RESUMEN
BACKGROUND: Studies have shown that mesenchymal stem cells can reduce inflammation, promote wound healing, and reduce scar formation in wound healing. However, previous two-dimensional culture environment can lead to differences in gene expression, signal transduction, and morphology because of intracellular contact inhibition. OBJECTIVE: To investigate whether the ability of wound healing related factors secreted by human amniotic mesenchymal stem cells is affected by two-dimensional or three-dimensional culture environment. METHODS: The human amniotic mesenchymal stem cells cultured by traditional enzyme digestion method were inoculated in traditional cell culture flask (two-dimensional culture group) and ShakeGelTM 3D hydrogel (three-dimensional culture group) and induced to differentiate into adipocytes, osteoblasts, and chondrocytes, respectively. The direction of cell differentiation was determined by immunofluorescence staining. Human amniotic mesenchymal stem cells were fused to 70%-80% in two culture environments, and the growth characteristics and morphology of cells were observed under inverted phase contrast microscope and laser confocal microscope. After 24 hours of culture, relative mRNA expression of wound healing-related factors was detected by reverse transcription-quantitative polymerase chain reaction. After 48 hours of culture, the protein expression of wound healing-related factors was detected by the enzyme-linked immunosorbent assay. RESULTS AND CONCLUSION: (1) Human amniotic mesenchymal stem cells cultured in two-dimensional culture group were flat and spindle-shaped, which was a typical mesenchymal stem cell-like morphology. Human amniotic mesenchymal stem cells in the three-dimensional culture group were round and evenly dispersed in each layer of the hydrogel. (2) Human amniotic mesenchymal stem cells in the three-dimensional culture group exhibited the potential to differentiate into adipocytes, osteoblasts, and chondrocytes. (3) The mRNA expression of interleukin-6, interleukin-8, epidermal growth factor, basic fibroblast growth factor, hyaluronic acid, hepatocyte growth factor, and vascular endothelial growth factor in the three-dimensional culture group was significantly higher than that in the two-dimensional culture group (P 0.05). (4) The protein expression of interleukin-6, interleukin-8, interleukin-10, epidermal growth factor, basic fibroblast growth factor, hepatocyte growth factor, transforming growth factor β1 and vascular endothelial growth factor in the three-dimensional culture group was significantly higher than that in the two-dimensional culture (P 0.05). (5) These findings suggest that human amniotic mesenchymal stem cells cultured in three-dimensional hydrogel show better morphology and more encouraging paracrine effect of wound healing related factors than those cultured in traditional two-dimensional culture environment.
RESUMEN
ABSTRACT OBJECTIVE：To evaluate the in vitro and in vivo genotoxicity of emodin-8-O-β-D-glucoside（EG），and to compare the difference of in vitro cell test and in vivo test of rats. METHODS：2D and 3D hepatocyte models were established by in vitro two-dimensional（2D）and three-dimensional（3D）cell culture. After modeling，2D and 3D hepatocyte were divided into blank control group（0.5％ DMSO），mitomycin C group（positive control，0.1 μg/mL），EG low-dose，medium-dose and high-dose groups（10，50，200 μg/mL），respectively. The micronucleus ratio and tail DNA％ of HepaRG cells were detected. SD rats were divided into blank control group（0.5％ sodium carboxymethyl cellulose），ethyl methanesulfonate group（positive control，200 mg/kg），EG low-dose，medium-dose and high-dose groups（100，300，1 000 mg/kg），with 6 rats in each group. They were given medicine intragastrically for consecutive 15 d，once a day. 15 days later，the micronucleus formation rate of bone marrow polychromatic erythrocytes and hepatocytes，the tail DNA％ and tail distance of peripheral blood lymphocytes and hepatocytes were measured. RESULTS：In the in vitro 2D HepaRG hepatocyte model，compared with blank control group，the micronucleus formation rate and tail DNA％ of HepaRG cell were increased significantly in mitomycin C group （P＜0.01）. There was no statistical significance in micronucleus formation rate and tail DNA％ of HepaRG cell among EG groups（P＞0.05）. In 3D HepaRG cell model， compared with blank control group， micronucleus formation rate and tail DNA％ of HepaRG cell were increased significantly in mitomycin C group （P＜0.01 or P＜0.001）， while tail DNA％ of HepaRG cell wasincreased significantly in EG high-dose group（P＜0.01）. In the in vivo test，compared with blank control group，the micronucleus formation rate of bone marrow polychromatic erythrocytes and hepatocytes，the tail DNA％ and tail distance of peripheral blood lymphocytes and hepatocytes were all increased significantly in ethyl methanesulfonate group（P＜0.01）. Tail DNA％ of peripheral blood lymphocytes was increased significantly in EG high-dose group （P＜0.01）. There was no statistical significance in the micronucleus formation rate of bone marrow polychromatic erythrocytes and hepatocytes，the tail DNA％ and tail distance of hepatocytes among EG groups（P＞0.05）；with the increase of dose，there was an increasing trend. CONCLUSIONS：The results of this study suggest that in 2D cell model，EG not lead to chromosome breakage and DNA damage，but the long-term administration and repeated administration in vivo of 3D cell model show that EG has a certain risk of DNA damage，so the evaluation results of 3D HepaRG cell model are more similar to those of rats in vivo. KEYWORDS Emodin-8-O-β-D-glucoside；Genotoxicity；Two-dimensional culture；Three-dimensional culture；Rat；Micronucleus test