Objective To discuss the applycation possibility of themicroscopic stripping technique used in the primary culture of human embryonicesophagus squamous epithelial cells, and of the methodds for the isolation, depuration and subculture of the esophagus epithelial cells in vitro. Methods The squamous epithelial cells wereobtained from the esophagus mucous membrane of the 20-week abortion fetus through the microscopic stripping technique, and were digested with trypsin. Then, the morphological, immunohistochemical observation and the growth curve of the isolated cells were studied. Results The isolated cells were spherical in the cell suspension and spherical-like or polygon-like after attachment to the culture flask.The squamous epithelial specialized cytokeratin staining was bly positive. And the morphological studies by the transmission electron microscopy indicated that the cultured cells were squamous epithelial cells. The squamous epithelial cells reached the peak level 3-4 days after the transfer of the culture. The absorbanceat 3 and 4 days was significantly higher than that at 1,2,5 and 6 days (P＜0.05). Conclusion A large mumber of squamous epithelial cells can be available with the microscopic stripping technique and the digestion method. Thecultured squamous epithelial cells can be proliferated quickly, and fit for the tissue engineering study.
OBJECTIVE: To investigate the biological characteristics of human muscle satellite cell cultured in vitro. METHODS: Human muscle satellite cells were obtained from skeletal muscle biopsies of six patients during corrective orthopedic surgery, cultivated in growth medium for ten days, then in differentiation medium for additional five days. Human satellite cells were identified with monoclonal antibody against desmin. Cells were observed under phase contrast microscopy. RESULTS: Human muscle satellite cells proliferated in growth medium, and fused to form myotubes in differentiation medium. After 24 hours in differentiation medium, the confluent satellite cells began to fuse actively and achieved the top level at 72 hours. CONCLUSION: Human muscle satellite cell can proliferate and differentiate in appropriate culture condition. Immunocytochemical detection of desmin is the effective early method to determine satellite cell.
Objective Dexamethasone is one of the basic agents which could induce osteogenic differentiation of mesenchymal stem cells. To investigate the optimal concentration of dexamethasone in osteogenic differentiation of adiposederivedstem cells (ADSCs) so as to provide the theoretical basis for further bone tissue engineering researches. Methods FiveNew Zealand rabbits (2-3 kg) of clean grade, aged 3 months and male or female, were obtained. ADSCs were isolated from the subcutaneous adipose tissue of inguinal region, and cultured with collagenase digestion, then were detected and identified by CD44, CD106 immunofluorescence staining and adi pogenic differentiation. ADSCs at passage 3 were used and the cell density was adjusted to 1 × 105 cells/mL, then the cells were treated with common cultural medium (group A) and osteogenic induced medium containing 0 (group B), 1 × 10-9 (group C), 1 × 10-8 (group D), 1 × 10-7 (group E), 1 × 10-6 (group F), and 1 × 10-5 mol/ L (group G) dexamethasone, respectively. The cell prol iferation and the mRNA expressions of osteocalcin (OC) and core binding factor α1 (Cbfα1) were detected by MTT and RT-PCR, respectively. The activity of alkal ine phosphatase (ALP) was measured, and the percentage of mineral area was calculated. The mineral nodules were also detected by al izarin red staining. Results ADSCs mostly presented fusiform and polygon shape with positive expression of CD44 and negative expression of CD106. The result of oil red O staining was positive after ADSCs treated with adipogenic induced medium. The result of MTT revealed that the absorbance (A) value decl ined with the ascending of the concentration of dexamethasone, and there was significant difference in A value between groups D and E at 5 and 7 days after osteogenic induction (P lt; 0.05). The mRNA expressions of OC and Cbfα1 reached the peak in groups E and D at 7 days after osteogenic induction, respectively. The activity of ALP and the percentage of mineral area had the maximum value in group D at 14 days, then decl ined gradually. There was no significant difference in the mRNA expressions of OC and Cbfα1, the activity of ALP, and the percentage ofmineral area between groups D and E (P gt; 0.05), but significant differences were found between groups D and E and other groups (P lt; 0.05). After 14 days, the cells of group G died, and the result of al izarin red staining was positive in groups B, C, D, E, and F. Conclusion When the concentration of dexamethasone in osteogenic medium is 1 × 10-8 mol/L, it could not only reduce the inhibitive effect on cells prol iferation, but also induce osteogenic differentiation of ADSCs more efficiently.
Objective To verify whetheriris pigment epithelial cells(IPECs)possess the similar potential of specific phagocytosis to retinal outer segments(ROS) with retinal pigment epithelialcells(RPECs). Methods IPESc were isolated from neonatal bovines with Hu's method,and were cultured.The cultured cells were identified by immunohistochemical methods with antibodies to cytokeratin and s-100.Total RNA of IPECs was extracted by Trizol.The specific primers for mannose-receptor andbeta;-actin were designed according to their sequence from Genbank.The mRNA expression of these proteins in the IPECs was analyzed by reverse transcription polymerase chain-reaction (RT-PCT).Results The Cultured IPECs have no contamination of other cells .The extracted RNA was ideal and had no degradation.RT-PCR analysis showed that mannose-receptor's mRNA was expressed in cultured IPECs in vitro.ConclusionCultured IPECs may express the mannose-receptor,and may have similar potential of phagocytosis to ROS with REPCs.
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 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.
Objective
To investigate the bloodretinal barrier（BRB）function of porcine retinal pigment epithelial（RPE）cells cultured in vitro.
Methods
Primary porcine RPE cells were cultured，and the third generation were inoculated in a microporous filter with the filter membrane of polyvinylpyrrolidone（PVP）-free polycarbonate membrane.After 1，2，3 and 4 weeks of culture，the surface of filter membrane was observed by light microscope，and after 2 weeks of culture，the section of filter membrane was observed by light microscope and transmission electron microscope.Transepithelial electrical resistance（TER）was detected and the permeability was measured with fluorescein sodium（FS）and horseradish peroxidase（HRP）.
Results
Primary porcine RPE cells were cultured successfully.RPE cells converged1week after inoculation; 2 and 3 weeks after inoculation，the density of RPE cells did not changed obviously; 4 weeks after inoculation，the density of RPE cells decreased.The characteristics of polarized growth of monolayer were found in RPE cells on the surface of filter membrane; 2 weeks after inoculation，the TER of RPE cells was（97.44plusmn;11.36）Omega;/cm2 which maintained till the 3rd week after inocubation.After incubated for 30 minutes，only 0.27% of FS and 0.17% HRP reached the inferior filter membrane，and the permeability rate of SF with low molecular weight was higher than which of HRP with high molecular weight.
Conclusions
The filter with PVPfree polycarbonate membrane may be used to set up the model of RPE cells with polarized growth of monolayer and investigate the barrier function of RPE cells.
(Chin J Ocul Fundus Dis, 2006, 22: 188-191)
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 study the effects of advanced glycation end (AGEs) products induced by bovine serum albumin (BSA) on the survival and the morphology of bovine retinal endothelial cells (BREC) and pericytes (BRP).
Methods
BSA with the final concentration of 50 mg/ml was incubated in PBS, containing 500 mmol/L D-glucose, for 12 weeks under 37℃. AGEs-BSA was purified by Sephacryl S-300 column chromatography and was confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of AGEs-BSA was determined by the method of commassie protein assay. In order to detect the toxic effects of AGEs-BSA on cultured BREC and BRP, groups of AGEs-BSA and BSA with different concentration and untreated control were set up. Phase contrast microscope was used to observe the effect of AGEs-BSA and BSA (with the concentration of 500mu;g/ml and actuation duration of 48 hours) on morphology of BREC and BRP.
Results
As the dosage of AGEs-BSA increased, the number of inhibited cells increased. When the concentration of AGEs-BSA was 500mu;g/ml, the inhibited BREC in AGEs-BSA group was (72.8plusmn;15.9)% of which in untreated control group, and the inhibited BRP was (64.8plusmn;9) % of which in untreated control group. AGEs-BSA with low concentration promoted the proliferation of endothelial cells, but there was no significant difference between AGEs-BSA and the control group (P=0.231). Inhibited proliferation and abnormal morphology were seen under the phase contrast microscope while the normal morphology of cells was found in BSA and control group.
Conclusion
AGEs-BSA with the high concentration may inhibit the growth of both BREC and BRP, which leads the loss of BRP and damage of vascular function. These results suggest that nonenzymatic glycosylation plays a major role in diabetic complications.
(Chin J Ocul Fundus Dis, 2006, 22: 11-15)
Objective
To describe cultured human retinal pigment epithelial (RPE) cells transdifferentiation and investigate the effects of human vitreous fluid on the morphologic and cytoskeleton changes of RPE cells in vitro.
Methods
Cytoskeleton characteristics in the 2nd, 5th, 8th passage of RPE cells in normal culture, which included cytokeratin 18 (CK18) and α-smooth muscle actin (α-SMA) were analyzed by Western blot. RPE cells were cultured in human vitreous-conditioned medium (VCM) at the concentration of 1∶4 for 6 days, morphologic changes were examined by light and electron microscopy, and cytoskeleton characteristics were analyzed by imunocytochemistry and Western blot.
Results
During culture in vitro, RPE cells lost epithelial characteristics and aquired fibroblast-like phenotype. The expression of CK18 was the highest at the 5th passage, and it decreased in the following passage, but α-SMA increased gradually. The morphologic transdifferentiation from epithelial to fibroblast-like cells of RPE was accelerated by VCM. Ultrastructural changes such as decreased microvilli and gradually increased rough endoplasmic reticulum and Golgi complex were found during the cultivation. CK18 produced by RPE cells decreased in VMC (P＜0.05), and α-SMA increased (P＜0.01).
Conclusion
Morphologic changes in epithelialmesenchymal transdifferenetiation of RPE cells are stimulated by VCM and accomplied by the shift of cytoskeleton proteins, The results imply that cells migration may be decreased and contraction may be enhanced in VCM. It may suggest that vitreous accelerates the pathogenesis of PVR and RPE cells play an important role.
(Chin J Ocul Fundus Dis, 2002, 18: 289-292)
