OBJECTIVETo investigate the sensitivity to bortezomib of RPMI8226 cells after co-cultured with down-regulated Caveolin (Cav)-1 expression of HUVECs by transfection with Cav-1 shRNA (HUVECs(Cav-1 low)).

METHODSExposure to bortezomib with or without 50 nmol/L dexamethasone at different concentration, the proliferation of RPMI8226 was analyzed by MTT assay when it was cultured alone or co-cultured with HUVECs(Cav-1 low). Cav-1 expression was detected by using of Western blot and cell cycle, apoptosis and the level of reactive oxygen species (ROS) were analyzed by flow cytometry.

RESULTSCav-1 expression was notably down-regulated in HUVECs(Cav-1 low) (0.2199±0.0288 vs 1.3195±0.2393) (P<0.01). The IC(50) of bortezomib for RPMI8226 cultured alone, co-cultured with HUVECs orHUVECCav- 1 low were 20 nmol/L, 50 nmol/L and 65 nmol/L, respectively. The percentages of G₀/G₁ phase in RPMI8226 cultured alone, co-cultured with HUVECs and HUVECs(Cav-1 low) were 28.49%, 30.41%, and 36.15% respectively. The protection of RPMI 8226 against apoptosis by HUVECs was demonstrated that the apoptosis/death rates were 66.8%, 10.7% and 8.6% in RPMI8226 cultured alone, co-cultured with HUVECs and HUVECs(Cav-1 low) after exposure to 20 nmol/L bortezomib for 24 h. RPMI8226 could induce the oxidative stress of HUVECs before and after co-culture. The ROS level was raised from 15.0% to 35.2% in RPMI8226, from 80.4% to 91.0% in HUVECs, and from 84.6% to 96.8% in HUVECs(Cav-1 low).

CONCLUSIONThe down-regulated Cav-1 expression of HUVECs could promote proliferation and induce apoptosis of RMPI8226 cells, lead to G₀/G₁ phase arrest, and reduce the sensitivity to bortezomib.

Apoptosis ; Boronic Acids ; pharmacology ; Bortezomib ; Caveolin 1 ; metabolism ; Cell Line, Tumor ; drug effects ; Cells, Cultured ; Coculture Techniques ; Down-Regulation ; Human Umbilical Vein Endothelial Cells ; cytology ; metabolism ; Humans ; Multiple Myeloma ; Pyrazines ; pharmacology

China ; epidemiology ; Colonic Neoplasms ; epidemiology ; mortality ; Female ; Humans ; Incidence ; Male

Objective To construct an expression plasmid of human papillomavirus type 11 E7 (HPV11-E7)/hurnan IFN?-2b fusion gene, to express the fusion gene in E.coli BL21, and pave way for further immunological study. Methods The recombinant plasmid was introduced into E.coli BL21, then the expression product was analyzed by SDS-PAGE and Western blotting after induction with isopropy-?-D-thiogalactoside (IPTG). Results The fusion gene of HPV11-E7 and human IFN?-2b was successfully cloned into pET-32a by a linker with the same sequence as we expected. The expressed fusion protein was confirmed by SDS-PAGE and Western blotting. Conclusions The successful construction of prokaryotic expression plasmid and expression of HPV11-E7/human IFN?-2b fusion gene enable further immunological study.

OBJECTIVEFMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is constitutively activated in (70-90)% pediatric patients with acute myeloid leukemia (AML) and appears to confer an adverse prognosis. Although several FLT3-selective small molecule inhibitors and antibodies were developed with varied degrees of success, to address the specificity and resistance, new approaches for specifically targeted FLT3 are needed and RNA interference is a promising choice. The aim of the present study was to investigate the efficacy of suppression of FLT3 induced by small hairpin interfering RNA (shRNA) on myeloproliferation and apoptosis in an acute monocytic leukemia (AMOL) cell line THP-1.

METHODSFLT3-targeted small hairpin interfering RNA (FLT3-shRNA) was designed and synthesized by transcription system in vitro was transfected into THP-1 cells. Firstly FLT3 mRNA level was detected by semi-quantitative RT-PCR and FLT3 protein level was detected by flow cytometry (FCM) to verify the efficacy on FLT3-shRNA interference at 48 h after transfection. Cell growth viability was measured at 24 h, 48 h and 72 h after treatment with CCK-8. The distribution of cell cycle was assayed by FCM, and apoptosis was analyzed by DNA Ladder and Annexin V-FITC Staining at 48 h.

RESULTSFLT3 targeted shRNAs was synthesized successfully and the concentration of 15 nmol/L for 48 h could obtain desirable downregulation of FLT3 expression, the inhibitory percentages of FLT3 mRNA and protein were (72.95 +/- 2.07)% and (65.39 +/- 5.57)%, respectively. The suppression of FLT3 induced by FLT3-shRNA resulted in marked inhibition of cell growth and the inhibitory percentages were (36.66 +/- 3.67)% at 48 h, (35.56 +/- 0.73)% at 72 h. FLT3-shRNA induced the inhibition of cell cycle from G(0)/G(1) phase to S phase, the percentage of sub-G(0)/G(1) phase (65.71 +/- 4.47)% was higher than those in the PBS-control group (52.23 +/- 2.98)%, NC-shRNA control group (51.81 +/- 1.44)%, P < 0.01; the percentage of S phase (25.11 +/- 2.70)% was lower than those in the PBS-control group (34.41 +/- 4.07)% and NC-shRNA control group (32.50 +/- 1.46)%, P < 0.05. Furthermore treatment with FLT3-shRNA for 48 h resulted in clear apoptosis ladder, the percentage of early apoptosis detected by Annexin V-FITC was (18.59 +/- 2.07)% which was significantly higher than that in the PBS-control group (4.00 +/- 0.50)% and the NC-shRNA control group (6.06 +/- 0.70)%, P < 0.001.

CONCLUSIONThe suppression of FLT3 induced by the shRNA can effectively inhibit cell proliferation, and apoptosis induction on THP-1 cells, which indicates that this approach may bear the therapeutic potential on childhood AMOL.

Apoptosis ; drug effects ; genetics ; Cell Proliferation ; drug effects ; Child ; Humans ; Leukemia, Monocytic, Acute ; enzymology ; pathology ; Protein-Tyrosine Kinases ; metabolism ; RNA Interference ; physiology ; RNA, Small Interfering ; pharmacology ; Receptor Protein-Tyrosine Kinases ; metabolism ; fms-Like Tyrosine Kinase 3 ; metabolism

FMS-like tyrosine kinase 3 (FLT3) is a receptor of tyrosine kinase that is constitutively activated in most of acute myeloid leukemia patients and seems to give an adverse prognosis. In order to explore the silencing effect of FLT3 targeted short hairpin RNA (FLT3-shRNA) on acute leukaemia cell line THP-1, three FLT3-shRNAs (shRNA1, shRNA2, shRNA3) were designed and synthesized by transcription system in vitro and then transfected into THP-1 cells. FLT3 mRNA was analyzed by semi-quantitative RT-PCR, FLT3 protein was detected by Flow cytometry and immunofluorescence. The results indicated that FLT3 expression was downregulated by shRNA1 and shRNA3, and shRNA1 showed stronger inhibitory effect. At 48 hours following transfection, the inhibitory rate of 25 nmol/L shRNA1 was 72.95 +/- 2.07%, lasting 72 hours. The 5 nmol/L and more concentration of FLT3 shRNA1 could downregulate FLT3 mRNA level, which displayed a quantity-effect relation; the inhibitory rate of 15 nmol/L shRNA1 was 67.53 +/- 0.66%. FLT3 protein was located on THP-1 cell membrance, its expression was downregulated obviously by shRNA1, at 72 hours following transfection the inhibitory rate of shRNA1 was 79.67 +/- 0.66%. shRNA1 showed the best inhibitory effect on FLT3 protein, the optimal time of which was 72 hours with an inhibitory rate of 79.67%. It is concluded that FLT3-shRNA1 shows a desireable FLT3-targeted inhibitory effect, which can be used for further investigation of FLT3 mechanism or FLT3 targeting treatment.
Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; RNA Interference ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Transcription, Genetic ; Tumor Cells, Cultured ; fms-Like Tyrosine Kinase 3 ; genetics

OBJECTIVE: To study the changes of liver phosphofructokinase-2 (PFK-2) level at different postmortem intervals as well as due to different causes of death. METHODS: One hundred and five rats were randomly divided into 3 groups and the rats were sacrificed by either bleeding, suffocating, and neck breaking, respectively. The content of liver PFK-2 at 0, 1, 2, 4, 8, 12 and 24 hours following death were studied using immunohistochemishtry and image analysis. RESULTS: PFK-2 content of the rat's liver in all 3 groups showed a linear decrease at different postmortem intervals with no significant statistical differences found between the different groups. CONCLUSION PFK-2 level in rat liver appears to decrease linearly in correlation with prolonged PMI.
Animals ; Asphyxia/metabolism* ; Cause of Death ; Cervical Vertebrae/injuries* ; Female ; Immunohistochemistry ; Liver/enzymology* ; Male ; Phosphofructokinase-2/metabolism* ; Postmortem Changes ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Shock, Hemorrhagic/metabolism* ; Staining and Labeling ; Time Factors

BACKGROUNDFew studies have evaluated late clinical outcome of no-patch technique in patients with large left ventricular aneurysms. The objectives of this study were to evaluate a no-patch surgical technique to reconstruct the left ventricle in patients with left ventricular aneurysm and to assess early and late clinical outcomes.

METHODSIn 1995, we began using a no-patch technique in patients with dyskinetic left ventricular aneurysms. A total of 145 patients underwent left ventricular reconstruction with this technique and were followed up for (59 ± 29) months (range, 1 - 127 months). Risk factors for early mortality were analyzed by bivariate analyses. Cox's proportional hazards model was used to calculate risk factors for all-cause mortality and hospital readmission. Kaplan-Meier methodology was used to analyze late survival.

RESULTSOne week after operation, left ventricular end-diastolic diameter had decreased from (61 ± 8) mm to (55 ± 8) mm, and geometry of the left ventricle was restored to a more normal conical shape. Early mortality was 3% and late mortality 11%. Over a 5-year follow-up period, hospital readmission was 28%. One-, 5-, and 10-year survival estimates were 95% (95% confidence interval (CI) 91% - 99%), 86% (95%CI 78% - 94%), and 74% (95%CI 60% - 88%). Readmission-free survival at 1 and 5 years after operation was 87% (95%CI 81% - 93%) and 60% (95%CI 50% - 70%), respectively.

CONCLUSIONThe no-patch technique for left ventricular reconstruction is an effective and simple procedure that can achieve satisfactory early and late clinical outcomes in patients with left ventricular aneurysms.

Aged ; Cardiac Surgical Procedures ; methods ; Female ; Heart Aneurysm ; surgery ; Heart Ventricles ; surgery ; Humans ; Male ; Middle Aged ; Reconstructive Surgical Procedures ; Treatment Outcome

BACKGROUNDTo explore the cut-off period of subclassification and pathological features of severe hepatitis (SH).

METHODSBased on combined clinical and pathological analyses, the complete clinical and biopsy or autopsy liver tissues data from 196 cases of patients with severe hepatitis were investigated. Meanwhile, proliferative hepatocytes, cholangioepithelia and collagens were identified by a panel of monoclonal antibodies such as those against albumin, cytokeratin 18,19 and collagen I, III with immunohistochemical method.

RESULTSThe clinical and pathological analyses indicated the cut-off periods of acute, subacute and chronic SH (ASH,SSH and CSH) were (13.4+/-7.2) d, (77.4+/-69.3) d and (80.5+/-63.2) d, respectively. Among all SH cases, one case of ASH patient presented clinical manifestation and pathological changes of ASH for 21 days, however, one patient with SSH was demonstrated 12 day course by histological examination. The time of cut-off period between ASH and SSH in child cases was shorter than that in adult cases. Histologically, ASH liver tissues showed massive and/or submassive necrosis caused by one attack, with congestive sinusoid frameworks and proliferative cholangioepithelium-like hepatocytes, while SSH liver tissues presented combined fresh and old submassive or massive necrosis caused by multiple attacks, accompanied by obviously proliferative bile ducts and sinusoid framework collapse.However, the pathological changes of CSH showed ASH- or SSH-like lesions on the background of chronic liver injury.

CONCLUSIONOur data indicated that the cut-off period between ASH and SSH is in accordance with the Scheme of Viral Hepatitis Prevention and Therapy, China, published in 2000, but excluded a part of child SH cases. In our study, the authors found a few pathological features in ASH and SSH.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; Collagen ; metabolism ; Female ; Hepatitis ; classification ; metabolism ; pathology ; Humans ; Keratins, Type I ; metabolism ; Liver ; pathology ; Male ; Middle Aged ; Young Adult

Objective:To evaluate the safety and efficacy of balloon-assisted tracking (BAT) technique in guiding the catheter through the spastic radial artery via percutaneous coronary intervention.Methods:The data of patients who received coronary intervention through the transradial approach in Department of Cardiology of Zhongshan Hospital Affiliated to Fudan University from June 2014 to September 2016 were retrospectively analyzed.69 cases of radial artery and / or brachial artery spasm were selected, of which 24 cases were treated with BAT technique (group BAT), and 45 cases were treated by the conventional method (conventional group).The success rate of the catheter through the spastic segment and the incidence of related complications were compared between the two methods.Results:In the BAT group, the guide catheter was successfully negotiated across the spastic segment or dissecting vessels in all 24 cases (100%), while in only 14 cases (31.1%) in the conventional group (P=0.000).Guide catheter traversing the spastic segment within 5min, between 5min and 15min, and more than 15min was seen in 20 (83.3%), 3 (12.5%) and 1 (4.2%) patients in the BAT group, while in 2 (14.3%), 6(42.9%) and 6 (42.9%) patients in the conventional group, respectively (P=0.000).Incidence of forearm hematoma was 8.3% and 20% in the BAT group and the conventional group, and the difference was not statistically significant.Conclusions:The BAT technique is a safe and effective way to guide the catheter through the spasm of radial and/or brachial artery via percutaneous coronary intervention.BAT is superior to the conventional technique.

Objective: To describe the incidence,clinical characteristics,therapy strategy and outcomes of spontaneous coronary artery dissection based on single-center experience in China.Methods:We performed retrospective case-identification study in 16 526 patients underwent coronary angiography in Zhongshan Hospital of Fudan University between March 2015 to December 2016,and identified 17 patients with spontaneous coronary artery dissection.Risk factors,clinical features,angiographic features,therapy strategy,and clinical outcomes were analyzed.Results:The incidence of SCAD was 17 of 16 526(1.03/1 000).The mean age was(49.06 ± 10.73)years old(range:26-67 years old).In these 17 cases,4 cases were males,and others were females.Females constituted 13 of 17(76.5%).All SCAD patients presented with acute coronary syndrome,including 10 patients with acute ST-elevated myocardial infarction,3 patients with acute non-ST-elevated myocardial infarction and 4 patients with unstable angina.Twenty dissection sites were identified in 17 SCAD patients. Dissection was predominantly located at the left descending artery(50%)and the right coronary artery(35%).All lesions fell into three types:type Ⅰ(n=5),type Ⅱ A(n= 7),type ⅡB(n= 6),and type Ⅲ(n= 2).The TIMI flow in the distal segment of the coronary dissection was classified as follows:class 0(n=4),class 1(n=2),class 3(n=14).Conservative medical treatment was adopted by 7 of 17(41.1%)patients,and percutaneous transluminal coronary angioplasty(PTCA)in 1 of 17(5.9%)patients.No recurrent angina and other cardiovascular events was observed during clinical follow up. Percutaneous coronary intervention(PCI)was performed in 9 of 17(52.9%)patients,and the mean number of deployed stent was(2.44 ± 1.13).Intramural hematoma was extended during PCI in 5 of 9(55.6%)patients,resulting in new-onset nonfatal myocardial infarction in one patient and cardiac death in another patient.Conclusions:SCAD should be considered in young and middle-aged female patients presented with acute coronary syndrome,especially in those with few coronary risk factors. Interventional cardiologist should be familiar with the angiographic characteristics of SCAD,and turn to intravascular ultrasound if necessary.Conservative treatment should be the first choice in most patients with SCAD,while PCI intervention could be considered in high risk patients.Be caution to prevent interventional complications such as dissection expansion in the patients with high-risk.