Establishment and characterization of high-throughput culture platform for kidney organoids / 中华肾脏病杂志

Objective:To establish and identify a high-throughput culture platform for induced pluripotent stem cells to differentiated kidney organoids.Methods:Human urine-derived induced pluripotent stem cells were selected and plated at a suitable cell density, and differentiated using small molecule compounds such as CHIR99021/fibroblast growth factor 9/heparin during day 1-6. On day 7, cells with appropriate density were digested and resuspended, than added to a 96-well 3D culture plate for 24 hours. After the cells formed spheroids, fibroblast growth factor 9 and heparin were added to induce differentiation till day 24. The immunofluorescence and transmission electron microscopy were used to compare the differences of kidney organoids obtained by the reported differentiation protocol (transwell protocol) method and high-throughput culture platform.Results:Kidney organoids were successfully differentiated by two protocols. Immunofluorescence results showed that LTL, GATA-3, and synaptopodin, which were major kidney cell markers, were all expressed, and mature renal organoids were formed. The results of transmission electron microscopy showed that the kidney organoids successfully developed foot processes, the unique cellular feature of the glomerular podocytes, which were evenly distributed and neatly interspersed with each other. At the same intermediate mesodermal cell count of 1.0×10 7, approximately 7 renal organoids were obtained by the transwell protocol, while approximately 1 000 renal organoids were obtained by the high-throughput culture platform. Conclusion:A high-throughput culture platform for kidney organoids is successfully established, and a large amount of mature kidney organoids with complete structure and function can be obtained. The differentiation efficiency of kidney organoids is greatly improved.

Application of Luminex liquid chip technology in diagnosis and monitoring for patients with Crohn's disease / 局解手术学杂志

Objective This study aimed to evaluate the clinical significance of detecting cytokine expression with Crohn's disease(CD) by Luminex liquid chip.Methods A total of 76 patients with CD and 50 healthy volunteers as healthy control group were recruited in this study.The expression of cytokines IL-2,IFN-γ,TNF-α,IL-4,IL-6,IL-10 and IL-17A were detected by Luminex liquid chip according to the instruction of MILLIPLEX MAP Kit.Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of anti-saccharomyces cerevisiae antibodies(ASCA).Then the relationship and differences among these groups were analyzed.Results Except IL-17A,the level of IL-2,IFN-γ,TNF-α,IL-4,IL-6,IL-10 was higher in the CD group than those in control group.The serum levels of IFN-γ,TNF-α and IL-6 in patients with active stage of CD group were significantly higher than those with remission stage and healthy control group.The level of TNF-α and IL-6 in moderate and severe stages were higher than those in slight stage.The serum level of ASCA had significant difference between CD group and control group.Compared with ASCA negative group,the ASCA positive patients have a higher level of IL-6 and TNF-α.ConclusionThe cytokines like IL-6 and TNF-α is correlated with CD severity,which is important for inflammatory activity and progression of CD.Altogether,these results demonstrated that the dynamic change of cytokines had a clear relativity in stage of CD,which is very imperative for diagnosis and clinical appraisal of CD.

Construct the recombinant plasmid of recombinant human basic fibroblast growth factor / 中国医师杂志

Objective To obtain the sequence of recombinant human basic fibroblast growth factor (rhbFGF) with endonuclease sites of BamHI and Pst Ⅰ by PCR based gene assembly and construct the recombining vector of rhbFGF gene by TA cloning technique.Methods The rhbFGF gene sequence which was designed for lactococcus with endonuclease sites of BamHI and Pst Ⅰ was divided into 22 oligonucleotides by DNASTAR 6.0 (bFGFl-bFGF22).The 22 oligonucleotides were spliced by PCR based gene assembly to get the rhbFGF eDNA with endonuclease sites of BamHI and Pst Ⅰ.The PCR product was inserted into the PMD18-T VECTOR.The recombining vector were converted to the competent E.coli TOP10.The clones generated from LAB were analyzed by miniprep isolation from LAB host.They were identified by the restriction enzyme cuuing and sequencing.Results The rhbFGFcDNA synthesized by PCR based gene assembly with endonuclease sites of BamHI and Pst Ⅰ was verified by 2％ agarose electrophoresis.The recombining vector of rhbFGF gene by TA cloning technique was identified by enzyme digestion and gene sequencing.Conclusions The TA cloning vector of recombining hbFGF with endonuclease sites of BamHI and Pst Ⅰ was constructed successfully.

The problems of media literacy in students and the improving paths / 中华医学教育探索杂志

Along with the mediated society,there are the problems in students'current media literacy,such as the alienation of media using,inability to distinguish'media reality'and'objective reality',lacking of media ethics and consciousness of self-discipline.Based on the analysis of these problems,in combination with the actual college education,we put forward the realization ways and the corresponding strategies to improve the students'media literacy.

High-level transcription of human mitochondrial tRNA(Trp) based on ribozyme and characterization on its aminoacylation activity / 生物工程学报

In vitro transcription systems with T7 RNA polymerase (T7 RNAP) were widely used in preparation of RNA because of their simplicity and high efficiency. The transcripts would have additional 5' sequence since T7 promoter spans the transcription start site, while deletion of the transcription start site would severely reduce the T7 RNAP transcriptional activity. We successfully developed an in vitro transcription by combining of T7 RNAP high efficient transcription system and highly specific self-splicing technology of ribozymes, in this system, ribozyme self-splices at the designed specific site and releases the aim RNA without affecting transcription efficiency of T7 RNAP, the aminoacylation activity of human mitochondrial tRNA(Trp) (HmtRNA(Trp) (UCA)) is 113.6 pmol/microg. This method with its high efficiency on transcription and good repeatability is very suitable for preparation of accurate RNA in large scale.

Biological properties study and animal experiment of polylactide/polytrimethylene carbonate blends membrane / 生物医学工程学杂志

Polylactide is a biodegradable and biocompatible biomaterial. Based on the PLA (Polylactide) membrane, we have produced a new PLA/PTMC (Polytrimethylene carbonate) blends membrane. The biological properties of this membrane were studied by cell toxicity experiment, acute toxicity experiment, skin irritant experiment, sensitization test, hemolytic test, micronucleus test and subcutaneous implantation test. The results demonstrated that the blends membrane has no toxicity and it does not cause skin irritation, hypersensitive reaction and hemolysis. The micronucleus ratio of the membrane is 1.3% +/- 1.0%, being less than 3%. The result of medullary micronucleus test was reported negative. The wounds were free from suppuration and necrosis after subcutaneous implantation in all periods. In the experimental application of this member to preventing adhesion after rabbit intestine operation, the membrane demonstrated good effect. In conclusion, PLA/PTMC blends membrane is a material with good biocompatibility.

The relationship between use of the antibiotics in a burn unit and the change of the drug-resistance of Staphylococcus aureus / 中华烧伤杂志

<p><b>OBJECTIVE</b>To investigate the relationship between the use of antibiotics in a burn unit and the change in the drug - resistance of Staphylococcus aureus (S. aureus).</p><p><b>METHODS</b>By calculating the defined daily doses (DDD) of accumulated consumption of antibiotics per unit time and expense consumption, the use of different kinds of antibiotics in a burn unit in recent five years was analyzed, and correlation analysis between the change of antibiotics consumption amount and the change in drug - resistance level of S. aureus were carried out.</p><p><b>RESULTS</b>Amikacin, gentamycin and cephazolin were the commonest antibiotics used in our burn unit. They were relatively cheaper than some other antibiotics. The consumption amount of compound antibiotics application was negatively correlated with the penicillin resistance level of S. aureus. Seven correlation coefficients between the consumption of first generation cephalosporins and seven coefficiences of resistance rate of S. aureus were negative. The consumption amount of the 3rd generation of cephalosporin application was positively related to the resistance of S. aureus to erythromycin and oxacillin.</p><p><b>CONCLUSION</b>Accumulated DDD might be one of the ideal indices of reflecting antibiotic use. The changes in the consumption amount of the 1st and 3rd generation of cephalosporins containing beta-lactamase-inhibitor might affect the drug-resistance levels of S. aureus to some degree.</p>

Application of Systems Software for Hospital Infection Control / 中国药房

OBJECTIVE:To improve information management of hospital infections.METHODS:The hospital infection monitoring and management software developed by the hospital where the authors work was applied to monitor the risk factors of hospital infections at real time and selectively.RESULTS:The software could be applied to investigate the situation of antibacterial use in hospital,evaluate the rationality of drug action,monitor the incidence of hospital infections,and analyze the risk factors of hospital infections.CONCLUSIONS:The hospital infection monitoring and management software is con-venient and easy to operate,which can enhance the efficiency and effect of the management of hospital infection.

Construction of cloning vector of c-myc-TFF2 fusion gene by PCR based gene assembly / 西安交通大学学报(医学版)

Objective To assemble fusion gene of c-myc tagged human trefoil factor family 2(hTFF2)(in vitro) and construct a cloning vector of this fusion gene.Methods Based on amino acid sequence of hTFF2 and codon of lactococcus lactis,cDNA of htff2 sequence was designed and extended at their 5' ends with a sequence encoding the c-myc tag;and the sequence of fusion gene c-myc-htff2 was designed.According to restriction enzyme sites of pBluescript Ⅱ sk(+),the SalⅠ and BamHⅠ were arranged at 5′ and 3′ ends of the fusion gene respectively.Instructed by DNAWORKS program,the fusion gene sequence of c-myc-htff2 was designed as 14 oligonucleotides that overlapped each other.The target gene fragment of cmyc-htff2 fusion gene was obtained by the means of polymerase chain reaction(PCR) based gene assembly.Then,the c-myc-htff2 was subcloned into vector of pBluescript II sk(+) for identification of the fusion gene by restricting enzyme excision and DNA sequencing.Results Assembly of c-myc-TFF2 fusion gene(in vitro) and construction of pBS-TFF2 had been completed successfully;and DNA sequencing showed that the sequence of synthetic gene accorded with the expectation.Conclusion With the help of DNAWORKS program,the design and assembly of oligonucleotides (in vitro) is effective to synthesize a target gene,and the cloning vector of c-myc-htff2 fusion gene has been successfully constructed.