OBJECTIVE To prevent early closure of growth plate and developmental deformities of limbs by allografts of cultured cartilages into growth plate defects of rabbits. METHODS Chondrocytes isolated from articular cartilage of 1-month rabbits formed cartilage after cultivation in centrifuge tubes. The cartilages cultured for two weeks were implanted into growth plate defects of proximal tibiae of 6-weeks rabbits. At 4th and 16th weeks, X-ray, histologic and immunohistochemical examination were performed. RESULTS The tibiae had no marked deformities after 4 weeks of operation. Histologic examinations showed that the defects were filled with cartilage. Immunohistochemical results of type II collagen were positive. The tibiae with allografts of cultured cartilages had no evident deformities after 16 weeks of operation. Histologic examination showed nearly closure of growth plates. On the contrary, the tibiae on control side formed severe deformities and growth plate were closed. CONCLUSION Allograft of cultured cartilages into growth plate defects may replace lost growth plate tissues, maintain normal growth of limbs and prevent developmental deformity.
Objective To study the migration of Schwann cells from the nerve autograft in the acellular nerve allograft of the rats in vivo. Mehtods The sciatic nerves (20 mm long) of the SD rats were harvested and prepared for the acellular nerve grafts by the chemical extraction. Then, they were observed by the gross view, HE staining, and Antilamininstaining, respectively. Another 32 female SD rats weighing 250-300 g were obtained for the study. A 2-mm-long nerve autograft was interposed between the two 10-mm-long nerve allografts to form a 22-mm-long composite. Then, the composite was placed in the muscle space, together with a sole 22-mm-long nerve allograftas a control. They were harvested at 5，10，15 and 20 days, respectively, and were then given the HE staining and the S-100 staining. Results The acellular nerve graft was semitransparent under the gross view. HE staining showed that no cell was observed within the nerve graft. Anti-laminin staining showed that the basal membrane was partially interrupted, with a positive result (dark brown). All the nerve grafts in both the groups exhibited the existenceof the cells. The S-100 positive cells were observed from the 15th day at the far ends of the two allografts of the composite; however, there were no suchcells observed within the sole nerve allograft. Conclusion Schwann cells from the sciatic nerves (2 mm- long) of the rats can migrate in the acellular nerve allograft to the far ends of the neighboring 10-mm-long nerve allografts at 15 days after operation, which offers the theoretical basis forthe repair of the longrange nerve defect by the composite of the acellular nerve allografts with the interposed nerve autograft.
Objective To establish a purified model of rat retinal ganglion cells （RGCs） cultured by serum-free medium，and provide a good cell model to investigate the damage of RGCs in glaucoma，retinal ischemia，and degenerative retinopathy. Methods Two monoclonal antibodies，anti-rat SIRP（OX-41）against rat macrophage and antibody against rat Thy-1（OX-7），were used to purify and characterize RGCs from 1-3-day old Sprague-Dawley（SD）rats by means of two-step filtration.Purified RGCs were cultured in serum-free neurobasal medium containing B27 and ciliary neurotrophic factor（CNTF） meeting the neuronal cellrsquo;s special requirements.Photomicrographs illustration，immunfluorescence staining of Thy-1，calcein-acetoxymethyl ester（calcein-AM）fluorescence images were used to observe and identify cultured retinal cells and purified RGCs. Results Among the primary cultured rat retinal cells，91% were retinal neurons.Protuberances of RGCs were seen after cultured for 24 hours.At the4th to 8th day，many cells had uniform configuration，large body，and long protuberances. At the 14th day，over 60% cells maintained viability.Immunoflurescence staining of Thy-1 showed the purity of RGCs was about 90%. The results of calcein-AM staining，which stained the living cells only，showed large cell body of RGCs and most of RGCs had a protuberance whose length was twice longer than the diameter of the cells. Conclusion RGCs cultured by serum-free medium has uniform size，good configuration，and high purity，which is adapt to the research of damage of RGCs caused by various factors and to evaluate the protective effects of neuroprotective agents. (Chin J Ocul Fundus Dis, 2006, 22: 200-203)
Human fibroblasts and human epidermal keratinocytes were used for culture. Chitosan solution were added in the culture solution(DMEM). After 72 hours, the fibroblasts showed rapid growth in the control culture without Chitosan, But the numbers of human fibroblasts from growth was decreased as the concentration of Chitosan was increasing. On the contrary the human epidermal keratinocytes growed more rapidly in the culture with Chitosan than in the culture without Chitosan. The results showed that Chitosan inhibited the growwth of human fibroblast and stimulated the growth of human epidermal keratinocyte .
Objective To compare biological characteristics between articular chondrocyte and meniscal fibrochondrocyte cultured in vitro andto investigate the possibility of using cultured cartilage as a substitute for meniscus.Methods Chondrocytes isolated from articular cartilage and meniscus of rabbits aged 3 weeks were respectively passaged in monolayer and cultured in centrifuge tube. Cartilages cultured in centrifuge tube and meniscus of rabbit aged 6 weeks were detected by histological examination and transmission electron microscopy. Growth curves of articular chondrocytes and meniscalfibrochondrocytes were compared; meanwhile, cell cycles of articular chondrocytes and meniscal fibrochondrocytes in passage 2and 4 were separately measured by flow cytometry.Results Articular chondrocytes in passage 4 were dedifferentiated. Articular chondrocytes formed cartilage 2 weeks after cultivation in centrifuge tube, but meniscal fibrochondrocytes could not generate cartilage. The differences in ultrastructure and histology obviously existed between cultured cartilage and meniscus; moreover, apoptosis of chondrocytes appeared in cultured cartilage. Proportion of subdiploid cells in articular chondrocytes passage 2 and 4 was markedly higher than that in passage 2 and 4 fibrochondrocytes(Plt;0.05). Conclusion Meniscal fibrochondrocytes can not form cartilage after cultivationin centrifuge tube, while cartilage cultured in centrifuge tube from articular chondrocytes can not be used as graft material for meniscus. Articular cartilage ismarkedly different from meniscus.
【Abstract】Objective To investigate the effects of ethanol on polarization of cultured hepatocytes.Methods Sandwich hepatocytes were used to study the effects of ethanol on polarization of shortperiod cultured hepatocytes. Expression of albumin mRNA and P450-b mRNA,specific distribution of plasma membrance protein and the morphology were observed before and after ethanol was given. Results After treated ethanol, hepatocytes became flat and polynuclear. Its cellular plasma became watery and anatomical networks like board structure disappeared. Immunohistochemical method proved that the distribution of specific plasma membrane protein was damaged, and in situ hybridization studies demonstrated that the levels of albumin mRNA, P450-b mRNA expression decreased after ethanol was given.Conclusion Ethanol induces significant disturbance of cell polarization in cultured hepatocytes to result in injury. The current study is important for exploring the mechanism of alcohol liver disease from the cellular plane to provide a model.
Objective To explore an effective method of culturing the canine bladder smooth muscle cells, observe the morphological characteristics of the bladder smooth muscle cells growing on acellular small intestinal submucosa（SIS） and offer an experimental basis for reconstruction of the bladder smooth muscle structure by the tissue engineering techniques. Methods The enzymetreatment method and the explant method were respectively used to isolate and harvest the canine bladder smooth muscle cells, and then a primary culture of these cells was performed. The canine bladder smooth musclecells were seeded on the SIS scaffold, and the composite of the bladder smooth muscle cells and the SIS scaffold were co cultured for a further observation. At 5,7 and 9 days of the co culture, the specimens were taken; the bladder smooth muscle cells growing on the SIS scaffold were observed by the hematoxylin staining, the HE staining, and the scanning electron microscopy. The composite of the bladder smooth muscle cells on the SIS scaffold was used as the experimental group, and the bladder smooth muscle cells with no SIS were used as the control group. In each group, 9 holes were chosen for the seeded bladder smooth muscle cells, and then the cells were collected at 3, 5 and 7 days for the cell counting after the enzyme treatment. Morphological characteristics of the cells were observed under the phase contrast microscope and the transmission electron microscope. Expression of the cell specific marker protein was assessed by the immunohistochemical examinaiton. The proliferation of the cells was assessed by the cell counting after the seeding on the SIS scaffold. Results The primary bladder smooth muscle cells that had been harvested by the enzyme treatment method were rapidly proliferated, and the cells had good morphological characteristics. After the primary culture in vitrofor 5 days, the bladder smooth muscle cells grew in confluence. When the bladder smooth muscle cells were seeded by the explant method, a small amount of the spindleshaped bladder smooth muscle cells emigrated from the explant at 3 days. The cells were characterized by the welldeveloped actin filaments inthe cytoplasm and the dense patches in the cell membrane under the transmissionelectron microscope. The immunohistochemical staining showed the canine bladdersmooth muscle cells with positive reacting α actin antibodies. The bladder smooth muscle cells adhered to the surface of the SIS scaffold, growing and proliferating there. After the culture in vitro for 5 days, the smooth muscle cells covered all the surface of the scaffold, showing a singlelayer cellular structure. The cell counts at 3, 5 and 7 days in the experimental group were（16.85±0.79）×105，（39.74±2.16）×105 and （37.15±2.02）×105, respectively. Thecell counts in the control group were（19.43±0.54）×105，（34.50±1.85）×105 and （33.07±1.31）×105, respectively. There was a significant difference between the two groups at 5 days （P＜0.05）. ConclusionWith the enzyme treatment method, the primarily cultured canine bladder smooth muscle cells can produce a great amount of good and active cells in vitro. The acellular SIS can offer an excellent bio scaffold to support the bladder smooth muscle cells to adhere and grow, which has provided the technical foundation for a further experiment on the tissue engineered bladder reconstruction.
We cultured retinal g[ial cells(RGC)from immature rats and observed the migratory responses to fetal bovine serum(FBS).We found thai FItS stimulats the migration of RGC in a dose response manner. We also observed the inhibition of heparin on RGC cben,otaxis,and found that heparin(10U/ml)decreased significantly the RGC migration stimulated by serum(0%to 10%)(all Plt;0.0001).but 1U/ml of heparin bad no effect on RGC chemotaxis(P=0.5118).These results showed that FBS contains chemoattractants for RGC,and heparin can inhibit RGC chemotaxis stimulated by serum. （Chin J Ocul Fundus Dis,1994,10:170-173）
Objective To study a simple and practical method of isolation, culture and identification of hepatic oval cells from adult rat. Methods Wistar adult rats were fed by 2-acetaminofluorere （AAF） and were stimulated by partial hepatectomy to activate the proliferation of hepatic oval cells. After operation 12 days, the livers were resected for isolating oval cells. Hepatic tissue was digested by 0.10% collagenase Ⅳ and the obtained heterogeneous liver cells were then isolated and purified by density gradient centrifugation. The expressions of albumin and CK19 mRNA in hepatic oval cells were analyzed by immuno-fluorescence and RT-PCR. Results The survival rate of the newly isolated oval cells was more than 90%. The hepatic stem cells were shown by immuno-fluorescence of stem cell’s antigen c-kit. The expressions of mRNA CK19 and albumin of the oval cell were also detected by PCR. The proliferation activity of the newly isolated oval cells was significantly high and they could be induced to differentiate into both hepatic and bile ductal cells by some growth factors. Conclusion The successful development of the simple and feasible isolation and purification procedure as well as the identification method for hepatic oval cells may provide a fundamental for further studies about bionomics of the hepatic stem cell and the relation between stem cells and hepatic carcinoma.
OBJECTIVE: To study the technique and method of urethral epithelium culture in vitro, so as to lay the groundwork for reconstructing a tissue engineering urethra and to provide an experimental model of urethral mucosa in physiological, pathological, toxicological and microbiological study. METHODS: The urethral mucosa from a young male New Zealand hare that had just been out of milk, was digested into single cell liquid with Dispase II and mixed enzyme, and the fibroblast were removed. After being seeded, the cells were cultured by using L929 cells as trophoderm. The medium was changed regularly and the cells were subcultured when they grew to mix together 80% to 90%. The cultured cells were analyzed with histochemistry, immunohistochemistry dyeing and flow cytometry examination. We observed the ultrastructure of cells with scanning electron microscope and transmission electron microscope. RESULTS: The primary cultured cells fused when they had been cultured for about ten days. They were the same in size like road rocks. The cultured cells were all epithelial cells without fibroblasts and were diploid cells. The cells could be subcultured 11-13 generations, and could survive 50-60 days. CONCLUSION: The urethral epithelium of young New Zealand hare can be cultured in vitro and maintain the ability to proliferate within a certain time. The study result not only sets a role in reconstructing a tissue engineering urethral mucosa, but also provides an experimental model for the research of urethral mucosa in vitro.