[ ABSTRACT] AIM:To observe the effect of high glucose on the protein expression of calreticulin ( CRT) and its association with cell apoptosis and mitochondrial dysfunction in the cardiomyocytes.METHODS: AC-16 cardiomyocytes were randomly divided into normal glucose group, high glucose group, high glucose+CRT siRNA group and isotonic con-trol group.The cell apoptotic rate, reactive oxygen species (ROS), mitochondrial membrane potential level, respiratory enzyme activity, and protein expression of CRT were observed.RESULTS: Compared with the cardiomyocytes in normal glucose group, the apoptotic rate and ROS production of cardiomyocytes increased in high glucose group, accompanying with the decreases in the mitochondrial membrane potential level and enzyme activitiy of the respiratory chain.The protein expression of CRT was significantly increased in high glucose group.However, compared with high glucose group, high glucose+CRT siRNA decreased the expression of CRT and attenuated the damage of mitochondria, but CRT siRNA did not reduce the ROS level in cardiomyocytes.CONCLUSION:High glucose brings about CRT over-expression to induce mito-chondrial injury, thus increasing myocardial apoptosis.

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.

BACKGROUND: Magnalium which is potential to be the medical biodegraded metal implant is more and more interesting,but it must be well biocompatibility to human body.OBJECTIVE: To evaluate the sensitization of magnalium (AZ31B).METHODS: A total of 35 guinea pigs were randomly divided into saline group (negative control group,n=10),5% volume of formaldehyde (positive control group,n=10),and AZ31B group (n=15).Sensitization test at the maximal dosage was performed according to "Biological evaluation of medical devices-Part 10: Tests for irritation and delayed-type hypersensitivity",including intracutaneous induction,local induction,and provocation.Patch was removed after 6,24,48,and 72 hours,and the skin response was classified accordingto Magnusson and Kligman criteria.Patch was removed after 72 hours,and skin was performed with biopsy,stained with FIE staining,and observed under optic microscope.RESULTS AND CONCLUSION: Sensitization response was not tested in both negative control group and AZ31B group at 24,48,and 72 hours after patch removal; however,moderate erythema was observed in the positive control group.Optic microscope demonstrated that criteria of allergy such as spongiosis,edema,and diffuse as well as perivascular mononuclear infiltration was not observed in the AZ31B group,but a few basophilic calls ware observed.This suggested that AZ31B was biologically safe for sensitization.

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.

Objective:To determine whether ox-LDL (oxdized low-density lipoprotein) is highly deposited on the uremic vessel wall and its influence on the vascular remodeling. Methods: Segments of radial arteries were obtained from 21 uremic subjects during the operation of A-V fistula prior to hemodialysis. Segments of internal thoracic arteries of similar diameter were obtained from patients with benign chest tumors as control.The vascular lesions and ox-LDL, CD68,MCP-1, eNOS,ET-1, PCNA,FN on the vessel wall were determined by means of H-E stain and immunohistochemistry. Results： With H-E stain,atherosclerotic plaques were found in the radial arteries of 4 uremic patients. The middle layer of the arteries in uremic patients were obviously thickened, and the T/D　(thickness of the wall/external diameter) ratio was significantly higher than those in control group(P<0.01). ox-LDL,CD68,MCP-1, ET-1, PCNA,FN on the vessel wall in uremic patients were much higher than those in control group (P<0.01). Moreover, ox-LDL on the vessel wall was positively related to the expression of other above mentioned substances on the vessel wall (P<0.01). Whereas the expression of eNOS on the vessel wall was lower than control group (P<0.01),and was negatively related to ox-LDL on the vessel wall(P<0.01). Conclusion: ox-LDL is an important factor contributing to uremic vascular remodeling by increasing the migration,adhesion and infiltration of monocyte,the proliferation of vascular smooth muscle cell and dysfunction of endothelia.

Objective:To determine whether ox-LDL (oxdized low-density lipoprotein) is highly deposited on the uremic vessel wall and its influence on the vascular remodeling. Methods: Segments of radial arteries were obtained from 21 uremic subjects during the operation of A-V fistula prior to hemodialysis. Segments of internal thoracic arteries of similar diameter were obtained from patients with benign chest tumors as control.The vascular lesions and ox-LDL, CD68,MCP-1, eNOS,ET-1, PCNA,FN on the vessel wall were determined by means of H-E stain and immunohistochemistry. Results： With H-E stain,atherosclerotic plaques were found in the radial arteries of 4 uremic patients. The middle layer of the arteries in uremic patients were obviously thickened, and the T/D　(thickness of the wall/external diameter) ratio was significantly higher than those in control group(P<0.01). ox-LDL,CD68,MCP-1, ET-1, PCNA,FN on the vessel wall in uremic patients were much higher than those in control group (P<0.01). Moreover, ox-LDL on the vessel wall was positively related to the expression of other above mentioned substances on the vessel wall (P<0.01). Whereas the expression of eNOS on the vessel wall was lower than control group (P<0.01),and was negatively related to ox-LDL on the vessel wall(P<0.01). Conclusion: ox-LDL is an important factor contributing to uremic vascular remodeling by increasing the migration,adhesion and infiltration of monocyte,the proliferation of vascular smooth muscle cell and dysfunction of endothelia.

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 .

Objective To investigate the influence of anti-depression therapy on cardiac function and short-term prognosis in elderly patients with congestive heart failure (CHF) and the safety and effectiveness of fluoxetine in these patients.Methods Ninety six elderly hospitalized patients with CHF accompanied with depressive disorder were randomly divided into two groups:fluoxetine (20mg once daily)and placebo treated groups based on the routine cardiac drug therapy for 3 months. Results The prevalence rate of depressive disorder in elderly patients with CHF was 31.1%.The length of stay of fluoxetine group was shorter than that of placebo group.The post-treatment depressive exponents of self-rating depression scale(SDS)in fluoxetine group (51.39%?8.63)was lower than those in pretreatment and in placebo group.The improvement of cardiac function in fluoxetine group(53.4%?4.5%) was much better than that of placebo group (P

To examine the clinical features of male patients with eating disorders in Singapore; and determine the differences in clinical features between the patients across the diagnostic categories. Methods: This is a database analysis of all male cases presenting to the Eating Disorders Clinic at Singapore General Hospital between 2003 and 2012. Results: 72 cases were identified; 36.1% were diagnosed with anorexia nervosa, 33.3% had bulimia nervosa and 30.5% had the diagnosis of eating disorder not otherwise specified. The mean presenting age was 19.9 years. 63.9% were heterosexual, while 15.3% were homosexual/bisexual. 61.1% had comorbid psychiatric diagnoses, with depression being the most common. 59.7% recorded premorbid obesity, while 66.7% reported excessive exercise. The patients in the various diagnostic categories had more similarities than differences. Conclusion: With more male cases over the years, it is important to further understand this condition, to better refine prevention, detection and treatment strategies.
Eating Disorders ; Asian Continental Ancestry Group ; Men

This study was designed to explore the influence of STI571, a tyrosine kinase inhibitor, on the expression of c-kit in the bone marrow cells from patients with acute non-lymphocytic leukemia (ANLL). The cells were exposed to various concentration of STI571 for 72 hours, the expression of c-kit mRNA and CD117 was assayed by RT-PCR and flow cytometry, respectively. The results showed that STI571 treatment induced concentration-dependent decrease of c-kit and CD117 expression, which was significant lower than that in group before treatment and untreated control groups (P < 0.05) and 0.1 micro mol/L STI571 group was significantly higher than that in 10 micro mol/L group (P < 0.05). It is concluded that STI571 has an obvious effect on the restraint of c-kit in ANLL cells.
Adolescent ; Adult ; Aged ; Antineoplastic Agents ; pharmacology ; Benzamides ; Bone Marrow Cells ; drug effects ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Imatinib Mesylate ; Leukemia, Myeloid, Acute ; blood ; genetics ; pathology ; Male ; Middle Aged ; Piperazines ; pharmacology ; Proto-Oncogene Proteins c-kit ; genetics ; Pyrimidines ; pharmacology ; RNA, Messenger ; drug effects ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction