ObjectiveTo study the pathogcncsis of gastrointestinal vascular malformation (GIVM) and the potential mechanism of thalidomide in the treatment of gastrointestinal bleeding due to GIVM.Methods We collected the surgical intestinal specimens from 10 patients who suffered from massive hemorrhage of gastrointestinal tract owning to GIVM and the normal intestinal mucosa around the lesions,as well as normal intestinal mucosa from healthy subjects.Immunohistochemical(IHC) staining was carried out to investigate the differences of angiopoietin 2 ( Ang2 ),Notch1 and delta like ligand 4 (Dll4) in the above three intestinal mucosa to find the relationship with the pathogenesis of GIVM. Human umbilical vein endothelial cells(HUVECs) were cultured with 0,25,50,100 and 200 mg/L thalidomide for 24 or 48 hours to observe their mRNA and protein expressions of Ang2,Notch1,Dll4 by real-time PCR and Western blot.ResultsBy IHC staining,more expressions of Ang2,Notch1 and Dll4 in the lesions were detected than those in the normal intestinal mucosa around the lesions and the normal intestinal mucosa in healthy people.The expressions of Ang2,Notch1 and Dll4 were significantly correlated (P =0.016,r =0.732),and the expressions of Notch1 and Dll4 were absolutely correlated ( P =0.000,r =1.000).Real-time PCR and Western blot showed that thalidomide could down-regulate the expressions of them,which were in a concentration-dependent manner.ConclusionAng2,Notch1 and Dll4 may correlate with the pathogenesis of GIVM,while thalidomide can concentration-dependently down-regulate the expression of Ang2,Notch1 and Dll4,which may be one of the mechanism that thalidomide play a therapeutic role in GIVM.

Objective To study the expressions of hypoxia inducible factor (HIF)-1 and angiopoietin (Ang)-2 in repeated gastrointestinal bleeding due to vascular malformation, and the efficacy of treatment with thalidomide. Methods Twenty-five patients with repeated gastrointestinal bleeding due to vascular malformation confirmed by capsule endoscopy or enteroscopy were collected and 18 subjects without severe diseases were served as controls. Ten patients with gastrointestinal vascular malformation, who received 25 mg of thalidomide 4 times daily for 4 months and were followed up for at least one year, were also enrolled. The serum samples from all participauts were detected for expressions of HIF-1 and Ang-2 using enzyme-linked immunosorbent assay (ELISA).The expressions of HIF-1 and Ang-2 were compared between angiodysplasia group and control group.The expressions of HIF-1 and Ang-2 were comparatively evaluated before and after treatment with thalidomide in treatment group. Results The expressions of HIF-1 and Ang-2 in vascular malformation group [( 113. 84 ± 26. 66 ) ng/ml and ( 652. 11 ± 140. 39) ng/ml, respectively] were significantly higher than that of control group [(43.28±17.30) ng/ml and (265.60±53.88) ng/ml,respectively, P=0. 000]. The expression of HIF-1 was positively associated with that of Ang-2. (r=0. 700, P= 0. 000). There was no difference in expressions of HIF-1 and Ang-2 before and after treatment with thalidomide (P=0. 498 and =0. 136, respectively). However, significant reduction of Ang-2 [(113. 80±73. 60) ng/ml(P=0. 003)] was found in 8 effectively treated patients after thalidomide treatment. Conclusions HIF-1 and Ang-2 might play an important role in the formation of vascular malformation. The extent of Ang-2 reduction after thalidomide treatment may be helpful in evaluating its efficacy or prognosis.

Objective To study the long-term effect of argon plasma coagulation (APC) combined with proton pump inhibitor (PPI) on Barrett esophagus (BE). Methods A total of 36 patients, histologically proven as having BE from 2004 to 2007, were enrolled to underwent a therapy of APC plus PPI. The patients were re-examined on endoscopy at 1, 6 and 12 months after first APC and once a year thereafter.Results A total of 48 APC sessions were given to 36 patients with a mean number at 1. 33 per patient. The effective rate of reversal of BE was 100%. The follow-up was accomplished for all patients in 14-51 months with a median of 36months. The total recurrence rate (RR) of BE reached 16. 7% (6/36). The 1-year and 2-year RRs were 2. 8% (1/36) and 11.1% (4/36), respectively. The logistic regression analysis suggested that 2-year and total RRs were related to APC sessions ( P ＜ 0. 01 ). Conclusion The therapy of APC combined with PPI for BE is safe and of long-term effects.

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.

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 .

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

AIM:To investigate the effect of microRNA-214 ( miR-214) on cardiomyocyte hypertrophy and the expression of the potential target genes .METHODS:A cell model of hypertrophy was established based on angiotensin-Ⅱ( Ang-Ⅱ)-induced neonatal mouse ventricular cardiomyocytes ( NMVCs) .Dual luciferase reporter assay was performed to verify the interaction between miR-214 and the 3’ UTR of MEF2C.The expression of MEF2C and hypertrophy-related genes at mRNA and protein levels was determined by RT-qPCR and Western blot , respectively .RESULTS:The expression of ANP, ACTA1,β-MHC and miR-214 was markedly increased in Ang-Ⅱ-induced hypertrophic cardiomyocytes .Dual lu-ciferase reporter assay revealed that miR-214 interacted with the 3’ UTR of MEF2C, and miR-214 was verified to inhibit MEF2C expression at the transcriptional level .The protein expression of MEF2C was markedly increased in the hypertro-phic cardiomyocytes .Moreover, miR-214 mimic, in parallel to MEF2C siRNA, inhibited the expression of hypertrophy-re-lated genes in Ang-Ⅱ-induced NMVCs.CONCLUSION:MEF2C is a target gene of miR-214, which mediates the effect of miR-214 on attenuating cardiomyocyte hypertrophy .

Humans ; HIV Infections/drug therapy* ; HIV-1/genetics* ; Tenofovir/therapeutic use* ; Anti-HIV Agents/therapeutic use* ; RNA ; Drug Combinations

Objective:To identify the classification characteristics and quality of life (QOL) of breast cancer (BC) patients during chemotherapy, so as to provide basis for improving the sleep and QOL of this group.Methods:A cross-sectional investigation was completed among 421 BC patients in 5 tertiary hospitals in Shanghai, Wuhan, Tangshan and Nanning in 1-12 months of 2016 using validated instruments including self-made general information questionnaire, Pittsburgh Sleep Quality Index (PSQI) and Functional Assessment of Cancer Therapy-Breast (FACT-B).Results:Four latent class of patients were identified through latent profile analysis (LPA), named by badly worse sleep quality(SQ) (C1, n=23), medium-SQ with difficulty to fall asleep (C2, n=127), medium-SQ with worse sleeping process (C3, n=30), none sleep disorders (C4, n=241). Total points of SQ among C1-C4 had significant difference ( χ2 value was 309.28, P<0.05). Age, BMI, job status, whether had surgery and course of chemotherapy between classes had statistically significant differences ( χ2 values were 9.57-25.28, all P<0.05). It had significant difference between C2 and C3, C2 and C4, C3 and C1, C3 and C4 on QOL ( χ2 values were 5.96-52.73, all P<0.05). Conclusion:SQ of BC patients during chemotherapy has heterogeneity among population. Different features of SQ of BC patients have different performance on QOL. Health professionals should keep an eye on patients with features of older age, high BMI, in job status, already received surgery and during early-stage chemotherapy, provide personal nursing intervention to improve SQ and QOL.