OBJECTIVETo gain stable genetic modification of neural stem cells (NSC) that constitutively secrete brain-derived neurotropic factor (BDNF) and to explore the biological role on the survival and neurite outgrowth of cultured dorsal root ganglion (DRG) neurons.

METHODSBDNF gene fragment from human brain cDNA libraries was obtained by using PCR. With molecular cloning technique, the recombinant stem cell viral vector with report gene was constructed, which is that MSCV-BDNF-IRES(2)-EGFP vector could encode Polycistronic mRNA. Viral particle was packaged by PT67 cell line and infected neural stem cells (mouse clone: C17.2). After selection with cloning cylinder, the expression of BDNF was assessed by immunohistochemistry enzyme linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR). The effects of stable gene-modified neural stem cells on embryonic mouse DRG neurons were evaluated in a dual-chambered cocultivation system at 3th, 10th day.

RESULTSRT-PCR analysis demonstrated expression of mRNA for human-BDNF. ELISA confirmed the presence of secreted BDNF 24 h after transfection and showed that the level of BDNF production by NSC-BDNF transfected C17.2 was at a rate of (14.6 +/- 0.8) ng x d(-1) x 10(-6) cells even after 3 months. With immunohistochemical analysis, compared with the control, the longer neurite outgrowth of cultured DRG cells and the more survival neurons were observed in NSC-BDNF transfected cells group.

CONCLUSIONSBDNF gene could be stably expressed in C17.2 cell line by MSCV, and the BDNF gene-modified NSC markedly enhances the survival and neurite outgrowth of cultured DRG neurons.

Animals ; Brain-Derived Neurotrophic Factor ; biosynthesis ; genetics ; Cell Culture Techniques ; Cell Survival ; Cells, Cultured ; Coculture Techniques ; Ganglia, Spinal ; cytology ; Humans ; Mice ; Neurons ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley ; Stem Cells ; cytology ; metabolism ; Transfection

OBJECTIVETo investigate the feasibility of life span extension of transformed human embryonic tendon cells (THETC) by reconstitution of the telomerase activity.

METHODSTHETC were transfected by pGRN145 plasmid containing the human telomerase reverse transcriptase (hTERT) cNDA in vitro by molecular cloning technique. The biological characteristics of transfected cells were detected and compared by morphological observation, plate cloning efficiency, soft agar culture, growth curve of cells cultured in different conditions, immunohistochemistry, telomerase activity assay by telomeric repeat amplification protocol (TRAP).

RESULTSThe THETC transfected by pGRN145 plasmid (telT) could express the telomerase activity with extension of life span. The telT maintained the original characteristics of temperature-dependant and serum-dependant, as well as secretion of type I collagen normally and without tendency of malignant transformation.

CONCLUSIONSThe life span of THETC can be prolonged by reconstitution of telomerase activity, which provides the novel experimental methods to establish the standard cells line.

Cell Line ; Cell Survival ; Embryo, Mammalian ; Humans ; Plasmids ; genetics ; RNA-Directed DNA Polymerase ; Telomerase ; genetics ; metabolism ; Tendons ; cytology ; enzymology ; Transfection

Objective To explore the effects of epithelial cell adhesion molecule(EPCM) expression downregulation on the proliferation,invasion and drug sensitivity of ECAM+/cluster of differentiation 44 (CD44) + colorectal cancer stem cells.Methods Colorectal cancer stem cells (ECAM) were obtained from human colorectal cancer cell line LoVo by the method of tumor microspheres.The specific markers of colorectal cancer stem cells were identified and studied.Lipofectamine 2000 liposome was used to complete the transfection,which was divided into three groups:blank group (ECAMhjgh/CD44 + LoVo),control group (transfection of common inhibitors,lipofectamine 2000-inhibitors) and experimental group (ECAM) according to different treatment methods.They were cultured in the same way,the three group of cells in logarithmic phase were treated with irinotecan (drug concentrations were 1,5,10,15,20,25,30,35,40,45,50 mg · L-1) and capecitabine (drug concentrations were 50,100,150,200,250,300,350,400,450,500,550,600mg· L-1).Then,they continued to be cultured.The expression levels of ECAM mRNA in three groups of ceils were detected by real-time fluorescence quantitative detection (RT-PCR).The thiazolyl blue tetrazolium bromide (MTI)assay was used to detect the proliferation and drug sensitivity of the three groups before and after treatment with irinotecan and capecitabine.Transwell cells were used to detect the invasion ability of three groups of cells.Results The percentage of ECAMhjgh/CD44 + colorectal cancer stem cell in LoVo cell line was 0.95％,85.78％ before and after enrichment,and the difference was significant (P ＜ 0.05).The expression of ECAM mRNA in blank group,control group and experimental group were 8.17 ±0.64,7.94 ±0.83,2.16 ±0.12.Compared with blank group,the difference had significantly in two groups (P ＜ 0.05,P ＜ 0.01).Compared with control group,the difference in experimental group had significantly in two groups (P ＜ 0.05).It indicated that model was succeed prepared.Invasive cell in the three groups were 79.22 ± 5.25,80.12 ± 4.89,31.23 ± 2.36.Compared with blank group,the difference had significantly in two groups (P ＜0.05,P ＜0.01).Compared with control group,the difference in experimental group had significantly(P ＜0.05).The IC50of irinotecan on colorectal cancer stem cells in the three groups were (20.25 ±4.35),(19.22 ±3.99),(10.24 ± 2.04) mg · L-1.The IC50of capecitabine on colorectal cancer stem cells in the three groups were (320.13 ± 23.65),(315.79 ± 21.03),(250.22 ± 15.45) mg · L-1.Compared with control group,the difference in experimental group had significantly(P ＜0.05).Conclusion Targeted down-regulation of ECAM can effectively inhibit the proliferation and invasion of colon cancer stem cells,and enhance their sensitivity to drugs at the same time.

The microRNA (miRNA) regulation mechanisms associated with atherosclerosis are largely undocumented. Specific selection and efficient validation of miRNA regulation pathways involved in atherosclerosis development may be better assessed by contemporary microarray platforms applying cross-verification methodology. A screening platform was established using both miRNA and genomic microarrays. Microarray analysis was then simultaneously performed on pooled atherosclerotic aortic tissues from 10 Apolipoprotein E (apoE) knockout mice (apoE-/-) and 10 healthy C57BL/6 (B6) mice. Differentiated miRNAs were screened and cross-verified against an mRNA screen database to explore integrative mRNA-miRNA regulation. Gene set enrichment analysis was conducted to describe the potential pathways regulated by these mRNA-miRNA interactions. High-throughput data analysis of miRNA and genomic microarrays of knockout and healthy control mice revealed 75 differentially expressed miRNAs in apoE-/- mice at a threshold value of 2. The six miRNAs with the greatest differentiation expression were confirmed by real-time quantitative reverse-transcription PCR (qRT-PCR) in atherosclerotic tissues. Significantly enriched pathways, such as the type 2 diabetes mellitus pathway, were observed by a gene-set enrichment analysis. The enriched molecular pathways were confirmed through qRT-PCR evaluation by observing the presence of suppressor of cytokine signaling 3 (SOCS3) and SOCS3-related miRNAs, miR-30a, miR-30e and miR-19b. Cross-verified high-throughput microarrays are optimally accurate and effective screening methods for miRNA regulation profiles associated with atherosclerosis. The identified SOCS3 pathway is a potentially valuable target for future development of targeted miRNA therapies to control atherosclerosis development and progression.
Animals ; Aorta/metabolism/pathology ; Apolipoproteins E/*deficiency/metabolism ; Atherosclerosis/genetics/pathology ; Down-Regulation/genetics ; Gene Expression Profiling ; *Gene Expression Regulation ; Gene Regulatory Networks/genetics ; Immunohistochemistry ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; MicroRNAs/*genetics/metabolism ; Models, Biological ; *Oligonucleotide Array Sequence Analysis ; RNA, Messenger/genetics/metabolism ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction/*genetics ; Suppressor of Cytokine Signaling Proteins/genetics/metabolism ; Up-Regulation/genetics

OBJECTIVETo kinds of establish a HPLC method for determining contents of indole alkaloids and bufadienolides contained in toad medicines, and analyze two kinds of components contained in toad venom, toad skin and toad periostracum.

METHODAs for alkaloids, Nucleosil C18 column was adopted with acetonitrile and water containing 0.5% potassium dihydrogen phosphate (6: 94, adjust pH to 3.2 with phosphate acid) as the mobile phase. The flow rate was 0.8 mL x min(-1), the detection wavelength was 275 nm, and the column temperature was 30 degrees C. As for bufadienolides, Alltima C18 column was adopted with acetonitrile and water containing 0.3% acetic acid (B) as the mobile phase. The gradient process was as follows: a linear gradient from 28% to 54% acetonitrile in the first 15 min, then kept at 54% for additional 20 min. The flow rate was 0.6 mL x min(-1), the detection wavelength was 296 nm, and the column temperature was 30 degrees C.

RESULTThe linear ranges were 0.079 6-0.796 microg for serotonin, 0.097 2-1.945 microg for N-methylserotonin, 0.074 4-0.744 microg for N,N-dimethylserotonin, 0.103-2.05 microg for N,N,N-trimethylserotonin, and 0.067 2-0.672 microg for bufothionine, respectively. The average recoveries of serotonin and N-methylserotonin were 98.6% and 91.3%, respectively. The linear ranges of gamabufotalin, bufotalin, bufalin, cinobufagin and resibufogenin were 0.004 83-0.614, 0.007 9-1.006, 0.007 95-1.016, 0.009 7-1.24 and 0.009 6-1.22 microg, respectively, and their average recoveries were 101.6%, 102.5%, 101.0%, 99.1% and 98.9%, respectively.

CONCLUSIONToad venom has the highest contents of indole alkaloids and bufadienolides, followed by toad skin, and toad periostracum showed the lowest contents and even no detection result.

Amphibian Venoms ; chemistry ; Animals ; Bufanolides ; chemistry ; isolation & purification ; Bufonidae ; Chromatography, High Pressure Liquid ; Indole Alkaloids ; chemistry ; isolation & purification ; Skin ; chemistry

Amino Acid Sequence ; Base Sequence ; Genes, Bacterial ; Humans ; Molecular Sequence Data ; Polymerase Chain Reaction ; Staphylococcus epidermidis ; enzymology ; genetics ; beta-Lactamases ; chemistry ; genetics

OBJECTIVETo study CXCR3 and CCR5 chemokine receptor expression in spleens of patients with primary immune thrombocytopenia (ITP) and its clinical significance.

METHODSThe splenectomy specimens from 10 ITP patients (ITP group) and 8 patients with traumatic splenic rupture (normal control group) were studied. Immunohistochemistry (IHC) was used to study the positive rate of CXCR3 and CCR5. Western blot was performed to detect CXCR3 and CCR5 protein expression, while real-time polymerase chain reaction (RT-PCR) was conducted to analyze their mRNA expression.

RESULTSThe positive rate of CXCR3 and CCR5 were both higher in ITP group (90% and 100%, respectively) than those in control group (75% and 87.5%, respectively)(P < 0.05). The differences were statistically significant (P < 0.05). Protein and mRNA level of CXCR3 in ITP group were 3.0 and 3.5 times as high as those in control group, respectively. Those of CCR5 in ITP group were 1.2 and 1.7 times as high as those in control group, respectively.

CONCLUSIONHigh expression of CXCR3 and CCR5 may play a part in the splenic immune disorders in patients with ITP.

Adolescent ; Adult ; Case-Control Studies ; Child ; Female ; Humans ; Male ; Middle Aged ; Receptors, CCR5 ; metabolism ; Receptors, CXCR3 ; metabolism ; Spleen ; metabolism ; Thrombocytopenia ; immunology ; metabolism ; Young Adult

Objective To clarify the understanding of types of laboratories and manufacturers for GB 9706.1-2020 Medical electrical equipment-Part 1:General requirements for basic safety and essential performance by laboratory proficiency testing for creepage distance and electrical clearance test.Methods An operation guide was formed according to the testing program in GB 9706.1-2020,and the homogeneity and stability of the samples were evaluated according to CNAS-GL003:2018 Guidance on Evaluating the Homogenneity and Stability of Samples Used for Proficiency Testing.Robust statistic methods were used to assess the quantitative parameters of the test results of the participating laboratories according to the requirements in GB/T 28043-2019 Statistical methods for use in proficiency testing by interlaboratory comparison;the results reported by the expert laboratories were used as the specified values of the qualitative parameters.SPSS 25.0 statistical software was used for data analysis.Results All the results of the crreepage distance and electrical clearance tests met the requirements for homogeneity and stability.Of the 46 laboratories involved in,37 ones did have comprehensive satisfactory determinations while the remained 9 ones not.Conclusion Some laboratories don't behave well in understanding the standard,which have to be reformed accordingly to enhance their proficiencies.[Chinese Medical Equipment Journal,2024,45(10):54-59]

Objective To investigate the clinical phenotypes and genotypes of children with congenital fibrinogen disorder(CFD).Methods A retrospective analysis was conducted on the clinical data of 16 children with CFD.Polymerase chain reaction was used to amplify all exons and flanking sequences of the FGA,FGB,and FGG genes,and sequencing was performed to analyze mutation characteristics.Results Among the 16 children,there were 9 boys(56%)and 7 girls(44%),with a median age of 4 years at the time of attending the hospital.Among these children,9(56%)attended the hospital due to bleeding events,and 7(44%)were diagnosed based on preoperative examination.The children with bleeding events had a significantly lower fibrinogen activity than those without bleeding events(P<0.05).Genetic testing was conducted on 12 children and revealed a total of 12 mutations,among which there were 4 novel mutations,i.e.,c.80T>C and c.1368delC in the FGA gene and c.1007T>A and C.1053C>A in the FGG gene.There were 2 cases of congenital afibrinogenemia caused by null mutations of the FGA gene,with relatively severe bleeding symptoms.There were 7 cases of congenital dysfibrinogenemia mainly caused by heterozygous missense mutations of the FGG and FGA genes,and their clinical phenotypes ranged from asymptomatic phenotype to varying degrees of bleeding.Conclusions The clinical phenotypes of children with CFD are heterogeneous,and the severity of bleeding is associated with the level of fibrinogen activity,but there is a weak association between clinical phenotype and genotype.

OBJECTIVETo observe the clinical effect of the treatment for complex fractures of the tibial plateau through the application of the external fixator and the locking plate.

METHODSFrom Feb. 2006 to Oct. 2008,12 patients with tibial plateau fractures were treated with external fixator and locking plate included 8 males and 4 females with an average age of 38 years ranging from 23 to 59. According to Schatzker type, 7 cases were type V and 5 cases were type VI. Using an anteromedial incision and an anterolateral approach, the locking plate were fixed in the tibia lateral. The collapse and height lossing of tibial plateau was observed through X-ray film before and after operation. The function of knee joint was evaluated according to HSS scoring.

RESULTSThese patients were followed up for 4 to 18 months (means 9.79 months). Eleven cases had bone primary union,and 1 delayed union. No deep phlebothrombosis and osteofascial compartment syndrome occurened. The average healing time was 3.1 months. Between the preoperative and postoperative X-ray film there were no second stage depression fracture of the tibial plateau,postoperative reduction loss and bad alignment. The range of knee flexion was 90 degrees to 110 degrees. The HSS knee functional scoring was(75.50 +/- 10.01)scores after operation and (21.50 +/- 11.68) scores before operation.

CONCLUSIONThe treatment with the external fixator and the locking plate for complex fractures of the tibial plateau could provid continuous stability of fixation,prevent the fracture from second stage displacement and the knee force line change, protect the soft-tissue around the knee, reduce the postoperative complications. The knee joint function is satisfied.

Adult ; Bone Plates ; External Fixators ; Female ; Humans ; Knee Joint ; physiopathology ; Male ; Middle Aged ; Tibial Fractures ; physiopathology ; surgery