Objective To investigate the role of MHC class Ⅱ transactivator(CⅡTA) in upregulating the expression of HLA class Ⅱ antigen in HepG2 cells.Methods Eukaryotic expression vector EBS-NPL-CⅡTA containing CⅡTA cDNA or the empty vector EBS-NPL was transfected into HepG2 cells respectively.CⅡTA mRNA and HLA class Ⅱ antigen(HLA-DR,DP,DQ) were respectively detecued by RT-PCR,indirect cell immunofluorescence technique and flow cytometry in original HepG2 cells,HepG2 cells transfected with EBS-NPL-CⅡTA or the empty vector EBS-NPL.Results The expression of CⅡTA mRNA and HLA class Ⅱ antigen(HLA-DR,DP,DQ) were not observed in original HepG2 cells and HepG2 cells transfected with empty vector,but in the HepG2 cells transfected with EBS-NPL-CⅡTA.Conclusion CⅡTA is a switching factor of mastering the expression of HLA class Ⅱ antigen in HepG2 cells.The lack of CⅡTA expression in HepG2 cells contributes to no expression of HLAⅡ antigen.

Objective To study the function of 4 different haplotypes cDNA which are constructed by two non-homonymy single nueleotide polymorphism (SNP) sites C19170G (Leu45Val) and C30799G (Ala500Gly) in the coding region of human CⅡTA gene. Methods HeLa cells were transfeeted with eu-karyotic expression vectors containing four different haplotypes cDNA. C Ⅱ TA mRNA and HLA classⅡanti-gen (HLA-DR, DP, DQ) were respectively detected by RT-PCR and indirect cell immunofluoreseence tech-nique in the untransfected and transfeeted with four eukaryotic expression vectors and empty vectors HeLa cells. The quantity of HLA classⅡ antigen were analyzed by flow eytometry. Results No expression of CⅡTA mRNA and HLA class Ⅱ antigen were observed on original HeLa cells and empty vector transfected cells. CⅡTA mRNA expression was emerged, and the expression of HLA class Ⅱ antigen were observed in the HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA. And there were not significantly different with the levels of HLA class Ⅱ antigen expression among HeLa cells transfected with eukaryotic expression vectors containing four different haplotypes cDNA ( P > 0.05 ). Con-dusion The SNP of Chinese at the sites C19170G(Leu45Val) and C30799G(Ala500Gly) in the coding site of C Ⅱ TA gene did not influence capability of CⅡTA trans-aetivating HLA class Ⅱgene expression.

[ABSTRACT]OBJECTIVETo explore the method on reconstruction of long and special tracheal defects which can mostly match with natural airway: pedicle rib cartilage with cilia endothelial wall.METHODS8 experimental model of rabbits were trained with cervical double belt blood supply fascia embedding and autologous costal cartilage and nasal septum mucos in the first period, then followed by transplantation in the second period. After the operation, we would assess the physiology, breathing and histopathology index of the rabbits, etc. After the animal experiment, we tried to apply the method to an appropriate clinical case.RESULTS8 cases of experimental rabbits dead after the second period operation with the average survival time of 21.9 days and caused by asphyxia. Histopathological results: rib cartilage and trachea ring up of cartilage cells and fibers have high similarity in histology; cartilage of all cases under the cultivation of the pedicle fascial package has not been absorbed; all cases' nasal septum mucosa in the body and blood supply to differentiation under fascia nutrition. Then we applied the method on a clinical case.CONCLUSIONTrachea ring rib cartilage had a higher similarity to the tracheal cartilage on the histology and biological characteristics that can be used as the preparation of artificial trachea shaping material or cell culture to tissue engineering materials. Package of rib cartilage pedicle fascial can provide adequate blood supply to make up for a free training rib cartilage defect to its absorption. Nasal septum mucosa of pressure in the body and blood supply of the fascia nutrition can simulate the trachea ciliated epithelium, which can play its biological characteristics similar to the inner wall of the trachea.

Objective To investigate the relationship between the non-homonymy single nucleotide polymorphism(SNP)of C19170G,C30799G in the coding area of class Ⅱ transaetivator(CII TA)and the different clinical phenotypes of chronic hepatitis B virus(HBV)infection.Methods Six hundred and twenty-seven patients with chronic HBV infection and 101 healthy blood donors were enrolled in this study.Genotyping of C19170G,C30799G in C Ⅱ TA gene coding region were done by sequence-specific primer polymerase chain reaction(PCR-SSP).Hardy-Weinberg balance of the genotypes was analyzed using chi-square test.Differences between two sets were tested by contingency table chi-square test and unconditional Logistic regression was performed. Results The frequencies of G allele and GG+GC genetypes at 19170 site were significantly higher in patients with liver cirrhosis than those with non-progressive liver diseases(X2=7.128,P=0.008;X2=6.404,P=0.011,respectively).There were significantly differences of the allele frequencies between patients with liver cirrhosis and non-progressive liver diseases(OR:0.742,95%CI:0.552～0.998,P=0.048),and the main differences were observed in G dominant model(OR:0.581,95% CI:0.353～0.954,P=0.032).The frequencies of C allele and CC genotype at 30799 site were significantly higher in patients with hepatocellular carcinoma than those in patients with liver cirrhosis(X2=4.861,P=0.027;X2=4.993,P=0.025).There were significant differences of the genotype frequencies at 30799 site between patients with liver cirrhosis and hepatocellular carcinoma(OR:0.557,95% CI:0.334～0.930,P=0.025),and the differences were mainly observed in C recessive model(OR:0.491,95% CI:0.269～0.898,P=0.021).Conclusions The polymorphisms at 19170 site are associated with liver cirrhosis in chronic HBV infection,and the G allele carriers are prone to progress into liver cirrhosis.The polymorphisms at 30799 site are associated with hepatocellular carcinoma in chronic HBV infection,and CC genotype carriers are prone to progress into hepatocellular carcinoma.

OBJECTIVEThe plasmid construction and validation of RET point mutation in vitro.METHODS The RET gene exon in the mainland familial medullary thyroid carcinoma family was analized with muon capture two generation sequencing of. In vitro, the RET relative point mutationswere reconstructed in NIH3T3 cells and the expression levels were studied.RESULTSThe corresponding lentiviral plasmids of RET point mutations were successfully obtained after point mutating the wild RET plasmids, it was verified that the target genes were expressed in NIH3T3 cells stably by Western Blot. CONCLUSIONSStable cell lines carrying RET point mutations were reconstructedsuccessfully, which provide a good platform for studying various point mutations.

Self-biting is a chronic disease, which cause wound to take effect on mink growth and pelt quality. In this study, we firstly adopted RAPD (random amplification polymorphism DNA) technique based on the reproducible 26 polymorphism primers screened from 100 random primers to analyze hereditary constitution of the samples from healthy minks and self-biting minks, respectively, at molecular level to aim to discuss the causes of self-biting. The results showed that 29 straps showed polymorphism among amplified 105 straps, of which the polymorphism rate is 27.62%. Between healthy and sick mink groups, the amplified DNA fragment through different primers indicated different distribution frequency. The similarity coefficient of mink groups is 0.8471 and genetic distance (variation) index is 0.1529. Through primer S356 (whose sequence is CTGCTTAGGG), we amplified different straps between healthy and sick mink. The amplified 1000 bp DNA fragment in the sick mink groups can preliminarily serve as molecular genetic label to distinguish from healthy and sick mink groups to gradually remove the mink individual of self-biting, achieve to purify mink groups and reduce economy loss of mink breeding industry. This work provide theoretical basis for further study on molecular breeding and disease prevention of mink.
Animals ; Base Sequence ; Genetic Diseases, Inborn ; etiology ; genetics ; veterinary ; Mink ; genetics ; Molecular Sequence Data ; Random Amplified Polymorphic DNA Technique

Objective To knock out p21 gene in human malignant rhab doid tumor(MRT)cell line G401 by using CRISPR/Cas9 genome engineering technology. Methods The expression of p21 was detected by reverse transcription quantitative PCR (RT-qPCR) and Western blot assay in several MRT cell lines. The guide RNA was designed by targeting the third exon of p21 gene,which encoded its home domains, and then subcloned into lentiCRISPR v2 vector and validated sequencing. The validated plasmids were further used to package and produce the lentivirus in 293T cells, and the G401 cells were infected, then puromycin was used to screen positive cells, and the clusters of G401 monoclonal cells, were obtained by selecting monoclonal cells and culturing under the microscope. The RNA and protein of new clonal cell line were extracted, and RT-qPCR and Western blot assay were applied to confirm whether p21 was successfully knocked out. Results The p21 was highly expressed in MRT tumor cells. The CRISPR/Cas9 lentivirus plasmids, targeted p21 gene were successfully constructed. Compared with negative control group,the expression of p21 was not detected in G401 monoclonal cells, which were successfully screened. Conclusion In view of the difficult transfection of cells such as G401, p21 knockout stable cell line has been successfully constructed by using CRISPR/Cas9 system, which lays the foundation for further study of the mechanism of p21 in MRT tumors .

A total of 169 patients with short-duration type 2 diabetic mellitus (DM) were divided into atherosclerosis (AS) group and non-AS group according to the intima-media thickness (IMT) of three conducting arteries.The level of serum amyloid A (SAA) was assayed by ELISA.The results showed that SAA level of type 2 DM patients increased significantly, patients in AS group showed higher SAA level than that in non-AS group, and SAA level was positively correlated with age, body mass index, waist hip ratio and IMT of common carotid artery.Age, C-reactive protein and SAA level are the major risk factors for IMT of common carotid artery.

OBJECTIVETo investigate the risk factors for post-stroke pneumonia and assess the value of A(2)DS(2) score in predicting post-stroke pneumonia in elderly stroke patients.

METHODSThe clinical data were retrospectively collected from elderly stroke patients from January, 2007 to December, 2012. A(2)DS(2) score was then assigned using the clinical information from the medical record. The ability of the score to discriminate between patients with post-stroke pneumonia and those without was quantified using ROC analysis. The calibration of the score was analyzed using Hosmer-Lemeshow goodness-of-fit test.

RESULTSA total of 131 elderly male stroke patients were enrolled in this study, among whom the incidence of post-stroke pneumonia was 29.01%. The independent risk factors for post-stroke pneumonia identified included moderate (P=0.0081, OR: 5.6089; 95%CI: 1.5663-20.0854) and severe (P=0.0048, OR: 44.4827; 95%CI: 3.1847-621.3126) neurological impairment, dysphagia (P=0.0005, OR: 7.5265; 95%CI: 2.4282-23.3292), and atrial fibrillation (P=0.0226, OR: 4.1778; 95%CI: 1.2221-14.2825). The incidence of post-stroke pneumonia ranged from 2.2% in patients with a A(2)DS(2) score less than 3 to 75% in those with a score higher than 8. The C-statistic of A(2)DS(2) score for predicting post-stroke pneumonia was 0.86 (95%CI: 0.784-0.911) by the ROC analysis. The A(2)DS(2) score was well calibrated to predict post-stroke pneumonia in elderly patients by Hosmer-Lemeshow test (7.083, P=0.528).

CONCLUSIONThe A(2)DS(2) score can be useful for predicting post-stroke pneumonia and for routine monitoring of high-risk elderly stroke patients in the clinical setting.

Aged ; Aged, 80 and over ; Atrial Fibrillation ; complications ; China ; Deglutition Disorders ; complications ; Humans ; Incidence ; Male ; Pneumonia ; epidemiology ; etiology ; prevention & control ; ROC Curve ; Retrospective Studies ; Risk Assessment ; Risk Factors ; Severity of Illness Index ; Stroke ; complications

Embryonic stem (ES) cells have the unique capacity to proliferate extensively and maintain the potential to differentiate into advanced derivatives of all three primary germ layers. ES cell lines can also be generated from human blastocyst embryos and are considered promising donor sources for cell transplantation therapies for diseases such as juvenile diabetes, Parkinson's disease, and heart failure. However, as for organ transplants, tissue rejection remains a significant concern for ES cell transplantation. Another concern is the use of human embryos. One possible means to avoid these issues is by reprogramming the nuclei of differentiated cells to ES cell-like, pluripotent cells. This review discusses the potential of these strategies to generate tailor-made pluripotent stem cells and the role of transcription factors in the reprogramming process.
Cell Culture Techniques ; Cell Differentiation ; physiology ; Cells, Cultured ; Cellular Reprogramming ; Humans ; Nuclear Transfer Techniques ; Pluripotent Stem Cells ; cytology