Objective To investigate the value of differential diagnosis of 5-hydroxytryptamine,dopamine and their metabolic productions in cerebral spinal fluid(CSF) in patients with viral encephalitis and schizophrenia.Methods The CSF concentrations of DA,5-HT,5-hydroxyindole acetic acid(5-HIAA) and homovanillic acid(HVA) were assayed by high-performance liquid chromatography(HPLC) in the patients with viral encephalitis and meningoencephalitis(67 cases),schizophrenia(37 cases) and other diseases(21 cases).Results(1) Compared with case control group,the concentrations of 5-HT,5-HIAA and the ratios of 5-HIAA/5-HT,5-HT/DA were significantly lower in viral encephalitis and meningoencephalitis group(P

Objective To investigate the risk factors for postoperative cognitive dysfunction (POCD)induced by patient-controlled intravenous analgesia(PCIA)in elderly patients. Methods 95 patients with POCD and 97 cognitive normal controls were included in the study. The cases and controls were matched for gender, type of operation and PCIA volume dose. Cognitive function was assessed by Mini-Mental-State test and the relationship between POCD and various factors was analyzed by univariate and multivariate analysis. Results Univariate analysis revealed that the education level and visual analog scale (VAS) score had significant differences between the two groups. Multivariate analysis showed that the VAS score and education level were significantly related to POCD induced by PCIA, with the odds ratios of 2. 379 (95%CI:1.205～4.698) and 0. 292 (95%CI:0.157～0.543), respectively. Conclusions Lower VAS score is an independent risk factor and higher education level seems to be a protective factor for POCD induced by PCIA.

Objective:To investigate the value of serum miR-214-5p level in predicting acute kidney injury(AKI)after cardiac surgery in children.Methods:From January 2015 to December 2019, 102 children with congenital heart disease underwent extracorporeal circulation in Haikou Maternal and Child Health Hospital were selected.The children were divided into AKI group( n=28)and non-AKI group( n=74). The levels of serum miR-214-5p, serum creatinine(Scr), cystatin C(Cys-C)and kidney injury molecule-1(KIM-1)were measured three hours after operation in both groups.Multivariate logistic regression was used to analyze the risk factors of AKI after cardiac surgery in children.The values of miR-214-5p, Scr, Cys-C and KIM-1 levels in predicting AKI in children after cardiac surgery were analyzed by receiver operating characteristic(ROC)curve. Results:The levels of serum miR-214-5p[(3.14±1.36)vs.(0.95±0.47)], Scr[(490.35±93.62)μmol/L vs.(108.26±22.40)μmol/L], Cys-C [(3.27±0.85)mg/L vs.(0.86±0.24)mg/L] and KIM-1 [(26.83±8.70)μg/L vs.(6.42±1.18)μg/L] in AKI group were significantly higher than those in non-AKI group(all P<0.05). Multivariate logistic regression analysis showed that serum miR-214-5p, Scr, Cys-C and KIM-1 levels were independent risk factors for AKI in children after cardiac surgery( OR=2.518, P<0.001; OR=1.630, P=0.035; OR=1.974, P<0.001; OR=2.902, P<0.001). ROC curve analysis showed that the area under the curve(0.958, 95% CI: 0.905-0.996)of miR-214-5p, Scr, Cys-C and KIM-1 combined prediction of AKI were the largest, and its sensitivity and specificity were 98.5% and 86.3%, respectively. Conclusion:The level of serum miR-214-5p is significantly higher in children with AKI after cardiac surgery, which is an independent risk factor for AKI after cardiac surgery, and the combination of Scr, Cys-C and KIM-1 levels can better predict the occurrence of AKI.

Objective To clarify whether the Cosmc gene down-regnhtion in lgA nephropathy (IgAN) patients is resulted from genetic disorders or external suppressions. Methods Forty IgAN patients, 16 non-IgAN glomerulonephritis patients and 21 healthy controls were enrolled in the study. Genomie DNA was extracted and then Cosmc gene was amplified and sequenced. Peripheral B lymphoeytes were isolated and cultured with RPMI-1640 alone or plus lipopolysaecharide (LPS). The Cosmc mRNA expression levels at baseline, after RPMI-1640 culture or RPMI-1640+LPS treatment were measured respectively by real-time RT-PCR. Results (1) Only 2 missense mutations and 2 silent mutations were detected in coding frame region of Cosine gene in 2 IgAN patients. (2) The baseline Cosmc gene expression level was significantly lower in IgAN patients (31% of that in healthy controls) than that in healthy controls. (3) Relative quantification PCR indicated that Cosmc mRNA expression level was significantly increased (219% of baseline) after RPMI-1640 culture, and treatment of LPS could strongly inhibit this effect. (4) The Cosmc gene expression of healthy control was not affected by RPMI-1640 or LPS. Conclusion It is not genetic disorders but external suppression to cause the down-regulation of Cosmc gene mRNA expression in IgAN.

Objective To investigate the effects of dextran sulfate on lung ischemia-reperfusion injury after lung transplantation in rats.Method A total of 32 male Wistar rats were subjected to unilateral left lung orthotopic transplantation.They were randomly divided two groups (n =16 each):DXS group [DXS (10 mg/kg) was given prior to the reperfusion],and the control group (the same volume of normal saline was given).After animals were sacrificed,the lung graft was harvested 2 h after reperfusion.Oxygenation indexes,wet/dry ratio (W/D),myeloperoxidase (MPO) activity,malondialdehyde (MDA) and endothelin 1 (ET-1) in the transplanted lung,and tumor necrosis factor a (TNF-α) and interleukin 8 (IL-8) in serum were measured.The lung injury scores were evaluated and complement deposition was observed.Result After 2-h reperfusion,compared to the control group,oxygenation indexes were improved significantly in DXS group (P＜0.05),but there were no significant differences in W/D between two groups.In DXS group,the activity of MPO was significantly reduced,and the contents of MDA and ET-1 in the lung tissue were significantly reduced as compared with the control group.DXS reduced the level of TNF-α and IL-8 markedly in serum (P ＜0.05).There was no significant difference in lung injury score between two groups (4.53 ± 0.46 vs.5.28 ±0.49,P＞0.05).Compared to the control group,DXS reduced the deposition of C3c (0.8 ±0.2vs1.5±0.3) andC6 (1.2±0.4vs.2.4±0.5) (P＜0.05).Conclusion Administration of DXS attenuated ischemia-reperfusion injury after lung transplantation by inhibiting complement deposition,and improved the oxygenation of the transplanted lung.This protection was associated with inhibition of inflammation and oxidation and endothelial cytoprotection.

Objective:To identify effective biomarkers for glioma patients.Methods:The mRNA expression profiles of 464 glioma patients with complete clinical follow-up information were downloaded from the Chinese Glioma Genome Atlas (CGGA). Weighted gene co-expression network analysis (WGCNA) was used to identify gene modules related to World Health Organization (WHO) grading of glioma, and univariate and multivatiate Cox regression analysis were performed to identify gliomas survival-related genes.Results:In weighted gene co-expression analysis, the module Brown was significantly positively correlated with glioma WHO stage ( r=0.55, P<0.05). In univariate analysis, five genes (TAGLN2, IGFBP2, METTL7B, ARAP3, PLAT) that were most significantly associated with clinical prognosis were selected for multivariate survival analysis, and the prognosis model was established to calculate the risk score. The receiver operating characteristic curve (ROC) confirmed that the risk score had high accuracy in predicting the 1-, 3-, 5-year survival rate of glioma patients. The above survival analysis results were verified in the Cancer Genome Atlas (TCGA) database. Conclusions:We use mRNA expression profiles to establish prognostic markers for gliomas to assess the overall survival of patients with glioma.

AIM: To establish human umbilical vein endothelial cells (HUVECs) to express green fluorescent protein (GFP), and to study the suppression of GFP by siRNA in HUVECs. METHODS: Using lipofectamine 2000 to transform plasmid pN_3-EGFP encoding GFP into HUVECs. The HUVEC containing pN_3-EGFP, named HUVEC-GFP, was screened and selected by antibiotic G418. Using in vitro transcription T7 kit, GFPsiRNA targeting GFP mRNA and control-siRNA used as control were synthesized. The siRNAs were transfected into HUVEC-GFP with oligofectamine. 48 h later, the expression levels of GFP protein and mRNA in HUVEC-GFP were determined. RESULTS: The HUVEC-GFP was screened to express GFP in the presence of G418. The agarose gel electrophoresis analysis showed that the siRNAs prepared were integrated. 48 h after transfection with siRNAs, compared to control group, the level of GFP fluorescence was obviously decreased in the HUVEC-GFP transfected with GFPsiRNA. The results of RT-PCR detection showed that GFP mRNA expression was obviously suppressed by GFPsiRNA at the rate of 40%, and no obvious suppression of GFP mRNA expression was found in the HUVEC-GFP transfected with control siRNA. CONCLUSION: The siRNA targeting GFP mRNA, synthesized in vitro, efficiently suppresses the GFP expression in HUVECs.

AIM:To investigate the effect of caspase-8 small hairpin RNA ( shRNA) on attenuating apoptosis of human mesenchymal stem cells ( hMSCs ) .METHODS: Two recombinant plasmids for over-expression of caspase-8 shRNA, pAd-Cap8 shRNA1 and pAd-Cap8 shRNA2, were constructed.Caspase-8 mRNA was determined in pAd-Cap8 shRNA-transfected human HEK293 cells by Q-PCR.The screened pAd-Cap8 shRNA was used to construct the recombinant adenovirus plasmid , which was linearized and transfected into HEK 293 cells for packaging and amplification of the recombi-nant adenovirus rAd-Cap8 shRNA.The expression of caspase-8 at mRNA and protein levels was determined by Q-PCR and Western blotting .Annexin V/PI staining and determination of caspase-8 activity were performed to assess apoptosis of hM-SCs under the conditions of serum deprivation and hypoxia .The mRNA expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), Bcl-2 and Bcl-xL was analyzed by Q-PCR.RESULTS:The pAd-Cap8 shRNA, which efficiently inhibited caspase-8 expression, was screened by Q-PCR.The recombinant adenovirus plasmid for caspase-8 shRNA was constructed and used to package and amplify the recombinant ad-enovirus ( rAd)-Cap8 shRNA successfully .rAd-Cap8 shRNA-mediated caspase-8 shRNA markedly inhibited caspase-8 ex-pression in hMSCs .Over-expression of caspase-8 shRNA by infection of rAd-Cap8 shRNA also efficiently decreased the ap-optotic rate and caspase-8 activity in hMSCs under the conditions of serum deprivation and hypoxia , with up-regulation of the mRNA expression of HGF, IGF-1 and Bcl-2.CONCLUSION:Caspase-8 shRNA attenuates hMSC apoptosis under the conditions of serum deprivation and hypoxia .

AIM:To construct recombinant adenovirus vector carrying human miR-133a and study its expression in human mesenchymal stem cells(hMSCs).METHODS:The PCR product containing miR-133a was amplified from human genomic DNA and inserted into the adenoviral shuttle vector pAdTrack-CMV.Then the recombinant shuttle plasmid linearized by pmeⅠwas cotransformed into competent E.coli.BJ5183 with the adenoviral backbone plasmid pAdEasy-1 to generate the recombinant adenovirus vector rAd-mir-133a.rAd-mir-133a was then packaged and amplified in human embryonic kidney 293(HEK293) cells.The purified rAd-miR-133a was used to infect the hMSCs and the expression of miR-133a was detected by non-quantitative RT-PCR and real-time PCR.RESULTS:The recombinant adenovirus shuttle vector pAdTrack-CMV-miR-133a was constructed and verified by restriction endonuclease analysis and DNA sequence analysis.rAd-miR-133a was successfully packaged and amplified in HEK293 cells.The transcriptions of primary miR-133a and mature miR-133a were over-expressed in the hMSCs infected with rAd-miR-133a.CONCLUSION:The recombinant adenovirus vector carrying human miR-133a is successfully constructed,which lay a foundation for miR-133a function study.

OBJECTIVETo investigate the association of desmoplakin with the distribution and function of Nav1.5 by RNA silencing technology in HL-1 cells.

METHODSHL-1 cells with desmoplakin expression suppression by RNA silencing were examined for desmoplakin and Nav1.5 protein expressions by Western blotting, and the distribution and co-location of desmoplakin and Nav1.5 protein were detected by immunofluorescence staining. Patch-clamp recording was applied to analyze the changes in whole-cell sodium current after desmoplakin silencing.

RESULTSCompared with the untreated group and negative control group, the cells with desmoplakin silencing showed obviously reduced expressions of desmoplakin and Nav1.5 proteins. Co-localization of desmoplakin and Nav1.5 was detected at cell-cell contact in untreated and control conditions, and desmoplakin expression silencing induced a drastic redistribution of Nav1.5 with decreased peak current density (156.3∓6.2 vs 41.8∓3.1, n=6, P<0.05), a shift in voltage dependence of steady-state inactivation (-42 mV vs -61 mV, n=5, P<0.05), and prolonged time of recovery from inactivation.

CONCLUSIONDesmoplakin silencing caused redistribution of Nav1.5 protein and also changes in its electrophysiological properties in HL-1 cells.

Animals ; Cell Line ; Desmoplakins ; genetics ; metabolism ; Gene Silencing ; Mice ; Mutation ; Myocytes, Cardiac ; metabolism ; NAV1.5 Voltage-Gated Sodium Channel ; metabolism