cells cultured with thecollagen sponge scaffolds increased at a different degree with an extension of the culturing time. Conclusion The collagen sponge has a satisfactory cell compatibility, and the SMG cells cultured with this kind of collagen sponge can keep their abilities of proliferation and differentiation and theirfunction of secretion. Therefore, this kind of cultured SMG cells can be used as the tissueengineered cells seeded in the scaffold.
Objective To investigate the effect of acid, basic fibroblast growth factor (aFGF, bFGF) and epidermal growth factor (EGF), andtheir combination on the proliferation of rabbit anterior cruciate ligament (ACL) and medial collateral ligament (MCL) in vitro. Methods Thecells of ACL and MCL were isolated and subcultured from the knee joints of tenweek-old New Zealand white rabbits. The cells were seeded into 96-well corning cluster plates. Three growth factors of different concentration alone or in combination were added into the culture medium respectively, which were 0, 1, 5, 10, 50 and 100 ng/ml for aFGF, bFGF and 0, 1.56, 3.13, 6.25, 12.5, 25 and 50 ng/ml for EGF. The proliferation of the fibroblasts was measured for 48 h with XTT method. Results All of the three growth factors alone promoted the cell proliferation of ACL and MCL fibroblasts. The concentration of aFGF hada significant effect on the proliferation of both ACL and MCL fibroblasts. The concentration of 1 ng/ml bFGF and 5 ng/ml EGF was most effective in promoting the proliferation of ACL, and both bFGF and EGF had a significant effect on MCL. 5ng/ml aFGF with 50 ng/ml EGF had effect on ACL. 1 ng/ml aFGF with 3.13 ng/ml EGF had effect on MCL. Conclusion The three growth factors may promote the cell proliferation of ACL and MCL. These findings suggest that topical application of aFGF, either alone or in combination with EGF may have the potential to promote the proliferation of rabbit ACL and MCL,and aFGF of low concentration in combination with EGF is more effective than single growth factor.
ObjectiveTo explore the distribution and features of the optic cup stem cells in embryonic rat at tailbud stage.MethodsThe distribution of optic cup stem cells in optic cup tissue in 12.5-embryonic-day-old rats was observed by immunohistochemistry. The separated cells from optic cup were cultured with serum-free media, and immunofluorescence technique was used to detect the ability of hyperplasia of stem cells and expression of CHX10 antigen and specific antigens of mature retinal cells before and after differentiation.ResultsThe optic cup stem cells in embryonic rat at tailbud stage were mainly located at inner, outer, and marginal layer of optic cup. No expression of specifically marked protein of mature retinal cells was detected. The cells separated from optic cup had the ability of single-cell clone, positive expression of CHX10 and expression of several specific antigens of mature retinal cells after the inducement, including Thy1.1, glial fibrillary acidic protein (GFAP), protein kinase C (PKC) α, and rhodopsin.ConclusionOptic cup of 12.5-embryonic-day-old rats composes of undifferentiated cells, and the stem cells are mainly located in optic cup inner and marginal. High ability of hyperplasia of the optic cup stem cells cultured in vitro is found. The cells, which are retinal stem cells, can express several specifically marked proteins of mature retinal cells after inducement and differentiation.(Chin J Ocul Fundus Dis, 2005,21:159-162)
Objective To observe the expression of proteins in light-injured retinal pigment epithelial (RPE) cells. Methods ARPE19 cells were exposed to the cool white light at the intensity of (2200plusmn;300) Lx for 6 hours to set up the light injured model. Cellular soluble proteins was extracted and analyzed by means of twodimensional electrophoresis to find out the changes of protein map of lightinjured RPE cells. Results Cellular soluble proteins had (390plusmn;10) spots on the map, in which 11 spots had obvious difference between the light injured group and the normal control group. In the lightinjured cells, the expressio of 8 proteins increased, 1 decreased, and 2 disappeared. Conclusion Twodimensional electrophoresis can find out the difference of expression of proteins in lightinjured and normal RPE cells.