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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

Application of enzyme histochemistry in evaluation of in vitro and in vivo biocompatibility of HA/TCP / 生物医学工程学杂志

In order to explore the possibility of applying enzyme histochemistry in biocompatibility evaluation, we investigated the effect of biomaterials on the activities of intracellular enzymes in this experiment. It was found that there was no obvious difference in morphology between osteoblasts co-cultured with HA/TCP and with Ti-alloy. However, transient down-regulation of NADH, SDH, LDH and CCO of the osteoblasts co-cultured with HA/TCP was detected by enzyme histochemistry, but these enzymes of osteoblasts the co-cultured with Ti-alloy were not down-regulated. It was indicated that something extracted from HA/TCP injured the co-cultured osteoblasts slightly. Similar early acute inflammatory reactions were observed after HA/TCP and Ti-alloy were separately implanted into the dorsal muscle of rabbit. There was also no obvious difference between the tissue response to HA/TCP and that to Ti-alloy. Activities of enzymes in tissues around implanted materials were down-regulated at the early injury period and recovered gradually within 30 days post-operation. But the mild toxicity of extracts from HA/TCP was demonstrated by the fact that the recovery period of HA/TCP group was longer than that of Ti-alloy group. It was proved that enzyme histochemistry is more sensitive than tissue morphology analysis in detecting the cell or tissue responses to biomaterials. Therefore, it is possible to use enzyme histochemistry in biocompatibility evaluation.

Bioartificial liver support system: state of the art / 生物医学工程学杂志

The temporary function replacement and intensive support play a pivotal role in the treatment of patients with acute or chronic end-stage organ failure. The hemodialysis and peritoneal dialysis have become routine techniques in the management of acute or chronic renal failure. But for the complexity of hepatic function, e.g. metabolism, biosynthesis and biotransformation for endogenous or exogenous substrates, the simulation or partial replacement of hepatic function is a great dream of bionic technologists. With the development of cell biology and biomedical material, the combination of biomaterial and the hepatocyte cultured ex vivo could provide a range of human liver-specific functions. The combination of biomaterial and viable cell was called hybrid or bioartificial liver support system (BALSS). It is a chimera of biomaterials and hepatocytes. The bionic technologists study the cell and its culture in vitro, which is the main component of BASLL. Many types of BALSS were translated into the early clinical stage. In this overview, we review the hepatocyte culture and the design of different bioreactors. It includes the immune obstacles in xeno-hemoperfusion and how to assess pre-clinical and clinical effectiveness.

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.

Study on rabbit mesenchymal stem cells differentiation to the adipogenic or osteogenic lineage in vitro / 生物医学工程学杂志

Rabbit bone marrow-derived mesenchymal stem cells(MSCs) are multipotent. We studied the adipogenic and osteogenic differentiation potent using adipogenic supplement (AS) or osteogenic supplement (OS) in vitro. Specific markers of this induced adipogenic and osteogenic lineage were identified. The findings showed that the rabbit MSCs are capable of differentiating into adipogenic and osteogenic lineages spontaneously. On the 21st day, approximately 75% rabbit MSCs were induced to adipogenic or osteogenic cells in medium containing AS or OS, respectively. These results demonstrated that the differentiation of MSCs could be regulated in vitro. The underlying molecular mechanisms of adipogenic or osteogenic differentiation await elucidation.

Advances and prospective application of stem cell technique in stomatology / 生物医学工程学杂志

In this paper the background and advances of stem cell technique in stomatology were reviewed, especially the lately research of repair of maxillofacial defects with bone marrow stem cells, repair or reconstitution of teeth with dental pulp stem cells and repair of other tissues such as parotid with embryonic stem cell. Stem cell technique provides a new choice and extensive prospect of application for stomatology, therefore, deserves further research.

Bone marrow mesenchymal stem cells: progress in bone/cartilage defect repair / 生物医学工程学杂志

Mesenchymal stem cells (MSC) are thought to be multi-potent cells that have the potential to differentiate into lineages of mesenchymal tissues, including bone, cartilage, tendon, fat, muscle, and marrow stroma during embryo morphogenesis. In recent years, cells that have the characteristics of mesenchymal stem cells were isolated from marrow aspirates of human and a few animals. It was found that these cells retain the characteristics of stem cells in vitro and could be induced to differentiate exclusively into the osteocytic, chondrocytic, myoblastic and adipocytic lineages. It was demonstrated that MSC could heal clinically significant bone and cartilage defects in animal models. The role of MSC in repairing tendon defect was also testified. In addition, for its multi-potential to differentiate into lineages of mesenchymal tissues, MSC could be used as gene vehicle for gene therapy of trauma care.

Osteogenesis of HA/TCP biphasic ceramics implanted into muscle: a long-term study / 生物医学工程学杂志

New bone formation in long-term intramuscle implant of Ca-P biomaterial was investigated in this experiment. After implanting into dog dorsal muscle for 15 months, a thin fibrous membrane that wrapped HA/TCP implant was still observed obviously. Three types of tissues, i.e. mesenchymal tissue, bone and bone marrow, regularly distributed in different pores of implant. Nearly all the pores of implants were occupied by bone. Bone in the pores located in the central region of implant was matured lamellar bone characterized by obvious lacuna and rich bone marrow. However, bone in the peripheral pores was immature woven bone without bone marrow formation. Furthermore, mesenchymal tissues only exist in the peripheral pores and usually were connected with immature woven bone. It was demonstrated that porous HA/TCP has bone inductivity and it could induce new bone formation at non-osseous site. Well-regulated distribution of mesenchymal tissue, bone and bone marrow in the pores suggest bone morphogenesis in the implant must obey a specific space-time program.

A preliminary study on chemical bile duct embolization for chemical hepatectomy / 中国普通外科杂志

Objective To investigate the feasibility, effect and mechanism of chemical bile duct embolization for chemical hepatectomy. Methods Phenol or absolute ethanol plus cyanonacrylate were employed as embolization agents. Histology, Fas and TGF-? 1 measurement were used to evaluate the result. Results Phenol plus cyanonacrylate effectively destroyed and embolized intrahepatic biliary duct, leading to complete disappearance of hepatocytes in the periphery of embolization lobe and thereby achieving the effect of chemical hepatectomy. Expression of Fas and TGF-? 1 in phenol embolism group (88.90?38.10, 185.22?70.39) and ethanol embolism group (72.39?29.51, 163.56?51.75) were higher than those in biliary duct ligated group (26.31?12.07, 74.84?40.73) ( P