Construction and Application of National Rare Diseases Registry System of China / 罕见病研究

Rare diseases have been a major challenge for clinical medicine and public health challenge in China. One of the effective measures is to conduct proactive research on rare diseases to deal with the disease burden of the diseases. However, low prevalence, disperse distribution of patients, lack of knowledge about the disease course, and phenotype heterogeneity hamper the development of research for rare diseases. Recently, it has been found that patients registry is effective in understanding the course of the disease and accu- mulating the cases and data of clinical research or clinical trial design. At present, most of developed countries or regions in the world have promoted clinical research and clinical trials of new medications on rare diseases by using the registration of rare disease. In 2016, Peking Union Medical College Hospital established China's first registry system at the national level-National Rare Disease Registry System of China(NRDRS). NRDRS has accumulated 68 137 cases data registered by the researchers from China's 101 collaborating hospitals in 29 provinces/municipalities/autonomous regions, covering 171 different, and forming 188 cohorts. To date, NRDRS complete the initial stage of resources buildup.Nex stage will be focused on clinical research and clinical trials related to rare diseases based on NRDRS. This article is on the process of building NRDRS, the potential support for conducting clinical research and clinical trials related to rare diseases, and the challenges will be faced.

Mucin-type O-glycosylation Landscapes of SARS-CoV-2 Spike Proteins

The densely glycosylated spike (S) proteins that are highly exposed on the surface of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) facilitate viral attachment, entry, and membrane fusion. We have previously reported all the 22 N-glycosites and site-specific N-glycans in the S protein protomer. Herein, we report the comprehensive and precise site-specific O-glycosylation landscapes of SARS-CoV-2 S proteins, which were characterized using high-resolution mass spectrometry. Following digestion using trypsin and trypsin/Glu-C, and de-N-glycosylation using PNGase F, we determined the mucin-type (GalNAc-type) O-glycosylation pattern of S proteins, including unambiguous O-glycosites and the 6 most common O-glycans occupying them, via Byonic identification and manual validation. Finally, 43 O-glycosites were identified in the insect cell-expressed S protein. Most glycosites were modified by non-sialylated O-glycans such as HexNAc(1) and HexNAc(1)Hex(1). In contrast, 30 O-glycosites were identified in the human cell-expressed S protein S1 subunit. Most glycosites were modified by sialylated O-glycans such as HexNAc(1)Hex(1)NeuAc(1) and HexNAc(1)Hex(1)NeuAc(2). Our results are the first to reveal that the SARS-CoV-2 S protein is a mucin-type glycoprotein; clustered O-glycans often occur in the N- and the C-termini of the S protein, and the O-glycosite and O-glycan compositions vary with the host cell type. These site-specific O-glycosylation landscapes of the SARS-CoV-2 S protein are expected to provide novel insights into the viral binding mechanism and present a strategy for the development of vaccines and targeted drugs.

Site-specific N-glycosylation Characterization of Recombinant SARS-CoV-2 Spike Proteins using High-Resolution Mass Spectrometry

SummaryThe glycoprotein spike (S) on the surface of SARS-CoV-2 is a determinant for viral invasion and host immune response. Herein, we characterized the site-specific N-glycosylation of S protein at the level of intact glycopeptides. All 22 potential N-glycosites were identified in the S-protein protomer and were found to be preserved among the 753 SARS-CoV-2 genome sequences. The glycosites exhibited glycoform heterogeneity as expected for a human cell-expressed protein subunits. We identified masses that correspond to 157 N-glycans, primarily of the complex type. In contrast, the insect cell-expressed S protein contained 38 N-glycans, primarily of the high-mannose type. Our results revealed that the glycan types were highly determined by the differential processing of N-glycans among human and insect cells. This N-glycosylation landscape and the differential N-glycan patterns among distinct host cells are expected to shed light on the infection mechanism and present a positive view for the development of vaccines and targeted drugs.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsACE2angiotensin-converting enzyme IICryo-EMcryoelectron microscopyEenvelope proteinHCoV-NL63human coronavirus NL63Mmembrane proteinMSmass spectrometryMERS-CoVMiddle East respiratory syndrome coronavirusNnucleocapsid proteinRBDreceptor-binding domainSspike proteinSARS-CoV-2severe acute respiratory syndrome coronavirusSCEstepped collision energyZic-HILICzwitterionic hydrophilic interaction liquid chromatographyView Full Text

Expression of miRNA-140 in Chondrocytes and Synovial Fluid of Knee Joints in Patients with Osteoarthritis / 中国医学科学杂志(英文版)

Objective To investigate the expression of miRNA-140 in chondrocytes and synovial fluid of osteoarthritis (OA) patients, and explore the relationship between the miRNA-140 expression and OA severity.Methods This study enrolled 30 OA patients who underwent total knee arthroplasty for chondrocytes sampling and 30 OA patients who underwent intra-articular injection for synovial fluid sampling. All OA patients were grouped into mild [Kellgren and Lawrence (KL) grade 1-2], moderate (KL grade 3) and severe (KL grade 4), with 10 in each subgroups for each sampling purposes. 7 non-OA patients and 10 patients with knee injury were collected for cartilage and synovial fluid sampling respectively as control groups. Chondrocytes were isolated from the cartilage tissue and cultured in vitro. Quantitative real time PCR for miRNA-140 in chondrocytes and synovial fluid were performed, and the U6 snRNA was used as internal control. The expression difference of miRNA-140 among groups and correlation between the expression and the KL grade of OA were analysed using one-way ANOVA and Spearman test respectively. Results The expression of miRNA-140 in chondrocytes of knees in OA patients was reduced than that in normal knees, and the between-group difference was statistically significant (F=305.464, P<0.001). miRNA-140 could be detected in synovial fluid of both normal knees and OA knees, its relative expression level was reduced in synovial fluid of OA group compared with normal group, and the between-group difference was statistically significant as well (F=314.245, P<0.001). The relative expression level of miRNA-140 in both chondrocytes and synovial fluid were negatively correlated with the KL grade of OA(r=-0.969, P<0.001; r=-0.970, P<0.001). Conclusion miRNA-140 could be detected in chondrocytes and synovial fluid of OA patients, and its expression was negatively correlated with the severity of OA.

Circulating miRNAs might be promising biomarkers to reflect the dynamic pathological changes in smoking-related interstitial fibrosis.

Cigarette smoking is the major risk factor for smoking-related interstitial fibrosis (SRIF). Despite recent advances, the molecular mechanisms involved in the initiation and progression of this disease remain elusive. We found 6 months of chronic mainstream smoking exposure induced SRIF in C57 mice, which was associated with pronounced enhanced oxidative stress, bronchoalveolar inflammation and fibrosis but not apoptosis of alveolar septal cell. We used Affymetrix microRNA (miRNA) arrays to determine the temporal alteration in global gene expression of peripheral blood during the progression of diffuse pulmonary interstitial fibrosis in C57 mice. Microarray analysis revealed the upregulation of 3 miRNAs (miR-92b, miR-700 and miR-668) and the downregulation of 5 miRNAs (let-7e, miR-142-5p, miR-350, miR-19a and miR191*) in the peripheral blood of mice exposed to mainstream smoking for 1, 2, 3 and 6 months. We proposed that circulating miRNAs might be promising biomarkers to reflect the dynamic pathological changes of SRIF related interstitial fibrosis.

Circulating biomarkers of hazard effects from cigarette smoking.

Smoking, leading to over 438,000 annual deaths in the US and 92 billion US dollars in lost productivity, is an important risk factor for several diseases. Global smoking statistics for 2002 have shown that 80,000 to 100,000 children worldwide start smoking every day. Human biomonitoring nowadays has provided an efficient and cost-effective means of measuring human exposures and biological effects of smoking. To review the utility of biomarkers in reflecting the hazard from cigarette smoking, we comprehensively searched the Cochrane Library, Medline and EMbase from 1966 to May 2010 with the language limit of English. We found that the currently used biomarkers, such as tobacco-specific metabolites, smoking-induced genotoxic products, may not scientifically reflect the hazard from cigarette smoking. More research is needed to find out more effective biomarkers for estimating hazards from cigarette smoking. This paper is expected to help researchers understand the current utilization of biomarkers related to cigarette smoking and to provide suggestive guides to tobacco companies in producing less-toxic cigarettes, thus to provide a safe way to smoke.

Expressed sequence tags analysis of a liver tissue cDNA library from rhesus monkey, Macaca mulatta / 生物医学工程学杂志

Rhesus monkeys (Macaca mulatta) are human's closest evolutionary relatives next to Chimpanzees, and they are widely used in biomedical researches. Analyses of the rhesus monkey trasnscriptome and the sequence divergence between monkey and human are of importantce to the development of scientific analyses and to the application and interpretation of the results from animal experiments. In this study, we analyzed the genetic and transcriptional information. Four hundred and one clones were randomly selected from a liver tissue cDNA library of rhesus monkey, and the expressed sequence tags (ESTs) were sequenced. We acquired 393 effective ESTs that were assembled into 221 Unigenes with Phrap software. Alignments of the sequences showed that 188 Unigenes matched with known proteins in Swiss_prot database, of which 16 Unigenes matched the known rhesus proteins, and 171 Unigenes had high homology with human proteins. Then the result of BLASTN comparison showed that 26 of another 33 Unigenes matched the known rhesus genes. Finally, the remaining Unigenes were aligned in dbEST and rhesus genome database, and the results suggested 3 Unigenes be newly discovered ESTs of rhesus.

In vitro study of strontium-calcium sulfate compounds as bioactive bone grafted substitute / 生物医学工程学杂志

This study was aimed to create strontium-calcium sulfate compounds for making a new bioactive material with osteoconductive and osteoinduceable activity for bone repairing. Its mechanics and degradation features were assessed in vitro. Powders of alpha-calcium sulfate hemihydrate (alpha-CSH) and SrCl2 were mixed completely to make Sr-calcium sulfate compounds materials with 6 different concentrations (0%, 0.1%, 0.3%, 0.5%, 1% and 2%) of Sr. Scanning electron microscope was used to observe the configuration of the new materials. The compressive strength of each material was tested. The materials were soaked into simulated body fluid (SBF) to test the features of degradation, which included pH, weight loss, declination of compressive strength and the changes of strontium ion concentration. The crystal appearances were influenced by incorporating of strontium. The compressive strength of non-strontium incorporating calcium sulfate was 36.65 +/- 2.22 MPa. When the concentration of strontium was increasing, the compressive strength measurements of the materials tended to decline. The compressive strength declined to 20.56 +/- 2.64 MPa when the strontium concentration reached to 2%. The pH value of the SBF declined when the time of degradation increased, but both of them were very stable. All of the materials got weight loss after being soaked in SBF for several weeks. The weight loss was slight within 4 weeks and it became dramatic after 4 weeks. When the concentration of strontium was increasing, the weight loss became more rapid and significant (P<0.05). During 0-4 weeks' degradation in SBF, the materials' compressive strength decreased much slower when the strontium concentration was below 0.5%; however, when the decrement of strength became faster, the strontium concentration became higher. The concentration of strontium ion in SBF began to increase faster after 4 weeks' soaking in SBF. As the concentration of strontium was increasing, the strontium ion concentration in SBF became higher (P = 0.000). The new compound materials made by the mixing of alpha-calcium sulfate hemihydrate and SrCl2 can provide efficient compressive strength. The features of degradation of the materials are very stable. The new materials can release lots of bone inducible substance-strontium ions to repair bone defection after 4 weeks of degradation.

Research and application of cell transplantation in the treatment of diabetes mellitus: a review / 生物医学工程学杂志

In the conventional treatments of type I diabetes, there are various problems. As a new adequate treatment of diabetes, cell replacement therapy of diabetes has been applied and given research priority. We have investigated the applications of cell transplantation in the treatment of diabetes and have retrieved the relevant articles on cells transplantation for the treatment of diabetes. In this paper, we review the history, development, merits and demerits of cell transplantation and the recent advances in pancreatic islet transplantation research. The latest progress in the induction of stem cell to differentiate into the insulin-producing cells was also introduced.

Cloning of Chinese Banna minipig inbred-line alpha1,3-galactosyltransferase gene and construction of its recombinant eukaryotic expression vector / 生物医学工程学杂志

This study sought to clone Chinese Banna minipig inbred-line (BMI) alpha1,3-galactosyltransferase (alpha1,3-GT) gene and construct its recombinant eukaryotic expression vector. Total RNA was isolated from BMI liver. Full length cDNA of alpha1,3-GT gene was amplified by RT-PCR and cloned into pMD18-T vector to sequence. Subsequently, alpha1,3-GT gene was inserted into pEGFP-N1 to construct eukaryotic expression vector pEGFP-N1-GT. Then the reconstructed plasmid pEGFP-N1-GT was transiently transfected into human lung cancer cell line A549. The expression of alpha1,3-GT mRNA in transfected cells was detected by RT-PCR. FITC-BS-IB4 lectin was used in the direct immunofluorescence method, which was performed to observe the alpha-Gal synthesis function of BMI alpha1,3-GT in transfected cells. The results showed that full length of BMI alpha1,3-GT cDNA was 1116 bp. BMI alpha1,3-GT cDNA sequence was highly homogenous with those of mouse and bovine, and was exactly the same as the complete sequence of those of swine, pEGFP-N1-GT was confirmed by enzyme digestion and PCR. The expression of alpha1,3-GT mRNA was detected in A549 cells transfected by pEGFP-N1-GT. The expression of alpha-Gal was observed on the membrane of A549 cells transfected by pEGFP-N1-GT. Successful cloning of BMI alpha1,3-GT cDNA and construction of its eukaryotic expression vector have established a foundation for further research and application of BMI alpha1,3-GT in the fields of xenotransplantation and immunological therapy of cancer.

Lovastatin inhibits adipogenesis and prevents osteonecrosis in steroid-treated rabbits.

OBJECTIVES: To investigate in vivo effects of lovastatin on inhibition of adipogenesis, an important mechanism of steroid-induced osteonecrosis, and on prevention of occurrence of steroid-induced osteonecrosis in rabbits. METHODS: Fifty-four rabbits were intramuscularly injected once with 20mg/kg of methylprednisolone acetate (MPSL). Then, they were divided into two groups: lovastatin was given in Group I and placebo was given in Group II. And another 16 rabbits were injected with physiologic saline (PS) as a control (Group III). Hematological examination was performed for serum lipid levels. Both the femora and the humeri were histopathologically examined for the presence of osteonecrosis before the injection and 1, 2, 4 and 12 weeks after the injection. RESULTS: The serum lipid levels were significantly higher in Group II than in Groups I and III 1, 2 and 4 weeks after the injection. The incidence of osteonecrosis was significantly higher in Group II (69%) than in Group I (36%) and Group III (0%). Pathological examination showed less serious adipogenesis and bone death in Group I than in Group II. The size and area of the fat cells in the bone marrow were significantly smaller in Group I (47.5+/-1.3 microm, 25%) and Group III (41.7+/-1.6 microm, 12%) than in Group II (59.8+/-6.3 microm, 51%) (P< 0.001). CONCLUSION: Lovastatin can prevent development of steroid-induced osteonecrosis in rabbits by inhibiting adipogenesis. Future evaluation on the effectiveness of lovastatin in the clinical practice is still necessary.

Isolation, culturing and growth characteristics of mesenchymal stem cells from bone marrow of Rhesus monkey, Macaca mulatta / 生物医学工程学杂志

Mesenchymal stem cells from bone marrow of Rhesus monkey (RhBMSCs) were isolated by density gradient centrifugation, purified by adherence separation, and further identified by phenotype and karyotype analysis. Growth characteristics of RhBMSCs were investigated by observation of cell proliferation and detection of apoptosis. Density gradient centrifugation and adherence separation revealed a simple method to obtain fairly pure RhBMSCs. Fusiform was the most common form of cell under observation, and cell karyotype was normal. Phenotyping assay revealed that the RhBMSCs are highly positive (99.2%) for CD29 when compared with the low positive rates (less than 3.2%) for CD34, CD45 and HLA-DR, which indicated a successful isolation of high purity RhBMSCs and a vigorous activity of cells to proliferate. But the proliferation activity of RhBMSCs gradually decreased following the increasing of cell generations. The methods and results of isolation, expansion, identification and growth characteristics of RhBMSCs were discussed in this paper, which may be helpful to understanding the bone marrow mesenchymal stem cells of human, and may serve as the groundwork for orientated differentiation of RhBMSCs and tissue repairment on experimental animal model of rhesus monkey.

Variation of host cell tropism of porcine endogenous retroviruses expressed in chinese Banna minipig inbred.

A serious donor-organ shortage urges the use of animal donors to treat a wide appropriate variety of major health problems including organ failure and diabetes. However, the promise of clinical xenotransplantation is offset at the present time by the potential of a public health risk due to the cross-species transmission of pathogens from animal donors to human patients. In particular, the transmission of porcine endogenous retrovirus (PERV) is a major concern. In this study, cell tropism of PERV was tested by in vitro infection of human primary cells and cell lines. Coculture of PERV supernatant derived from PK15 with human primary cells and cell lines resulted in the transfer and expression of PERV-specific sequences and the establishment of a productive infection. In the detection of tropism variation of PERV in pigs, 293 cells were cocultured with mitogenic-activated and lethally irradiated PBMC from 12 Banna minipig inbred (BMI). The results were that six coculture groups were PERV-positive. However, infectious virus was not detected when activated PBMC from the other 7 pigs were cocultivated with human cells known to be permissive for PERV, which indicated a tropism variation among the tested individuals. All these findings demonstrate that the presence of endogenous viruses in source animals needs to be carefully considered when the infectious disease potential of xenotransplantation is being assessed.

Evaluation of three in-vitro refolding methods for human-derived anti-CTLA4 scFv expressed in E. coli / 生物医学工程学杂志

To evaluate the efficiency of three in vitro refolding methods for a humanized single-chain Fv antibody against human CTLA4(CD152) expressed in E. coli, the denatured and purified inclusion bodies (IBS) were refolded by dilution, dialysis and in situ refolding via Immobilized Metal-Ion-Affinity Chromatography (IMAC), respectively. The concentration of refolded scFvs was examined by Bradford method. And the antigen binding activity of the refolded scFvs was analyzed by indirect cell-ELISA. The highest and lowest refolding yields could be obtained by dialysis and in situ refolding via IMAC, respectively. The binding activity of the refolded scFv by dialysis was 1.95-fold higher than that by dilution, 4.13-fold higher than that by in situ refolding via IMAC (GSH/GSSH excluded) and 3.63-fold higher than that by in situ refolding via IMAC (GSH/GSSH included), respectively. In conclusion, a high refolding yield and binding activity of scFv with natural conformation could be obtained by dialysis in the condition of 0. 15 mol/L sodium chloride, 50 mmol/L Tirs-HCl, pH 8. 0 buffer containing 3 mmol/L reduced glutathione and 1 mmol/L oxidized glutathione for 48 hours at 4 degrees C.

Gene chip application in organ transplantation and ischemia reperfusion injury / 生物医学工程学杂志

Gene chip has been an important way to detect gene changes of organism in different condition. It is a new tendency to use gene chip to research graft rejection at gene level. Ischemia reperfusion injury (IRI) accurs early in organ transplantation. Studying IRI with gene chip is beneficial to understand the mechanism of graft rejection and will provide guidance for surveying and treating graft rejection after organ transplantation.

An exploratory study of preoperative analysis on histocompatibility in porcine allo-kidney transplantation / 生物医学工程学杂志

Human tissue typing methods were employed in developing a porcine allotransplantation model. 23 Chinese Sichuan White Pigs(2-3 months old, 17.5+/-4.6kg, with clear family background) were selected for tissue typing, ABO blood type cross reaction, complement-dependent cytotoxicity (CDC) cross reaction and one way mixed lymphocyte reaction (MLR). 6 pairs of swine that showed better matching results were selected as donors and recipients. Single-kidney orthotopic transplantation was conducted after removing both kidneys of the recipient. Five recipients showed low matching results (MLR ranging from 2175 to 3560, CDC from 1 to 4); of them, 2 died of operation, 2 died of acute renal tubular necrosis and accelerating rejection 4 days after operation respectively, and 1 died of acute renal tubular necrosis 4 days after operation. 6 recipients showed high matching results (MLR ranging from 982 to 1916, CDC from 2 to 4); of them, 1 died of anaesthesia during operation, 3 died of accelerating rejection and acute rejection 2 weeks after operation respectively, 1 had good kidney function, and 1 presented weak rejection 1 week after operation but the kidney function came back to normal afterwards. Human tissue typing methods could be adopted in developing the porcine model. Hyperacute rejection could be avoided by screening ABO blood type, CDC and MLR tests. However, based on these primary data, it was hard to evaluate the predictive values of CDC and one-way MLR for accelerating rejection, acute rejection and graft chronic dysfunction. Further research by expanding experiments in these aspects is still going on.

Neonatal murine keratinocytes split express CD80/CD86 upon culture / 生物医学工程学杂志

It was previously thought that keratinocytes did not express the CD80 and CD86 which provide the most important costimulatory signals for the antigen-specific T-cell activation. The cultured keratinocytes allografts were initially accepted, but eventually, all grafted donor cells were gradually replaced by recipient cells. The precise mechanisms are not very clear. In this study, neonatal murine keratinocytes were cultured for 7 days, the results of flow cytometry and confocal microscopy showed that CD80 could be detected on keratinocytes, while CD86 could not be detected all the time. RT-PCR analysis confirmed this result. The expression level of the CD80 mRNA amplified significantly from day 1 to day 7, as expression of the control beta-actin, but CD86 was not detected. Mixed Lymphocyte Reaction (MLR) showed that keratinocytes cultured with 10% serum for 7 d stimulated effectively allogeneic rather than syngeneic T cell proliferation. This study demonstrated for the first time that costimulatory molecule CD80 can be expressed on keratinocytes in vitro. These data provided an alternative explanation for the ultimate rejection of allogeneic keratinocytes in which keratinocytes act as antigen-presenting cells.

An experimental study of the effect of biomechanical environment on the incorporation of cortical bone plates allografts / 生物医学工程学杂志

Sixteen goats with fractures of right femur received cortical bone plates allografts on both the sides of femurs. The right allograft strut endured the stimulation of physiological stress, and the left allograft strut did not. Groups of goats were sacrificed and specimens were procured at 3, 6, 12, and 24 week after surgery for histology observation and image analysis of the vessels after Chinese ink perfusion, the rate of bone porosity, the integrated optical density (IOD) of tetracycline fluorescence labeling and new bone formation were investigated in order to evaluate the incorporation of the allograft strut. The allograft strut revascularized at 6 weeks after surgery in the fracture group, but at 3 weeks in the control group. The rate of area of vessels after Chinese ink perfusion, the rate of bone porosity, the integrated optical density (IOD) of tetracycline fluorescence labeling and new bone formation in the fracture group were worse than control from 3 weeks to 6 weeks, but the observed and measured values were better in the fracture group than in the control group beyond 6 weeks after surgery (P < 0.05). The stimulation of stress would be harmful to the allograft strut if the strut endured the stress at an earlier period postoperation. Yet, it would be beneficial to the revascularization, new bone formation, substitution, and internal re-building on the strut provided that the extremity was immobilized for 6 weeks; and if the cortical graft endured the stimulation of physiological stress from 6 weeks postoperation till cancellous conjunction between the ends of fractures, the revascularization on the allograft strut and the bone conjunction between partially allograft strut and host would be faciliated.

Phylogenetic relationship of porcine endogenous retrovirus (PERV) in Chinese pigs with some type C retroviruses.

PCR amplification of proviral DNA extracted from peripheral blood lymphocytes of three Chinese pigs (Banna minipig inbreed (BMI), Wu-Zhi-Shan pig (WZSP) and Neijiang pig (NJP)), using primers corresponding to highly conserved regions of reverse transcriptase (RT) of pol gene and nucleocapsid sequence of gag gene. PCR products were then extracted and cloned into pGEM-T vector. Phylogenetic analysis of the nucleotide sequences of PERV-BMI, PERV-WZSP and PERV-WZSP revealed that they were of retroviral origin. Phylogenetic trees were constructed from the translated amino acids of PERVs and other type C retrovirus, as well as lentivirus of GenBank. The research demonstrated that PERVs of Chinese pigs and other PERVs were closely related to other pathogenic type C retroviruses. From the gag analysis, a novel subgroup of PERV was identified and this novel sequence described in this report would allow such investigation to be actively pursued.

Visualizing living fibroblast on co-cultured denture base resin by green fluorescent protein marker introduced into the cell / 生物医学工程学杂志

Visualizing living cells growing on co-cultured biomaterials is ideal for material biocompatibility evaluation in vitro. In this experiment, mouse fibroblasts L929 were labeled by introducing the gene coding enhanced green fluorescent protein (EGFP) marker into the cells. Morphology as well as proliferation of labeled cells surrounding or on the surface of co-cultured denture base resin slides were observed by use of phase-contrast microscope and fluorescent microscope directly. It was found that residual methyl methacrylate (MMA) in the denture base resin exhibited transient cytotoxicity to fibroblasts and this transient cytotoxicity could be eliminated by pre-extracting the resin with ddH2O for a short time. This fact demonstrated that even slight cytotoxicity of materials could be detected through imaging of living cells near material or material touched. And it was suggested that imaging of living cells co-cultured with biomaterial is helpful to understanding biocompatibility of materials more accurately.