Objective To investigate the possibility of creation of tissue engineered heart valve leaflets in vitro . Methods Aorta were obtained from 9 hybrid young pigs. The endothelial cell, fibroblast and smooth muscle cells were isolated and cultured to get enough cell. The expanded fibroblast, smooth muscle cell,and endothelial cells were seeded on the polymers sequentially. The cell polymer constructs were sent for scanning electron microscopy(SEM) examination after cultured for 7, 14, and 28 days. Histological examination were performed after the cell polymer constructs cultured for 28 days. Results SEM showed that the number of cells on the polymers increased as the culture time prolonged, with the formation of matrix. After 28 days, there were a great number of cells and large amount of matrix on the scaffolds. The confluent cell had covered a large area of the polymers. Hematoxylin and eosin(HE) stain showed large amount of cells attached to the polymers. Conclusion With the viability of the cultured cellular scaffolds,it is possible to create tissue engineered heart valve leaflets in vitro.
Objective To investigate the expression of Human leucocyte antigen(HLA)-DP, -DQ, -DR and CD40 in human retinal pigment epithelial (RPE) cells, to determine their molecule expression in immune response process, and their abilities to stimulate T lymphocyte activation. Methods Human RPE cells were cultured with or without (IFN respectively. Expression of HLA-DP, -DQ, -DR and CD40 was measured by immunohistochemical staining. Meanwhile, peripheral blood mononuclear cells (PBMC) were cocultured with RPE cells in vitro, and then the expression of activated lymphocytes CD69 was measured by fluorescence activated cell sorter(FACS). Results Expression of HLA-DP, -DQ, -DR and CD40 antigen were enhanced by gamma;-interferon inducement. Increasing amount of CD69 positive lymphocytes were found in the co-culture system of RPE cells and PBMC. Conclusion T-lymphocytes in the peripheral blood were activated by human RPE cells which is antigen presenting cells with immunological characteristics potential.
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 feasibil ity and effect of inducing adi pose-derived stem cells (ADSCs) treated with growth differentiation factor 5 (GDF-5) to undergo chondrogenic differentiation in vitro. Methods Six healthy Japanese rabbits aged 3 months (2-3 kg) of clean grade were chosen, irrespective of sex. ADSCs were isolated and cultured with collagenase digestion, then were detected and identified by vimentin immunohistochemistry and CD44, CD49d, CD106immunofluorescence staining. ADSCs at passage 3 were used and the cell density was adjusted to 1 × 106/mL, then the ADSCs were treated with 0, 10, 100 ng/mL GDF-5 and common cultural medium, respectively. The morphology changes of the induced ADSCs were observed by inverted contrast phase microscope and their growth state were detected by MTT. The mRNA quantities of Col II and proteoglycan expressed by the induced ADSCs were detected with RT-PCR. The Col II proteoglycan synthesized by the induced ADSCs were detected with alcian blue staining, toluidine blue staining, immunohistochemistry staining, and Western blot method. Results ADSCs mostly presented small sphere, fusiform and polygon shape with positive expression of CD44 and CD49d and negative expression of CD106 and vimentin. The ADSCs treated with 100 ng/mL GDF-5 presented sphere or sphere-l ike change and vigorous prol iferation. The mRNA quantities of Col II and proteoglycan synthesized by the induced ADSCs treated with 0, 10, 100 ng/mL GDF-5 and common cultural medium increased in a dose-dependent manner at 7 days. There were significant differences among all the groups (P lt; 0.05), except that no significant difference was evident between the 0 ng/mL group and the 10 ng/mL group (P gt; 0.05). When ADSCs were treated with 100 ng/mL GDF-5 for 14 days, the Col II and the mRNA and protein quantities of ptoteoglycan reached the peak, and the results of alcian blue, toluidine blue and Col IIimmunohistochemistry staining were positive. Conclusion ADSCs treated with certain concentration of GDF-5 have higher expression of Col II and proteoglycan and possess partial biological function of chondrocyte.
ObjectiveTo observe the effect of subretinal injection of retinal pigment epithelium (RPE) cells for RPE in mice. MethodsA total of 30 postnatal day 7 C57BL/6J mice were randomly divided into normal mice group, OIR model group and OIR model cell transplanted group, 10 mice in each group. The OIR model was induced in mice of OIR model group and OIR model cell transplanted group. The RPE cells were subretinal injected into the RPE of mice in OIR model cell transplanted group. At 20 days after the injection, the RPE thickness was evaluated by fluorescence microscope. The expression of RPE65, Bestrophin and zonula occludens-1 (ZO-1) were estimated by Western blot and real-time quantitative PCR (RT-PCR). ResultsThe thickness of RPE in OIR model mice was thinner than that in normal mice; the thickness of RPE in OIR model cell transplantation mice was significantly thicker than that in the OIR model mice. The results of Western blot and RT-PCR indicated that the differences of protein (F=8.597, 18.864, 25.691) and mRNA expression (F=39.458, 11.461, 34.796) of RPE65, Bestrophin, ZO-1 were statistically significant between OIR model group and OIR model cell transplanted group (P < 0.05). ConclusionsSubretinal injection of RPE cells can promote RPE thickening. RPE65 and Bestrophin protein relative expression levels increased, ZO-1 protein relative expression levels reduced; mRNA expression levels of RPE65, Bestrophin and ZO-1 genes increased.
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.
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 develop a reliable method for primary culture of normal human peritoneal mesothelial cells. Methods Human peritoneal mesothelial cells were dissociated by a mixture of pancreatin and ethylene diamine tetraacetic acid with a magnetic puddler. Inverted phase contrast microscope was used to observe the morphological structures of cells, approximate process of growth. Calretinin was used to identify the mesothelial cells. Results On the 4th d of culture, mesothelial cells adhered to the culture dish. After day 14, mesothelial cells confluenced gradually and grew well like the slabstone. Calretinin was positively expressed by mesothelial cells after 5 d of cultivation. The mesothelial cell population of subculture was less than that of the primary culture. Conclusion A reliable method for primary culture of normal human peritoneal mesothelial cells has been successfully developed, by which sufficient amount of highly purified normal human peritoneal mesothelial cells can be obtained.