DNMT1-mediated epigenetic suppression of FBXO32 expression promoting cyclin dependent kinase 9 (CDK9) survival and esophageal cancer cell growth.

Esophageal cancer (EC) is a common and serious form of cancer, and while DNA methyltransferase-1 (DNMT1) promotes DNA methylation and carcinogenesis, the role of F-box protein 32 (FBXO32) in EC and its regulation by DNMT1-mediated methylation is still unclear. FBXO32 expression was examined in EC cells with high DNMT1 expression using GSE163735 dataset. RT-qPCR assessed FBXO32 expression in normal and EC cells, and impact of higher FBXO32 expression on cell proliferation, migration, and invasion was evaluated, along with EMT-related proteins. The xenograft model established by injecting EC cells transfected with FBXO32 was used to evaluate tumor growth, apoptosis, and tumor cells proliferation and metastasis. Chromatin immunoprecipitation (ChIP) assay was employed to study the interaction between DNMT1 and FBXO32. HitPredict, co-immunoprecipitation (Co-IP), and Glutathione-S-transferase (GST) pulldown assay analyzed the interaction between FBXO32 and cyclin dependent kinase 9 (CDK9). Finally, the ubiquitination assay identified CDK9 ubiquitination, and its half-life was measured using cycloheximide and confirmed through western blotting. DNMT1 negatively correlated with FBXO32 expression in esophageal cells. High FBXO32 expression was associated with better overall survival in patients. Knockdown of DNMT1 in EC cells increased FBXO32 expression and suppressed malignant phenotypes. FBXO32 repressed EC tumor growth and metastasis in mice. Enrichment of DNMT1 in FBXO32 promoter region led to increased DNA methylation and reduced transcription. Mechanistically, FBXO32 degraded CDK9 through promoting its ubiquitination.

Interaction effect of chronic cough and ageing on increased risk of exacerbation in patients with asthma: a prospective cohort study in a real-world setting.

Background: Older adults with asthma have the greatest burden and worst outcomes, and there is increasing evidence that chronic cough (CC) is associated with asthma severity and poor prognosis. However, the clinical characteristics of older adult patients with both asthma and CC remain largely unknown. Methods: Participants with stable asthma underwent two cough assessments within 3 months to define the presence of CC. Patients were divided into four groups based on CC and age (cut-off ≥60 years). Multidimensional assessment was performed at baseline, followed by a 12-month follow-up to investigate asthma exacerbations. Logistic regression models were used to explore the interaction effect of CC and age on asthma control and exacerbations. Results: In total, 310 adult patients were prospectively recruited and divided into four groups: older CC group (n=46), older non-CC group (n=20), younger CC group (n=112) and younger non-CC group (n=132). Compared with the younger non-CC group, the older CC group had worse asthma control and quality of life and increased airflow obstruction. The older CC group showed an increase in moderate-to-severe exacerbations during the 12-month follow-up. There was a significant interaction effect of CC and ageing on the increased moderate-to-severe exacerbations (adjusted risk ratio 2.36, 95% CI 1.47-3.30). Conclusion: Older asthma patients with CC have worse clinical outcomes, including worse asthma control and quality of life, increased airway obstruction and more frequent moderate-to-severe exacerbations, which can be partly explained by the interaction between CC and ageing.

Self-Reported Insufficient Sleep Is Associated With Clinical and Inflammatory Features of Asthma: A Prospective Cohort Study.

BACKGROUND: A few studies have explored the association between short sleep duration and worse asthma outcomes in patients with self-reported asthma; however, all of them were cross-sectional. OBJECTIVES: To investigate the association between self-reported sleep duration and asthma-related clinical and inflammatory characteristics and whether sleep duration is associated with asthma exacerbations (AEs) in the following year. METHODS: A prospective cohort study consecutively recruited participants with asthma, who were classified into short (n = 58), normal (n = 380), and long (n = 84) sleep duration groups. We investigated the clinical and inflammatory characteristics and exacerbations within a 1-year follow-up. RESULTS: Patients with short sleep duration were older and had significantly lower total IgE and FeNO levels and higher airway inflammation, characterized by increased levels of IL-6 and TNF-α in sputum than those of patients with normal sleep duration. Furthermore, they had a significantly increased risk for poorly controlled asthma (adjusted odds ratio = 2.741; 95% CI, 1.379-5.447; P = .004) and moderate to severe AEs (adjusted incidence rate ratio = 1.798; 95% CI, 1.098-2.942; P = .020). CONCLUSIONS: Short sleep duration was associated with non-type 2 inflammation and is an independent risk factor for future AEs. Therefore, as a potentially treatable trait, sleep duration may have clinical implications for asthma management.

Esophageal metastases from primary lung cancer: a case report.

BACKGROUND: Primary lung cancer is one of the most frequently diagnosed cancers. The common metastatic sites are the liver, bones, brain, adrenal glands and central nervous system. However, gastrointestinal metastases, particularly esophageal metastases, from lung cancer are rare. There are no cases of esophageal metastases from lung cancer which refer to its particular treatment. CASE PRESENTATION: We report a case of esophageal metastases from lung cancer. The patient was a 55-year-old Han Chinese man who first attended our hospital due to dry cough and was diagnosed with late-stage lung cancer. Three months later, the patient complained of dysphagia. Endoscopic ultrasonography (EUS) and pathological examination of the biopsy specimen was performed to confirm the lesion was metastases from lung cancer. Thyroid transcription factor 1 (TTF-1), cytokeratin 7 (CK-7) and napsin A were positive by immunohistochemistry examination. These results reconfirmed the diagnosis of esophageal metastases from lung cancer. CONCLUSIONS: Esophageal metastasis from lung cancer is very rare. It may be alleviated with personalized chemotherapy. In addition, molecular targeted therapy for patients with epidermal growth factor receptor (EGFR) mutations may be reasonable.

Early Warning Factors of Death in COVID-19 Patients.

The infectious coronavirus disease 2019 (COVID-19) has spread all over the world and been persistently evolving so far. The number of deaths in the whole world has been rising rapidly. However, the early warning factors for mortality have not been well ascertained. In this retrospective, single-centre cohort study, we included some adult inpatients (≥18 years old) with laboratory-confirmed COVID-19 from Renmin Hospital of Wuhan University who had been discharged or had died by Apr. 8, 2020. Demographic, clinical and laboratory data at admission were extracted from electronic medical records and compared between survivors and non-survivors. We used univariable analysis, Cox proportional hazard model analysis and receiver operating characteristic (ROC) curve to explore the early warning factors associated with in-hospital death. A total of 159 patients were included in this study, of whom 86 were discharged and 73 died in hospital. Hypertension (52.1% vs. 29.1%, P=0.003) and coronary heart disease (28.8% vs. 12.8%, P=0.012) were more frequent among non-survived patients than among survived patients. The proportions of patients with dyspnoea (67.1% vs. 25.6%, P<0.001), chest distress (58.9% vs. 26.7%, P<0.001) and fatigue (64.4% vs. 25.6%, P<0.001) were significantly higher in the non-survived group than in the survived group. Regression analysis with the Cox proportional hazards mode revealed that increasing odds of in-hospital death were associated with higher IL-6 (odds ratio 10.87, 95% CI 1.41-83.59; P=0.022), lactate (3.59, 1.71-7.54; P=0.001), older age (1.86, 1.03-3.38; P=0.041) and lower lymphopenia (5.44, 2.71-10.93; P<0.001) at admission. The areas under the ROC curve (AUCs) of IL-6, lymphocyte, age and lactate were 0.933, 0.928, 0.786 and 0.753 respectively. The AUC of IL-6 was significantly higher than that of age (z=3.332, P=0.0009) and lactate (z=4.441, P<0.0001) for outcome prediction. There was no significant difference between the AUCs of IL-6 and lymphocyte for outcome prediction (z=0.372, P=0.7101). It was concluded that the potential risk factors of higher IL-6, lactate, older age and lower lymphopenia at admission could help clinicians to identify patients with poor prognosis at an early stage.

[Ecotoxicological effects of transgenic mCry1Ac maize (BT799) on zebrafish]. / 转mCry1AcåŸºå› çŽ‰ç±³BT799对斑马鱼的生态毒理学效应.

The safety of feed derived from genetically modified (GM) crops is one of the focuses of attention. To evaluate the ecotoxicological effects of transgenic mCry1Ac maize (BT799) on fish, zebrafish (Danio rerio) were fed extruded feeds containing either 20% GM maize (GMF) or its parental control maize (PF), GM maize meal (GMM) or its parental control maize meal (PMM), and a control commercial feed (CF), respectively. The growth performance, histopathology, reproduction, antioxidant enzyme activity and mRNA expression levels of sensitive protein in the liver were investigated over the course of a 98-day feeding trial. The results showed that transgenic mCry1Ac maize had no significant effect on growth, histopathology of the liver, brain and intestinal tract, fecundity, hatching rate of fertilized eggs, superoxide dismutase (SOD), catalase (CAT) activity, mRNA expression levels of SOD and CAT, or heat shock protein 70 (HSP70) and vitellogenin (VTG) in the liver. However, zebrafish fed the commercial feed exhibited significantly greater weight, longer length, and higher specific growth rate than those fed feeds (GMF and PF) and maize meals (GMM and PMM). The hatching rate of zebrafish in the feed groups was significantly lower than that of the maize meal groups and the commercial feed group. The mRNA transcriptional levels of VTG were significantly higher in the liver for the feed groups (3.85±0.76) than that for the maize meal groups (1.60±0.56). These results suggest that transgenic mCry1Ac maize has no ecotoxicological effects on zebrafish. However, the differences in nutrient composition and palatability between the extruded experimental feeds and the commercial feed would lead to significant diffe-rences in some parameters.

Mechanism of Cxc Chemokine Ligand 5 (CXCL5)/Cxc Chemokine Receptor 2 (CXCR2) Bio-Axis in Mice with Acute Respiratory Distress Syndrome.

BACKGROUND Acute respiratory distress syndrome (ARDS) is a common acute and severe disease in clinic. Recent studies indicated that Cxc chemokine ligand 5 (CXCL5), an inflammatory chemokine, was associated with tumorigenesis. The present study investigated the role of the CXCL5/Cxc chemokine receptor 2 (CXCR2) bio-axis in ARDS, and explored the underlying molecular mechanism. MATERIAL AND METHODS The pathological morphology of lung tissue and degree of pulmonary edema were assessed by hematoxylin-eosin staining and pulmonary edema score, respectively. Real-time PCR and Western blot analysis were performed to detect the expression levels of CXCL5, CXCR2, Matrix metalloproteinases 2 (MMP2), and Matrix metalloproteinases 9 (MMP9) in lung tissues. Enzyme-linked immunosorbent assay (ELISA) was performed to determine the expression levels of CXCL5 and inflammatory factors (IL-1ß, IL-6, TNF-alpha, and IL-10) in serum. RESULTS The results demonstrated that diffuse alveolar damage and pulmonary edema appeared in lipopolysaccharide (LPS)-induced ARDS and were positively correlated with the severity of ARDS. In addition, CXCL5 and its receptor CXCR2 were overexpressed by upregulation of MMP2 and MMP9 in lung tissues of ARDS. In addition, CXCL5 neutralizing antibody effectively alleviated inflammatory response, diffuse alveolar damage, and pulmonary edema, and decreased the expression levels of MMP2 and MMP9 compared to LPS-induced ARDS. CONCLUSIONS We found that CXCL5/CXCR2 accelerated the progression of ARDS, partly by upregulation of MMP2 and MMP9 in lung tissues with the release of inflammatory factors.

Real microgravity condition promoted regeneration capacity of induced pluripotent stem cells during the TZ-1 space mission.

Induced pluripotent stem cells (iPSCs) are reprogrammed somatic cells that gained self-renewal and differentiation capacity similar to embryonic stem cells. Taking the precious opportunity of the TianZhou-1 spacecraft mission, we studied the effect of space microgravity (µg) on the self-renewal capacity of iPSCs. Murine iPSCs carrying pluripotency reporter Oct4-GFP were used. The Oct4-EGFP-iPSCs clones were loaded into the bioreactor and exposed to µg in outer space for 14 days. The control experiment was performed in identical device but on the ground in earth gravity (1 g). iPSCs clones were compact and highly expressed Oct4 before launch. In µg condition, cells in iPSC clones spread out more rapidly than those in ground 1 g condition during the first 3 days after launch. However, in 1 g condition, as the cell density increases, the Oct4-GFP signal dropped significantly during the following 3 days. Interestingly, in µg condition, iPSCs originated from the spread-out clones during the first 3 days appeared to cluster together and reform colonies that activated strong Oct4 expression. On the other hand, iPSC clones in 1 g condition were not able to recover Oct4 expression after overgrown. Our study for the first time performed real-time imaging on the proliferation process of iPSCs in space and found that in µg condition, cell behaviour appeared to be more dynamic than on the ground.

Membrane-Binding Adhesive Particulates Enhance the Viability and Paracrine Function of Mesenchymal Cells for Cell-Based Therapy.

Understanding the fundamental cell-material interactions is essential to designing functional materials for biomedical applications. Although mesenchymal stromal cells (MSCs) are known to secrete cytokines and exosomes that are effective to treat degenerative diseases, the inherent property of biomaterials to modulate the therapeutic function of MSCs remains to be investigated. Here, a multivalent cell-membrane adhesive conjugate was generated through polyamindoamine (PAMAM) and an oligopeptide, IKVAV, and the conjugate was further complexed with hyaluronic acid (HA). The adhesive particulates were used to coat the surface of adipose-derived mesenchymal stromal cells (Ad-MSCs) and studied in the MSC spheroid culture. The analysis showed that the adhesive complexes formed via PAMAM conjugates and HA significantly promoted the proliferation and the gene expression of pro-angiogenesis cytokines in MSCs; the production of anti-inflammatory miRNAs in exosomes could also be elevated. The transplantation of the Ad-MSCs primed with PAMAM-IKVAV/HA composite particulates in a rat myocardial infarction model further demonstrated the beneficial effects of membrane-binding materials on improving the cell retention and tissue angiogenesis. The new function of membrane-binding adhesive materials potentially provides useful ways to improve cell-based therapy.

Efficient GSH delivery using PAMAM-GSH into MPP-induced PC12 cellular model for Parkinson's disease.

Glutathione (GSH) depletion has been an important contributor to the dysfunction of dopamine neurons. Polyamidoamine-GSH (PAMAM-GSH) was synthesized and the delivery effect of GSH into PC12 cells was tested. MTT assessment for cytotoxicity and reactive oxygen species (ROS) as well as nitrite oxide (NO) and intracelluar superoxide dismutase (SOD) detection for antioxidative ability were performed. Furthermore, the antiapoptotic ability was analysed by assessing caspase-3, JNK1/2 and Erk1/2 expression. Our data indicated that PAMAM-GSH is an effective agent to replenish GSH into PC12 cells. PAMAM-GSH developed its antioxidative and protective ability for 1-methyl-4-phenylpyridinium (MPP)-induced PC12 cells by reducing the intracellular levels of ROS and SOD activity as well as decreasing the release of NO. Meanwhile, PAMAM-GSH could inhibit caspase-3 activation and might show its antiapoptotic ability to MPP-induced PC12 cells through JNK2/Erk1/2 pathway. In summary, these studies suggest that PAMAM-GSH conjugate has an intrinsic ability to penetrate PC12 cells and deliver GSH into these cells which may provide a new strategy for clinical applications in the treatment of Parkinson's disease.

Covalent bonding of YIGSR and RGD to PEDOT/PSS/MWCNT-COOH composite material to improve the neural interface.

The development of coating materials for neural interfaces has been a pursued to improve the electrical, mechanical and biological performances. For these goals, a bioactive coating was developed in this work featuring a poly(3,4-ethylenedioxythiophene) (PEDOT)/carbon nanotube (CNT) composite and covalently bonded YIGSR and RGD. Its biological effect and electrical characteristics were assessed in vivo on microwire arrays (MWA). The coated electrodes exhibited a significantly higher charge storage capacity (CSC) and lower electrochemical impedance at 1 kHz which are desired to improve the stimulating and recording performances, respectively. Acute neural recording experiments revealed that coated MWA possess a higher signal/noise ratio capturing spikes undetected by uncoated electrodes. Moreover, coated MWA possessed more active sites and single units, and the noise floor of coated electrodes was lower than that of uncoated electrodes. There is little information in the literature concerning the chronic performance of bioactively modified neural interfaces in vivo. Therefore in this work, chronic in vivo tests were conducted and the PEDOT/PSS/MWCNT-polypeptide coated arrays exhibited excellent performances with the highest mean maximal amplitude from day 4 to day 12 during which the acute response severely compromised the performance of the electrodes. In brief, we developed a simple method of covalently bonding YIGSR and RGD to a PEDOT/PSS/MWCNT-COOH composite improving both the biocompatibility and electrical performance of the neural interface. Our findings suggest that YIGSR and RGD modified PEDOT/PSS/MWCNT is a promising bioactivated composite coating for neural recording and stimulating.

The paracrine effects of adipose-derived stem cells on neovascularization and biocompatibility of a macroencapsulation device.

The foreign-body response to biomaterials compromises the performance of many biomedical devices by severe fibrosis and limited neovascularization. Mesenchymal stem cells are known to secrete cytokines for treating inflammatory conditions. In this study, we aim to investigate whether the paracrine products of adipose-derived mesenchymal stem cells (ADSCs) can affect the microenvironment of biomaterials and improve tissue responses to biomaterial implants. A model system was built by loading ADSC spheroids into a macroencapsulation device composed of polytetrafluoroethylene (PTFE) filtration membranes. Soluble ADSC factors that diffused out of the device in vitro promoted the angiogenetic activity of endothelial cells and affected the secretion pattern of macrophages. In vivo study was carried out by subcutaneously embedding blank or ADSC-laden devices in rats. Following a 4 week implantation, the ADSC-laden devices were better vascularized and induced significantly less fibrotic tissue formation in comparison to the non-cellular controls. This study may facilitate our understanding of foreign-body responses and suggest new ways to improve the tissue reaction of biomedical devices for cell-based therapy.

The spatiotemporal development of intercalated disk in three-dimensional engineered heart tissues based on collagen/matrigel matrix.

Intercalated disk (ID), which electromechanically couples cardiomyocytes into a functional syncitium, is closely related to normal morphology and function of engineered heart tissues (EHTs), but the development mode of ID in the three-dimensional (3D) EHTs is still unclear. In this study, we focused on the spatiotemporal development of the ID in the EHTs constructed by mixing neonatal rat cardiomyocytes with collagen/Matrigel, and investigated the effect of 3D microenvironment provided by collagen/Matrigel matrix on the formation of ID. By histological and immmunofluorescent staining, the spatiotemporal distribution of ID-related junctions was detected. Furthermore, the ultra-structures of the ID in different developmental stages were observed under transmission electron microscope. In addition, the expression of the related proteins was quantitatively analyzed. The results indicate that accompanying the re-organization of cardiomyocytes in collagen/Matrigel matrix, the proteins of adherens junctions, desmosomes and gap junctions redistributed from diffused distribution to intercellular regions to form an integrated ID. The adherens junction and desmosome which are related with mechanical connection appeared earlier than gap junction which is essential for electrochemical coupling. These findings suggest that the 3D microenvironment based on collagen/Matrigel matrix could support the ordered assembly of the ID in EHTs and have implications for comprehending the ordered and coordinated development of ID during the functional organization of EHTs.

Real-time tracking of adipose tissue-derived stem cells with injectable scaffolds in the infarcted heart.

Adipose tissue-derived stem cells (ADSCs) has shown promise in the emerging field of regenerative medicine. Many studies have highlighted the importance of coadministering a "scaffold" for increasing intramyocardial retention of stem cells. In this work, an optimized method was developed for efficient transduction of ADSCs with a lentiviral vector carrying a triple-fusion reporter gene that consists of firefly luciferase, monomeric red fluorescence protein, and truncated thymidine kinase (fluc-mrfp-ttk). The transduced ADSCs were assessed on biological performance and transplanted into infarcted heart with fibrin scaffolds. In vivo cell retention was tracked by bioluminescence imaging (BLI) and micro positron emission tomography/computed tomography (PET/CT) imaging. Histological assessment was performed for regeneration potentials. The results showed that lentiviral transduction did not influence cell functions. In vitro imaging analysis showed a robust linear correlation between cell numbers and BLI signals (R (2) = 0.99) as well as between cell numbers and radiotracer uptakes (R (2) = 0.98). Transduced ADSCs were visualized in the heart under both BLI and PET/CT imaging, contributing to cardiomyocyte regeneration and angiogenesis in the implanted areas. Compared with BLI monitoring, PET/CT data provided precise localization for cell retention. Thus, a combination of imaging modalities can assist in reliable and efficient monitoring of transplanted cells, holding great potential for the transplantation of injectable scaffolds encapsulating stem cells in treating heart disease.

[Cordyceps sinensis enhances lymphocyte proliferation and CD markers expression in simulated microgravity environment].

This study was aimed to explore the effect of cordyceps sinensis enhancing lymphocyte proliferation and surface CD marker expression in simulated microgravity environment. The splenic lymphocytes were separated from mice and cultured in the rotary cell culture system simulated microgravity environment. The cells were treated with different concentration of cordyceps sinensis solution (0, 6.25, 12.5, 25 and 50 µg/ml) for 24, 48 and 72 h respectively, then the cells were harvested, and analyzed for cell proliferation and the expression of cell surface markers (CD4 and CD8). The results showed that under simulated microgravity environment, the lymphocyte proliferation was inhibited. When the concentration of cordyceps sinensis was 25 or 50 µg/ml, the lymphocyte proliferation, CD4 and CD8 expressions all increased, but 50 µg/ml cordyceps sinensis could inhibit the proliferation ability with the time prolonging. It is concluded that the suitable concentration of cordyceps sinensis displayed the ability to enhance the lymphocyte proliferation and CD marker expression in simulated microgravity environment. These results may be valuable for screening drugs which can be potentially against immunosuppression under simulated microgravity.

[Effect of lentinan against immunosuppression of lymphocytes cultured in simulated microgravity environment].

This study was aimed to evaluate the effect of lentinan on the immune function of splenic lymphocytes in rotary cell culture system (RCCS) microgravity environment. The splenic lymphocytes from mice were separated and cultured in the normal gravity and the microgravity environments. The cells were treated with lentinan solution (0, 10, 20 and 40 µg/ml). After incubated with lentinan for indicated times (24, 48 and 72 h), the cell proliferation, secretion of cytokine and the expression of cell surface markers were detected by MTT method, ELISA and flow cytometry respectively. The results indicated that lentinan of above mentioned concentrations did not obviously promote the lymphocyte proliferation, but increased the secretion of IL-2 and IFN-γ and enhanced the expression of lymphocyte surface markers CD4 and CD8 in microgravity environment. It is concluded that lentinan has the ability to enhance the lymphocyte immune function in microgravity environment.

Self-assembly of renal cells into engineered renal tissues in collagen/Matrigel scaffold in vitro.

To tissue engineer a kidney is a formidable task because of the complex cell composition and structures in the kidney. This study reconstructed renal tissues using mixed renal cells in collagen/Matrigel® scaffolds in vitro. Neonatal rat renal cells were seeded in collagen I supplemented with Matrigel in a casting mold that could exert static stretch when the renal constructs contracted. During in vitro culture, the renal constructs were observed under microscope and analyzed by histological and immunofluorescent examinations. Results showed that the mixed renal cells reconstituted renal tubular and glomeruli-like structures with different appearances at varying developmental stages. Tubular structures were formed by CK18-positive cells with similar appearances lining the surrounding hollow centres. The glomeruli-like structures were tufts of cell aggregates containing Flk-1-positive cells. These results show that neonatal rat renal cells self-assembled into engineered renal tissues containing both tubules and glomeruli-like structures when cultured in 3D collagen/Matrigel scaffold in vitro.

RCCS enhances EOE cell proliferation and their differentiation into ameloblasts.

In this article we report on the culturing of dental enamel organ epithelia (EOE) using a rotary cell culture system (RCCS) bioreactor associated with a cytodex-3 microcarrier. This culture system enhanced the proliferation and differentiation of the EOE into ameloblasts. Primary dental EOE trypsinized from 4-day old post-natal rat pups were cultured in the RCCS associated with Cytodex-3. The results were analyzed in comparison to a conventional plate system (control). Cells grown in RCCS have shown higher viabilities (above 90%) and final cell densities in terms of cells/ml than in the control system. In the case of RCCS, 46±2 manifold increases were obtained, while significantly lower yields of 10.8±2.5 manifod were obtained for control plates. Throughout the experiments, glucose levels were maintained within the accepted physiological range. In this case, LDH levels are kept low (below 150 mmol/ml), which is in accordance with the low cell death observed in the RCCS. Scanning electron microscopy revealed cells that were spread and forming three dimensional aggregates on the surface of cytodex-3. Cells cultured in the RCCS exhibited a stronger positive immunofluorescence staining for ameloblastin than those in control plates. RT-PCR results revealed that cells cultured in RCCS have higher amelogenin mRNA levels compared to controls. We have done an exploratory study on biological characteristics and self-assembling of epithelium cellula intersitialis, which demonstrated that the special 3D environment enhanced the rat dental EOE cell proliferation and differentiation into ameloblasts. The study has revealed that RCCS could be used to study the reaction of the EOE cells, tooth enamel organ cells and mesenchymal cells under the spacial 3D culture system, which will also provide a novel hypothesis for dental regeneration.