Effect of siRNA-mediated α-TAT1 gene silencing on migration behavior of endothelial cells in rats with hepatopulmonary syndrome / 陆军军医大学学报

Objective To investigate the effect of silencing alpha tubulin acetyltransferase 1(α-TAT1)on migration behavior of endothelial cells induced by hepatopulmonary syndrome(HPS).Methods Online database Tabula Muris was used to analyze the expression of α-TAT1 in various cell subsets in the lungs.Twenty-four male SD rats were randomly divided into control group(Sham group,n=6)and common bile duct ligation group(HPS group,n=18).The rats in HPS group were euthanasized at 2 and 4 weeks after modelling,and then the expression of α-TAT1 in pulmonary vascular endothelial cells was detected by immunofluorescence colocalization.The sera from the Sham and HPS rats were used to stimulate human umbilical vein endothelial cells(HUVECs)for 12 and 24 h,respectively.Then the obtained HUVECs were divided into 4 groups:Sham serum+siRNA NC group,Sham serum+siRNA α-TAT1 group,HPS serum+siRNA NC group,HPS serum+siRNA α-TAT1 group.The expression levels of α-TAT1 and Ace-α-tubulin in HUVECs were detected by Western blotting.Immunofluorescence assay was applied to observe the levels of polymerized microtubules of α-Tubulin in HUVECs after nocodazole(10 μmol/L)pretreatment to evaluate the stability of microtubule structure.Cell scratch assay combined with cell immunofluorescence assay was employed to observe the nuclear localization of Golgi apparatus and cell migration ability of HUVECs.The angiogenesis ability of HUVECs was tested by in vitro angiogenesis test.Results In vivo and in vitro experiments showed that the expression of α-TAT1 in endothelial cells was significantly increased after HPS inducement.The expression levels of α-TAT1 and Ace-α-tubulin were significantly down-regulated,and the stability of microtubules was weakened in the siRNA α-TAT1 interference group(P＜0.01).In addition,the distribution of GM 130 labeled Golgi apparatus in the protrusion of HUVECs was down-regulated in the siRNAα-TAT1 interference group,as well as the migration ability(P＜0.01).And the length of angiogenesis and network level were also significantly declined(P＜0.01).Conclusion Silencing α-TAT1 reduces the migrαtion and angiogenesis of endothelial cells in HPS,which was associated with weakened stabilization of microtubule.

Construction of WISP3 gene's mutants in SEDT-PA and their expression in COS-7 cells / 中南大学学报(医学版)

OBJECTIVE@#To construct two types of Wnt-inducible secreted protein 3(WISP3) gene's mutants(1000T/C,840delT) found in spondyloepiphyseal dysplasia tarda with progressive anthopathy (SEDT-PA) patients, and to observe their expression in COS-7 cells.@*METHODS@#Full-length cDNA of wild type WISP3 gene(WT-WISP3) was amplified from human chondrocytes by RT-PCR, and site-directed mutagenesis was used to obtain full-length cDNAs of the mutated WISP3 genes(MUT1000T/C and MUT840delT). The recombined plasmids WT-WISP3/pcDNA3.1(+), MUT1000T/C/pcDNA3.1(+) and MUT840delT/pcDNA3.1(+) were transfected transiently into COS-7 cells by liposome-mediated method, and pcDNA3.1(+) vector was used as a control. The total RNA and protein of the transfected COS-7 cells were extracted after 48 hours of transfection. The expression of WISP3 gene in the transfected COS-7 cells was detected by semi-quantitative RT-PCR and Western blot.@*RESULTS@#By restriction endonuclease analysis and sequencing, the sequence of MUT1000T/C and MUT840delT were consistent with that mutated in SEDT-PA, and the open reading frames matched with the vector sequence. Semi-quantitative RT-PCR and Western blot showed that the recombined plasmids were highly expressed in COS-7 cells.@*CONCLUSION@#WISP3 gene's mutants of SEDT-PA are successfully constructed by genetic recombination, and expressed in COS-7 cells, which lays the foundation for the further study on its molecular functions in SEDT-PA.

Evaluation of enzymatic method for determination of serum beta-hydroxybutyrate and its clinical application / 中国医学科学院学报

<p><b>OBJECTIVE</b>To evaluate an enzymatic method for determining serum beta-hydroxybutyrate (beta-HB) with the National Committee for Clinical Laboratory Standards (NCCLS) projects, and to discuss its clinical values in diabetic ketoacidosis (DKA).</p><p><b>METHODS</b>The precision, accuracy, specificity, linearity and interference of the enzymatic method were analyzed. This method was used to determine serum beta-HB in 60 cases of normals, 50 cases of diabetes, and 34 cases of DKA by autochemistry analyzer.</p><p><b>RESULTS</b>Enzymatic beta-HB assay was precise (within-run CV, day-to-day CV, and total CV < 5%). The linearity studies showed the method was linear up to 4 mmol/L. Recovery rate was 98.5%-104.1%. Hemolysis (Hemoglobin up to 18.2 g/L), icteric samples with total bilirubin up to 224 mumol/L, and lipemia up to triglyceride concentration of 2.28 mmol/L did not interfere with the beta-HB results in this method. Serum beta-HB levels were significantly elevated in DKA patients compared with DM patients and controls (P < 0.01). Positive rate of serum beta-HB in DKA patients was significantly higher than that of urinary ketone (P < 0.05).</p><p><b>CONCLUSIONS</b>Enzymatic method is convenient and reliable, allows full automation, and is rapid enough to be used for both routine and urgent determinations of serum beta-HB. It can be used in diagnosing and monitoring treatment of DKA.</p>