OBJECTIVE: To explore the effect of melatonin(Mel) on the proliferation, apoptosis and expression of bcl-2 in oxidized low-density lipoprotein(ox-LDL)-induced endothelial progenitor cells (EPC) from human umbilical cord blood in vitro. METHODS: Total mononuclear cells were isolated from human umbilical cord blood in vitro by Ficoll density gradient centrifugation, and the cells were plated on fibronectin-coated culture dishes. After 7 days, the attached cells were divided into 7 groups: a control group (normal cells), 3 ox-LDL groups[the attached cells were incubated with different concentrations of ox-LDL(5,10,and 20mg/L) for 24 hours], and 3 Mel groups[the attached cells were incubated with different concentrations of Mel (0.5,1.0, and 2.0 mmol/L) respectively for 24 hours before incubation with 10 mg/L ox-LDL]. EPC was identified by examining the expression of CD34, vascular endothelial growth factor receptor-2(VEGFR-2) and CD133 under a laser scanning confocal microscope. We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to detect the effect of Mel and ox-LDL on the multiplication ability of EPC. Flow cytometry was used to detect the apoptosis. The expressions of Bcl-2 mRNA and protein were detected respectively by RT-PCR and immunohistochemistry technology. RESULTS: After being exposed to the ox-LDL, the proliferation of EPC in the 3 ox-LDL groups was lower, and the apoptosis rate was higher than that in the control group in a dose-dependent manner (P<0.01); Mel was added at different concentrations before the ox-LDL incubation, and the cells in the 3 Mel groups showed higher proliferation and lower apoptosis rate than those of the 3 ox-LDL groups (P<0.01). Expression of Bcl-2 mRNA and protein of EPC in the 3 Mel groups was higher than that in the 3 ox-LDL groups (P<0.01). CONCLUSION Ox-LDL can inhibit the proliferation of EPC and promote the apoptosis of the cells by down-regulating the bcl-2 expression. Mel can inhibit these effects of ox-LDL.
Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; Humans ; Lipoproteins, LDL ; adverse effects ; Melatonin ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Stem Cells ; cytology ; drug effects

OBJECTIVE: To investigate the culture of endothelial progenitor cells (EPCs) from peripheral blood in patients with coronary heart diseases (CHD) before and after percutaneous coronary intervention (PCI), and to observe the cells shape and determine the cell number and proliferation activity. METHODS: Ninety-five patients were divided into a CHD group(n=65) and a control group (n=30). The mononuclear cells were isolated from peripheral blood of patients with CHD before, right after and 4 days after PCI by Ficoll-density centrifugation. The isolated cells were cultured in RPMI1640 medium supplemented with VEGF165 and bFGF.EPCs were characterized as adherent cells of double positive for DiL-acLDL uptake and FITC-UEA-I binding by direct fluorescent staining under a fluorescence microscope. The EPCs specific surface mark CD34 and KDR were assessed by fluorescence activated cell sorter analysis. The cell shapes were analysed and the number of colony-forming units(CFU) was counted by phase-contrast microscope. RESULTS: The number of EPCs reduced in patients with CHD before the PCI, but the cell number was significantly increased in patients with CHD after the PCI, and the number reduced in patients with CHD 4 days after the PCI. How-ever, the number of CFUs did not change in patients before and after the PCI. CONCLUSION PCI can increase endothelial progenitor cells in patients after the PCI; but 4 days after the PCI, this increase will not exist.
Aged ; Aged, 80 and over ; Angioplasty, Balloon, Coronary ; Cell Adhesion ; Cell Count ; Cell Movement ; Cells, Cultured ; Coronary Disease ; blood ; therapy ; Endothelial Cells ; pathology ; Female ; Humans ; Male ; Middle Aged ; Stem Cells ; pathology

OBJECTIVETo explore the effect of Yiqi Huoxue recipe (YHR), a Chinese herbal medicine for supplementing Qi, activating blood circulation to remove stasis, on vascular extracellular matrix (ECM) remodeling and its molecular mechanism.

METHODSVascular smooth muscle cell (VSMC) proliferation activity and collagen turnover rate were detected by 3H-TdR test and hydroxyproline amount determined by 3H-Pro incorporation. Expression activity of MMP-2 and osteopontin genes was detected by Northern blotting and MMP-2 zymography analysis.

RESULTSYHR could markedly inhibit VSMC collagen synthesis stimulated by blasic fibroblast growth factor (bFGF) and lower the collagen turnover rate induced by vascular de-endothelialization. The expression level of MMP-2 and osteopontin genes was down-regulated by YHR in cultured VSMC and vascular wall with endothelial injury, and VSMC proliferation was inhibited by the serum obtained from YHR treated rats. Removing protein from the drug serum made no change on the effect of YHR to VSMC.

CONCLUSIONYHR could inhibit and/or retard ECM remodeling through regulating the expression of MMP-2 and osteopontin genes and lowering the collagen turnover rate.

Animals ; Aorta ; cytology ; Cell Division ; drug effects ; Cells, Cultured ; Collagen ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Extracellular Matrix ; genetics ; metabolism ; Fibroblast Growth Factor 2 ; metabolism ; Gene Expression ; Male ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Osteopontin ; Rats ; Rats, Sprague-Dawley ; Sialoglycoproteins ; genetics ; metabolism

Objective: To observe the effect of STC-1 gene expression was inhibited on the apoptosis, IL-1β and TNF-α expression and JAK2/STAT3 signal in esophageal cancer cells. Methods: Compared with normal human esophageal squamous epithelial cells Het-1 A, STC-1 expression was detected in human esophageal squamous cell carcinoma KYSE170, Eca109, TE1 and TE10 cells by RTPCR and Western blot; the siRNA sequence that the synthesized STC-1 and the si NRA sequence without interference were transfected into Eca109 cells, which were labeled as STC-1-siRNA group and NC group, and the blank control group was set, cells were transfected for 48 h, the expression of STC-1 were detected by RT-PCR and Western blot. Eca109 cell viability and apoptosis rate were detected by CCK8 and flow cytometry. IL-1β and TNF-α expression were detected by RT-PCR; the expression of Ki67, p53, p-JAK2 and p-STAT3 protein were detected by Western blot. Results: Compared with Het-1 A cells, expression of STC-1 mRNA and protein in KYSE170, Eca109, TE1 and TE10 cells were increased significantly ( P＜0. 05); compared with the control group, STC-1 expression was decreased significantly in STC-1-siRNA group, cell viability was decreased significantly in STC-1-siRNA group, the apoptosis rate was increased significantly in STC-1-siRNA group; IL-1β, TNF-α, Ki67, p-JAK2 and p-STAT3 expression were decreased significantly in STC-1-siRNA group, p53 expression was increased significantly in STC-1-siRNA group ( P＜0. 05). Conclusion: STC-1 was highly expressed in esophageal cancer cells; inhibiting of STC-1 expression could significantly reduce the activity of cancer cells, and increase apoptosis rate, this effect may be related to the inhibition of JAK2/STAT3 signaling pathways and inflammatory factors as IL-1β and TNF-α.

Polysaccharide sulfate (PSS) is a new type of antiatherosclerotic medicine for its effects of anticoagulation, anti-thrombosis and modulation of dyslipidemia. However, it is still uncertain whether PSS could modulate the diabetic dyslipidemia or not. Here, the rat model of diabetic dyslipidemia was developed and the effects of PSS on glucose and lipid levels were investigated in this animal model. Wistar rats were iv injected with streptozotocin 20 mg x kg(-1) after feeding with high fat diet for one and a half month. Then, rats received orally PSS (30, 90, and 180 mg x kg(-1)) for 1 month. After oral treatment with PSS (90 and 180 mg x kg(-1)) for 1 month, the levels of triglyceride (TG), total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C) were significantly reduced and the level of high density lipoprotein-cholesterol (HDL-C) increased, compared with diabetic control rats. Moreover, PSS (30, 90, and 180 mg x kg(-1)) had a tendency to reduce glucose and insulin levels, and significantly increased insulin sensitivity index. Our results suggest that PSS could improve insulin sensitivity and relieve dyslipidemia in diabetic dyslipidemic rats.
Administration, Oral ; Animals ; Blood Glucose ; metabolism ; Cholesterol, HDL ; blood ; Cholesterol, LDL ; blood ; Diabetes Mellitus, Experimental ; blood ; chemically induced ; complications ; Dyslipidemias ; blood ; etiology ; Hypolipidemic Agents ; administration & dosage ; pharmacology ; Insulin ; blood ; Insulin Resistance ; Male ; Polysaccharides ; administration & dosage ; pharmacology ; Random Allocation ; Rats ; Rats, Wistar ; Streptozocin ; Sulfates ; administration & dosage ; pharmacology ; Triglycerides ; blood

AIM:To observe the effects of vitamin D on the apoptosis ,prolyl isomerase 1(Pin1)protein ex-pression and activity ,mitochondrial translocation of p 66Shc,and reactive oxygen species(ROS)production in high glu-cose-cultured human umbilical vein endothelial cells(HUVECs),and to explore the role of vitamin D receptor(VDR)in these processes.METHODS:HUVECs were treated with high glucose(33 mmol/L)in the presence or absence of vita-min D or Pin1 inhibitor juglone.The cell apoptosis was measured by flow cytometry and TUNEL staining.Intracellular ROS levels were examined by flow cytometry and fluorescence microscopy.The protein levels of Pin1,p66Shc,p-p66Shc,mito-chondria to cytoplasm ratio of p66Shc,and caspase-3 in HUVECs were measured by Western blot.Pin1 activity in HU-VECs lysate was assessed by a commercial kit.Knockdown of VDR by siRNA was conducted to evaluate the role of VDR in the regulatory effects of vitamin D on Pin 1 protein expression and activity in HUVECs under high-glucose condition.RE-SULTS:Vitamin D suppressed the apoptosis and intracellular ROS generation of HUVECs induced by high glucose(P<0.05).Vitamin D inhibited high glucose-induced upregulation of Pin1 protein expression and activity(P<0.05).Vita-min D inhibited the phosphorylation and mitochondrial translocation of p 66Shc and caspase-3 protein expression induced by high glucose(P<0.05).Knockdown of VDR by siRNA abolished the inhibitory effects of vitamin D on high glucose-in-duced upregulation of Pin 1 protein expression and activity.CONCLUSION:Vitamin D alleviates high glucose-induced endothelial cell apoptosis by inhibition of Pin 1 protein expression and activity ,and attenuation of p66Shc-mediated mito-chondrial oxidative stress ,which are dependent on VDR activation.

This study was aimed to investigate the feasibility of low dose of fludarabine, cyclophosphamide combined with donor derived alloreactive NK cells as a new nonmyeloablative conditioning regimen in the haploidentical hematopoietic stem cell transplantation (haploidentical HSCT). F1 derived-NK cells were enriched with MACS magnetic separation system, in which the proportions of the Ly49C+ and Ly49A+ cells were detected by flow cytometry and the alloreactivity was measured by LDH method. The haploidentical HSCT models were constructed, and the myeloablativity in vivo, donor engraftment and the intensity of GVHD were compared between different myeloablative and nonmyeloablative conditioning regimens, including 9 Gy TBI, 6.5 Gy TBI, flu + cy, and flu + cy + allo-NK. The results showed that the flu + cy + allo-NK conditioning was nonmyeloablative, but the rate of donor chimerism after haploidentical HSCT was significantly higher as compared with other nonmyeloablative methods, which were (28.70 +/- 5.90)% in bone marrow and (46.40 +/- 5.00)% in spleen at day 21 post-transplantation. When compared with the flu + cy conditioning, the intensity of GVHD was slight in the flu + cy + allo-NK group, in which only a half of C57BL/6 recipients experienced weight loss, and no distinct pathological damages observed in the liver, intestine, kidney and skin samples. It is concluded donor derived-alloreactive NK cells can facilitate engraftment of the haploidentical hematopoietic stem cells and mitigate GVHD. The flu + cy + allo-NK conditioning provides a new method for those elder patients with high-risk solid tumor undergoing haploidentical-HSCT.
Animals ; Cyclophosphamide ; administration & dosage ; Female ; Graft vs Host Disease ; prevention & control ; Haplotypes ; Hematopoietic Stem Cell Transplantation ; methods ; Killer Cells, Natural ; transplantation ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Models, Animal ; Transplantation Chimera ; Transplantation Conditioning ; methods ; Vidarabine ; administration & dosage ; analogs & derivatives ; Whole-Body Irradiation

OBJECTIVETo study the effect of RNA interference (RNAi) targeting E6AP on the proliferation and apoptosis of HeLa cells.

METHODSHeLa cells were cultured and divided into 3 groups: blank control group, cells transfected with nonsense siRNA (small interference RNA), and cells transfected with specific E6AP siRNA. The expressions of E6AP mRNA and protein were detected by RT-PCR and Western blot before and after the transfection respectively. Cell proliferation was determined by methylthiazolyl tetrazolium (MTT). The cell apoptosis index was assessed by flow cytometry.

RESULTSUpon treatment with E6AP siRNA for 24, 48 and 72 h, the expression level of E6AP mRNA decreased 33%, 72% and 70% than siRNA treated group. The protein expression levels in 48 h and 72 h E6AP siRNA groups decreased 38%, 59% comparing with those of the nonsense siRNA treated group (P < 0.05). The proliferative capacity of cells transfectd with E6AP siRNA was significantly lower than blank control group (F = 101.38, P < 0.05) and siRNA treated group (F = 38.64, P < 0.05). The apoptosis index of HeLa cells treated with E6AP siRNA was significantly higher than that of the nonsense siRNA (F = 41.48, P < 0.05) and the blank control group (F = 86.36, P < 0.05).

CONCLUSIONSiRNA targeting can effectively suppress the expression levels of E6AP mRNA, corresponding with a proliferation inhibition and an enhanced apoptosis of HeLa cells.

Cell Line, Tumor ; Cell Proliferation ; Female ; Gene Expression Regulation, Neoplastic ; genetics ; Gene Silencing ; HeLa Cells ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Ubiquitin-Protein Ligases ; antagonists & inhibitors ; metabolism ; Uterine Cervical Neoplasms ; genetics ; metabolism

Adolescent ; Adult ; Aged ; Chronic Disease ; Female ; Hepatitis B e Antigens ; analysis ; Hepatitis B virus ; genetics ; Humans ; Liver Diseases ; virology ; Male ; Middle Aged ; Mutation ; Oligonucleotide Array Sequence Analysis ; Promoter Regions, Genetic

OBJECTIVETo evaluate the value of the detection of a 4-marker (ER, VIM, CEA and p16) panel in the differential diagnosis of primary endocervical and endometrial adenocarcinomas.

METHODSImmunohistochemical EnVison method was used to detect the expressions of ER, VIM, CEA and p16 in paraffin-embedded tissues from 31 cases of primary endocervical adenocarcinomas and 30 cases of endometrial adenocarcinomas. The specificity, sensitivity, predictive value and accuracy were compared between the 4-marker and 3-marker (ER, VIM and CEA) panels.

RESULTSThe positivity rates of ER, VIM, CEA and p16 in endocervical adenocarcinomas were 35.5%, 19.4%, 77.4% and 67.7%, respectively; those in endometrial adenocarcinomas were 70%, 73.3%, 40% and 13.3%, respectively, showing significant frequency differences (P<0.05) between primary endocervical and endometrial adenocarcinomas. The specificity, sensitivity, positive predictive value and accuracy of the 4-marker panel in endocervical adenocarcinomas were significantly higher than those of the 3-marker panel (96.3% vs 90.2%, 65.1% vs 57.6%, 94.9% vs 89.4%, and 85.8% vs 80.6%, respectively). These values were almost similar for both panels in endometrial carcinoma except for better negative predictive value and accuracy value with the 4-marker panel (58.7% vs 51.9% and 75.4% vs 68.6%, respectively).

CONCLUSIONAdding the p16 marker to the traditional 3-marker panel may have significant clinical importance in the differential diagnosis of primary endocervical and endometrial adenocarcinomas to improve the diagnostic accuracy, although there is only a slight increase in the diagnostic sensitivity.

Adenocarcinoma ; diagnosis ; metabolism ; Adult ; Aged ; Biomarkers, Tumor ; analysis ; Carcinoembryonic Antigen ; analysis ; Cyclin-Dependent Kinase Inhibitor p16 ; Diagnosis, Differential ; Endometrial Neoplasms ; diagnosis ; metabolism ; Female ; Humans ; Middle Aged ; Neoplasm Proteins ; analysis ; Predictive Value of Tests ; Receptors, Estrogen ; analysis ; Sensitivity and Specificity ; Uterine Cervical Neoplasms ; diagnosis ; metabolism ; Vimentin ; analysis