Objective To examine the biological characteristic changes in thededifferenciated human articular chondrocytes by the bioreactor culturing in vitvo.Methods The cartilage tissue was obtained from the joints of the adult human. The chondrocytes were isolated from the cartilage tissue with the type Ⅱ collagenase digestion（0.2%, 37℃, 3 h）and were cultured in DMEMF12 supplemented with 20% fetal bovine serum (FBS) with 1 ng/ml of TGF-β1and 5 ng/mlof FGF-2. After about 20 passages by the monolayer culture，the cells were then transferred to the bioreactor culturing of the rotational cell culture system (RCCS) for a 3-week sequence culture. The cell counting was performed with the platelet counter, and the doubling time for each passage of thecells was determined. The frozen section was stained with HE. The differentiated phenotype was evaluated by histochemistry or immunohistochemistry. Results When the monolayer culture was performed without any growth factors, the chondrocytes were rapidly proliferated within 3 passages (average doubling time, 59 h),but at the same time, dedifferentiation was also progressing rapidly. After the4th passage, most of the cells were dedifferenciated and the proliferation was decreased. With the growth factors (TGF-β1/FGF-2), the speed of the expansion was accelerated (average doubling time, 47 h), but the speed of the dedifferentiation was slowed down. After 20 passages were performed with the monolayer culture, the dedifferentiated chondrocytes could be redifferentiated when they were cultured for 3 weeks with RCCS. Then, the Safranine-O staining was bly positive for the cells, positive for aggrecan and collagen Ⅱ, but negative for collagen Ⅰ, with a wellregained phenotype. Conclusion The bioreactor culturing of the dedifferenciated human articular condrocytes can regain the differentiated phenotype and it is a useful method of obtaining the human articular chondrocytes in large amounts and in a differentiated phenotype in vitro.
Objective To investigate the protective effect and mechanism of erythropoietin (EPO) on injury of human retinal pigment epithelial (hRPE) cell induced by hydrogen peroxide (H2O2). Methods Take subcultured hFRPE cells as study target. They were treated with 800 mu;mol/L of H2O2 for 3 hours to establish the cell injury model. The cultured cells were divided into three groups：control group, simply injury group and therapeutic group which again divided into 10 IU/ml, 20 IU/ml, 40 IU/ml,60 IU/ml subgroups according to the concentration of recombinant human erythropoietin(rhEPO). NF-kappa;B was measured by immunohistochemistry. The content of Malondialdehyde(MDA) which was the product of cellular lipid peroxidation and the releasing rate of lactate dehydrogenase（LDH）were estimated by chromatometry. Results H2O2 could elevate the level of MDA and the releasing rate of LDH, compared simply injury group with control group, the differences were significant.(tLDH＝29.746,tMDA＝20.426,Plt;0.05); Compared all of therapeutics groups with simply injury group, the releasing rate of MAD and LDH were decreased obviously, the differences were significant.（LDH t10IU＝5.770，t20IU＝12.774，t40IU＝19.818，t60IU＝24.833，Plt;0.05；MDA t10IU＝5.345，t20IU＝10.278，t40IU＝18.571，t60IU＝20.247，Plt;0.05）; The correlative analysis results of each therapeutic subgroup were: ①the concentration of rhEPO had negative correlation with the relation rate of LDH and the content of MDA(r=-0.976，P=0.024； r=-0.968，P=0.032) ; ②the concentration of rhEPO had positive correlation with the nuclear translative rate of NF-kappa;B(r=0.998，P=0.002); ③the nuclear translative rate of NF-kappa;B had negative correlation with the content of MDA(r=－0.954，P=0.046). Conclusion EPO can protect hFRPE cells from the injury of H2O2, the mechanism may be related to the activation of NF-kappa;B.
Objective To study the effect of transforming growth factor β1(TGF-β1) and insulin-like growth factor 1(IGF-1) during the induction course from marrow mesenchymal stem cells (MSCs) to chondrocytes and to observe the effect of cell density on cell induction. Methods Differential time adherent methods were used to purify MSCs obtained from the bone marrow of Kunming mice. MSCs were cultured under special conditionsto induce themto differentiate into chondrocytes. Toluidine blue staining and immunofluoresence were used to identify those induced chondrocytes.TGF-β1 and IGF-1 were used individually or in combination under two different culture patterns: pellet culture and monolayer culture. According to different growth factors, experiment included 3 experimental groups(TGF-β1+IGF-1 group,10 ng/mland 50 ng/ml respectively;TGF-β1 group, 10 ng/ml; and IGF-1 group, 50 ng/ml) and control group(without growth factor). In TGF-β1+IGF-1 group, toluidine blue staining and immunofluoresence staining were carried out at 14 days and 21 days. The effect ofTGF-β1 and IGF-1 on the expression of collagen Ⅱgene was detected by RT-PCR at 7, 14 and 21 days of induction; the expressionsof collagen Ⅱ were compared between two culture patterns. Results In TGF-β1+IGF-1 group, the histological examination and immunofluoresence showed that those inducted chondyocytes could express collagen Ⅱ at 14 days. The gel electrophoresis results showed that the fragment of collagen Ⅱ gene was seen in TGF-β1+IGF-1 group andTGF-β1 group and that no fragment ofcollagen Ⅱ gene was seen in IGF-1 group and control group. The expression of collagen Ⅱ gene was ber in TGF-β1+ IGF-1 group than inTGF-β1 group, showing significant difference(Plt;0.05). Cells expressed more collagen Ⅱ under pellet culture than under monolayer culture. Conclusion IGF-1 could enhance the effect ofTGF-β1 during the induction course from MSCs to chondrocytes. A certain extent of high cell density is more effective for MSCs to differentiate into chondrocytes.
The effects of pentagastrin (PG) on the viable cell count (Α value) and the synthesis of DNA (CPM value) of primary cultured large bowel carcinoma cells in 25 patients were evaluated in vitro by MTT assay,3H-TdR incorporation. The results showed that Α value and CPM value in well， moderately and poorly-differentiated carcinoma cells were higher than normal control (Ｐlt;0.01，Ｐ＜0.05). The proliferative effect was significant at a dose of 0.3907 μg/ml in well-differentiated carcinoma cells, and at a dose of 6.2500μg/ml in moderately and poorly-differentiated carcinoma cells. These indicat that PG has the proliferative effect on large bowel carcinoma cells. These results provide an experimental foundation for the endocrine therapy for patients with large intestine carcinoma, especially by using gastrin receptor antagonists for well-differentiated carcinoma.
Objective To observe the effects of culture medium of amniotic cells on NO and NOS in retinal tissues of rabbits in vitro in order to provide a protective method for antioxidation in retina transplantation. Methods Thirty adult healthy rabbits (30 right eyes) were divided into 3 groups. Group I: fresh retinal tissue; group II: routine culture medium; group III: culture medium of amniotic cells. The retinal tissues in group II and III were cultured in the corresponding culture medium for 1 week. The content of NO and NOS in retinal tissues in the 3 groups were determined. Results Compared with group I, the content of NO and NOS of group II increased obviously (t=3.821, 3.854; P＜0.001). There was no statistical difference of content of NO and NOS between group I and III (t=1.657, 1.745; P＞0.05). Conclusion Culture medium of amniotic cells may remove free radicals and enhance the ability of antioxidation. (Chin J Ocul Fundus Dis，2004，20：366-368)
Objective To isolate neural stem cells (NSCs) from rabbit retina and brain, and induce differentiation of those NSCs using different culture media. Methods Single-cell suspensions of retina and cerebral cortex were prepared from rabbit embryo, cultured in 5 types of different media to isolate the NSCs by continual passages. After 3 passages, NSCs were induced to differentiation in 2 types of different media for 8 to 10 days. NSCs and inducedretinal cells were examined by immunofluorescence and flow cytometry for the expression pattern of some specific antigens.Results Immunofluorescence showed that NSCs from retina and brain, cultured in serumfree media, both expressed Nestin partially. Flow cytometry showed that Nestin positive cells were significantly decreased while the Rhodopsin and Thy1.1 positive cells were increased after induction. Compared with the combined induction of alltrans retinoid acid (ATRA) and serum, 5%FBS (fetal bovine serum) led to higher expression of Rhodopsin（P＜0.01），but lower expression of Thy1.1（P=0.01）.Conclusion Serumfree media with N2, EGF, bFGF, LIF is the best for NSCs purification. Both induciton media can induce NSCs to differentiate.Retina NSCs have higher potentials to differentiate into retinal neuroepithelial cells than brain NSCs.
Osteoblasts were cultured and isolated from a piece of tibial pettiosteum of four New-Zealandrabbits. After subeultured,these cells Were incubatd in vitro with tritiated thvmidine for 36 hoursand then these labeled cells were implanted in the subeutaneous layer of the defects of the auriclarcartilage and the radial bone, After 2 weeks and 4 weeks respectively, these rabbits were killed andthe spoimens were obtained from the site where the cells had been transplanted. The transformation of these cells was observed by autoradiographic method. The results indicated that nearly all of the cultured cells were labeled. After 2 weeks, it was observed that many labeled osteoblasts were in different stages of differentiation, some were beried by extracellular matrix and resembled osteocyte, thers were differentiated into chondrocyte-like cell. In addition, some labeled osteoblasts were congregated in the form of multinucleated osteoclast. After 4 weeks , in the subcutaneous layer the labeled osteoblasts were changed to osteoid tissue and in the defect of the auricular crtilage these cells transformed into chondritic tissue; moreover, those labeled osteoblsts which had been implanted into the radial defect had differentiated into typical bone tissue. The results of this research indicated that the osteoblasts isolated from the periosteum if reimplanted to the same donor might be possible to repair the bone and cartilage defects.
Objective To investigate the feasibility of differentiation of invitro induced rat bone marrowderived mesenchymal stem cells（rMSCs） into retinal pigment epithelial （RPE） cells.Methods The rMSCs from BrwonNorway (BN) rats were isolated and cultured by adherent screening method. RPE cells lysate made by repeated freezethawing was put into the rMSCs culture system to identify whether the induced cells could express characteristic label cytokeratin（CK）and S-100 simultaneously or not.Results The growth rate of rMSCs induced by RPE cells lysate was slower and protuberant burr surrounded the fusiform cells. The results of immunoblotting and double immunofluorescence showed that partial induced cells expressed CK and S-100 simultaneously. The result of flow cytometry indicated that 14.1% induced cells expressed CK and S-100 simultaneously.Conclusion Induced by RPE cells lysate, rMSCs can differentiate into RPE cells.
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 investigate the feasibility oftissue engineered intervertebral disc for regeneration of discs. Methods A three-dimensional porous poly(L-lactic-co-glycolic acid) (PLGA) scaffold was fabricated by temperature induced phase separation method. Human fetal disc cells were isolated and cultured in vitro. The disc cells labeledwith a PKH-26 fluorescent dye were seeded into a threedimensional porous scaffold. The proliferation of disc cells with PKH-26 fluorescent labels was assessed by using MTT uptake, laser fluorescence microscopy and SEM. Results Human fetal disc cells displayed a polygonal shape in primary monolayer culture. A regular arrangement and microtubules orientationstructure scaffold with 50-300 μm in diameter was fabricated by thermal-induced phase separation technique. MTT uptake and fluorescent microscopy examination indicated that the seeded disc cells were viable and showed proliferation activity within a porous scaffold. Conclusion The above findings support potential applications of tissue engineered disc in treatment of disc degenerative diseases.