This study was aimed to establish the real-time fluorescent quantitative PCR (RT-qPCR) with erythrocyte Kidd blood group gene for detecting the hematopoietic chimera and to investigate the feasibility of this method. The TaqMan MGB probes and special primers were designed on basis of difference of erythrocyte Kidd blood group alleles, the hematopoietic chimerism was detected by RT-qPCR, the DNA chimerism was simulated by means of dilution of multiple proportions, and the sensitivity analysis was performed. The results showed that the RT-qPCR with erythrocyte Kidd blood group gene could effectively distinguish JK*A and JK*B alleles. There was no significant difference between the theoretic value and the practical measured value by this method (P > 0.05). As 156 donor's cells could be discriminated from 10(4) chimeric cells, this method may effectively detect donor's cells with correlation coefficient 0.998. It is concluded that the established RT-qPCR with erythrocyte Kidd blood group gene shows the feasibility for quantitative detection of hematopoietic chimera, and may be used to quantitatively detect chimera in a certain range.
Chimera ; Erythrocytes ; Humans ; Kidd Blood-Group System ; genetics ; Real-Time Polymerase Chain Reaction

OBJECTIVETo explore the associations between fasting serum lipids and high-sensitivity C-reactive protein (hsCRP).

METHODSSerum triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and hsCRP were measured in residents of Chengdu, China. Subjects with potential factors which might influence lipids and hsCRP were excluded, 580 subjects [mean age (62.3 ± 6.6) years; male: 58.7%] were finally recruited by random sampling methods.

RESULTSThere was a weak positive relationship between TG and hsCRP (r = 0.108, P = 0.01) and a weak negative relationship between HDL-C and hsCRP (r = -0.197, P < 0.001), this was also true in the sub-group with BMI < 24 kg/m(2) (r = 0.236, -0.140 respectively, all P < 0.001). In subjects with BMI < 24 kg/m(2), the hsCRP concentration was significantly higher in subjects with higher TG or lower HDL-C (all P < 0.05). hsCRP increased in proportion with the degree of dyslipidemia. After adjusting for gender, age, TC, LDL-C, fasting blood glucose, systolic blood pressure, diastolic blood pressure, history of hypertension and diabetes, smoking and alcohol drinking, logistic regression analysis showed that the odds ratio for increased hsCRP was 1.970 in subjects with either increased TG or lower HDL-C (P = 0.105) and 9.098 in subjects with both higher TG or lower HDL-C levels (P = 0.031). However, the observed relationship between TG, HDL-C and hsCRP in subjects with BMI < 24 kg/m(2) could not be observed in subjects with subjects with BMI > 24 kg/m(2) despite significant more cardiovascular risk factors in these subjects.

CONCLUSIONSA weak positive correlation between TG and hsCRP as well as a weak negative correlation between HDL-C and hsCRP was evidenced in the whole cohort suggesting dyslipidemia might be related to enhanced inflammatory status. However, this relationship is not observed in subjects with BMI > 24 kg/m(2) despite existence of more cardiovascular risk factors in these subjects.

Aged ; Alcohol Drinking ; C-Reactive Protein ; analysis ; Cardiovascular Diseases ; blood ; epidemiology ; China ; epidemiology ; Cholesterol, HDL ; blood ; Cholesterol, LDL ; blood ; Dyslipidemias ; epidemiology ; Female ; Humans ; Inflammation ; Male ; Middle Aged ; Smoking ; Triglycerides ; blood

Bone morphogenetic protein 2(BMP-2) is a member of the of BMPs family, its osteoinductive capacity has already been demonstrated. We tried to express hBMP-2 in CHO cell. In this study, we inserted hBMP-2 cDNA into vector pCDNA3.1(+) to construct hBMP-2 eukaryotic expression vector pCDNA3.1(+)-hBMP-2. Recombinant Chinese hamster ovary (rCHO) cell line expressing high-level recombinant human bone morphogenetic protein 2(rhBMP-2) was constructed by co-transfecting the expression vectors pCDNA3.1(+)-hBMP-2 and plasmid pSV2-dhfr into dihydrofolate reductase (dhfr)-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.1 and 1 micromol/L. Western blot analyses showed a specific band of about 18 kD in reduced sample lane and a specific band of about 32 kD in non-reduced sample lane, this indicated that rCHO cells secret rhBMP-2 as a homodimeric glycoprotein form. Finally, we obtained a single clone cell strain expressing a high level (7.83 microg/24 h/10(6) cells) of rhBMP-2 tested by ELISA. Biological activity of rhBMP-2 was tested by the induction of alkaline phosphatase(ALP) activity in C2C12 cells. We treated C2C12 with different concentration of rhBMP-2 condition medium(CM) for 5d. The results showed that the rhBMP-2 could significantly increase the ALP activity of C2C12.
Alkaline Phosphatase ; biosynthesis ; Animals ; Blotting, Western ; Bone Morphogenetic Protein 2 ; biosynthesis ; chemistry ; isolation & purification ; pharmacology ; CHO Cells ; Cell Line ; Cricetinae ; Cricetulus ; Enzyme Induction ; drug effects ; Gene Expression ; Genetic Vectors ; genetics ; Humans ; Mice ; Recombinant Proteins ; biosynthesis ; chemistry ; isolation & purification ; pharmacology ; Solubility

BMP6 is a member of TGF-beta superfamily, represent more effective osteogenic activity. Two recombinant plasmids were constructed to expression rhBMP6 in mammalian cells, one contained the cDNA encoding the signal peptide, propeptide and mature peptide of human BMP6, wich was named pcDNA-BMP6, the other contained the recombinant DNA encoding the signal peptide, propeptide of human BMP2 and the mature peptide of BMP6, which was named pcDNA-BMP2/6. Transient expression in Cos7 cells demonstrated that the pcDNA-BMP2/6 produced more rhBMP6 than pcDNA-BMP6. For stable expression, the CHO-dhfr- cells were transfected with pcDNA-BMP2/6 and pSV2-dhfr, then screened by G418 and treated with MTX for targeting gene amplification. The partially purified rhBMP6 by heparin affinity chromatography was shown to possess bone induction activity tested by the induction of alkaline phosphatase activity in C2C12 cells.
Alkaline Phosphatase ; metabolism ; Animals ; Blotting, Western ; Bone Morphogenetic Protein 2 ; genetics ; Bone Morphogenetic Protein 6 ; genetics ; metabolism ; pharmacology ; CHO Cells ; COS Cells ; Cell Line ; Cercopithecus aethiops ; Cricetinae ; Cricetulus ; Gene Expression ; Humans ; Myoblasts ; cytology ; drug effects ; enzymology ; Protein Precursors ; genetics ; Protein Sorting Signals ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVEThe aim of this study was to explore the possibility of creating a toxin, C-CPE-ETA', by fusing C-terminal high affinity binding domain of CPE (C-CPE) with a truncated form of Pseudomonas aeruginosa exotoxin A (ETA') and to examine whether C-CPE-ETA' could specifically target CLDN-3, 4 molecule and the targeted toxin was cytotoxic against CLDN-3,4-overexpressing ovarian cancer.

METHODSCLDN-3 and CLDN-4 expressions were analyzed at the mRNA level in three ovarian cancer cell lines and epithelial ovarian cancer tissues from 20 patients. After transforming an expression plasmid of C-CPE-ETA' into E. coli BL21 (DE3) plysS strain, the recombinant protein was purified using His-Bind resin chromatography column and analyzed by Western blot and Coomassie blue staining. The specific binding, proapoptotic and cytolytic activities were evaluated by flow cytometry, fluorescence microscopy with the JC-1 probe and MTT assay in CLDN-3,4-overexpressing ovarian cancer cells.

RESULTSQuantitive RT-PCR results showed there existed high levels of CLDN-3 and CLDN-4 in ovarian cancer cells, CAOV3, OVCAR3 and SKOV3. Moreover, high expressions of CLDN-3 and CLDN-4 were observed in 90.0% (18/20) and 60.0% (12/20) of ovarian cancer tissues, with an expression level 10-fold higher than that in the normal ovarian tissue. A 58 000 recombinant protein C-CPE-ETA' was demonstrated by Western blot and Coomassie blue staining. Purified and recombinant C-CPE-ETA' was bound with high affinity to CLDN-3,4-overexpressing ovarian cancer cells, CAOV3, OVCAR3 and SKOV3 cells. C-CPE-ETA' was strongly proapoptotic and cytotoxic towards the CLDN-3,4-overexpressing ovarian cancer cells. The concentration of IC(50) was 7.364 ng/ml for CAOV3 cells, 8.110 ng/ml for OVCAR3 cells and 22.340 ng/ml for SKOV3 cells, respectively. However, control CLDN-3,4-deficient cell line HUVEC was not susceptible to the recombinant C-CPE-ETA' at a concentration up to 10 µg/ml.

CONCLUSIONSThe C-CPE-ETA' protein exhibits remarkably specific cytotoxicity for CLDN-3,4-overexpressing ovarian cancer cells. Its therapeutic potential warrants further development for ovarian cancer molecular targeted therapy.

ADP Ribose Transferases ; metabolism ; physiology ; Apoptosis ; Bacterial Toxins ; metabolism ; Cell Line, Tumor ; Claudin-3 ; Claudin-4 ; Claudins ; genetics ; metabolism ; Enterotoxins ; metabolism ; physiology ; Exotoxins ; metabolism ; physiology ; Female ; Humans ; Immunotoxins ; metabolism ; Ovarian Neoplasms ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Recombinant Fusion Proteins ; metabolism ; physiology ; Virulence Factors ; metabolism ; physiology

BACKGROUNDIn China the ginseng root began to be used in medicine over 2000 years ago. Ginsenosides are the most important component isolated from ginseng. The authors investigated the effect of ginsenoside Rg1 on the spectrum of gene expression in the endothelial cells stimulated by TNF-alpha and further explored the potential molecular mechanism of endothelial protection by ginsenoside Rg1.

METHODSNitric oxide (NO) production in the cultured human umbilical vein endothelial cells (HUVECs) was measured by using an NO assay kit. A home-made oligonucleotide microarray containing approximately 400 cardiovascular disease-related genes was constructed. The alteration of the spectrum of gene expression induced by ginsenoside Rg1 in HUVECs which were activated by TNF-alpha were detected by oligonucleotide microarray analysis.

RESULTSNO production in HUVECs was decreased significantly after TNF-alpha treatment, while pretreatment with ginsenoside Rg1 enhanced NO production in TNF-alphastimulated HUVECs. Ginsenoside Rg1 affected the expression levels of genes involved in vascular constriction, cell adherence, coagulation, cell growth and signal transduction in TNF-alphastimulated HUVECs.

CONCLUSIONSGinsenoside Rg1 could enhance NO production and the expression of eNOS mRNA in TNF-alpha stimulated HUVECs. Ginsenoside Rg1 regulated sets of genes in endothelial cells and protected endothelial cells from TNF-alpha activation. Microarray analysis provided us with valuable insights into the atheroprotective mechanism by gingsenoside Rg1.

Endothelial Cells ; drug effects ; physiology ; Gene Expression ; drug effects ; Ginsenosides ; pharmacology ; Humans ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase ; analysis ; Nitric Oxide Synthase Type III ; Oligonucleotide Array Sequence Analysis ; Tumor Necrosis Factor-alpha ; pharmacology

BACKGROUNDBone morphogenetic protein-6 (BMP-6) is closely correlated with tumor differentiation and skeletal metastasis. Estrogen is considered as a stimulant for the initiation and promotion of breast cancer. Previous studies demonstrated that 17beta-estadiol (E2) can selectively increase the expression of BMP-6. This experiment is designed to detect the molecular mechanism of estrogen activating BMP-6 gene transcription in human estrogen receptor positive (ER+) breast cancer cell line MCF-7.

METHODSAfter the treatment of MCF-7 cells with E2 at different concentrations (10(-11) mol/L, 10(-9) mol/L, 10(-7) mol/L), the BMP-6 expression level was examined through real-time polymerase chain reaction. Through restriction enzyme digestion, human BMP-6 1.2 kb long promoter, BMP-6 0.7 kb long promoter was cloned into pGL-3 basic vector; after the treatment with 10(-7) mol/L E2, luciferase activities of the two promoters were detected. Site-directed mutagenesis was performed to obtain the mutant forms of estrogen response element half-site (1/2 ERE) element and Sp1 sites in the BMP-6 promoter, the activities of these mutant form promoters were detected following the methods mentioned above. Chromatin immunoprecipitation (ChIP) assay was also used to confirm the binding of estrogen receptor alpha (ERalpha) on BMP-6 promoter in the presence of E2.

RESULTSE2 dose dependently increased BMP-6 mRNA expression in human ER+ breast cancer cell line MCF-7. At a dose of 10(-7) mol/L E2, human BMP-6 1.2 kb promoter activity was increased by 90% compared with the control group treated with ethanol (P < 0.05). Both the 1/2 ERE response element mutant form and the Sp1 site mutant form of the BMP-6 promoter abolished the activation of the BMP-6 promoter's response to E2. Through ChIP assay, the binding of ERalpha on 1/2 ERE response element in BMP-6 promoter was further validated.

CONCLUSIONEstrogen induces BMP-6 expression in human ER+ breast cancer cell line MCF-7 through its receptor ERalpha binding on 1/2 ERE element in the BMP-6 promoter.

Bone Morphogenetic Protein 6 ; Bone Morphogenetic Proteins ; genetics ; Breast Neoplasms ; genetics ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Estradiol ; pharmacology ; Estrogen Receptor alpha ; physiology ; Female ; Humans ; Parathyroid Hormone-Related Protein ; secretion ; Promoter Regions, Genetic ; Transcriptional Activation ; drug effects

OBJECTIVETo observe the prophylactic effect of curcumin on hepatic fibrosis and the number, location, apoptosis of activated hepatic stellate cells (HSCs) in the livers and to discuss the relationship between the prophylactic effects and activated HSC.

METHODSA rat model of hepatic fibrosis was established by intraperitoneal injection of carbon tetrachloride. Curcumin doses of 5 mg, 10 mg, 20 mg per 100 gram per 100g of body weight were given to three groups of the model rats. No curcumin was given to one group of the model rats and it served as the control. After eight weeks, all rats were sacrificed and their left liver lobes were examined histopathologically with H.E and Masson stainings. Grades of hepatic fibrosis were evaluated according to the SSS system. Activated HSC was detected by the alpha-SMA immunohistochemistry staining. HSC apoptosis was detected by double-stainings of terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) and desmin immunohistochemistry staining.

RESULTSDegrees (SSS system scores) of hepatic fibrosis in the curcumin groups were all less severe in comparison with those of the control group. Activated HSCs in the livers of the rats of the control group increased significantly compared with that of the treatment groups, and also fewer apoptotic HSCs were detected in the control group. On the contrary, fewer activated HSCs and more apoptotic HSCs were detected in the curcumin groups compared with those of the control group. The degrees of the effects were curcumin dose-dependent.

CONCLUSIONSCurcumin can prevent hepatic fibrosis. It can inhibit activation and proliferation of HSCs and induce HSCs apoptosis, which may be the mechanism(s) contributing to the prophylactic effects of curcumin on hepatic fibrosis.

Animals ; Apoptosis ; drug effects ; Carbon Tetrachloride ; Carbon Tetrachloride Poisoning ; Curcumin ; therapeutic use ; Enzyme Inhibitors ; therapeutic use ; Hepatocytes ; pathology ; Liver Cirrhosis, Experimental ; pathology ; prevention & control ; Rats ; Rats, Sprague-Dawley

BACKGROUNDInflammation within vulnerable coronary plaques may cause unstable angina by promoting rupture and erosion. C-reactive protein (CRP) is the most reliable and accessible test method for clinical use for identifying coronary artery disease event. Matrix metalloproteinase 9 (MMP-9) is highly over-expressed in the vulnerable regions of a plaque. Our aim was to evaluate the plasma levels of MMP-9 and hsCRP in subjects with both unstable angina and coronary plaques, as well as in those with unstable angina without coronary plaques.

METHODSPatients with newly diagnosed unstable angina pectoris from clinical presentation and ECG, who were undergoing coronary angiography from April 2007 to April 2009, were included in this study. A total of 170 subjects were enrolled in the study. Before angiography, the baseline clinical data (mainly including conventional risk factors) was collected. These patients were divided into two groups, a non-plaque group (G1) which included 55 patients with no significant stenosis or less than 20% stenosis in at least one of the major coronary artery branches, and a plaque group (G2) which included 115 patients with at least one of the major coronary artery branches unstable angina pectoris with at least 50% stenosis of one major coronary artery. The patients presenting with calcified nodules of a major coronary artery were excluded from this study. We examined the serum levels of MMP-9 for all cases by multi-effect enzyme-linked immunosorbent assay.

RESULTSThere was a significant difference in the serum levels of MMP-9 between the two groups (P < 0.001). The percentage of patients with hypertension, diabetes and current smokers were significantly different between the two groups (P = 0.034, P = 0.031, and P = 0.044 respectively). The univariate Logistic regression analyses of risk factors showed that smoking was the main risk factor for angina in the non-plaque group with the OR being 1.95 (95%CI 1.02 - 3.75). Hypertension, diabetes mellitus were negatively related with the occurrence of angina in the non-plaque group with the ORs being 0.50, and 0.36, respectively (95%CI 0.26 - 0.96 and 0.14 - 0.94). The MMP-9 level was negatively related to the occurrence of angina in the non-plaque group with an OR of 0.59 (95%CI 0.47 - 0.81).

CONCLUSIONSThere is a significantly difference in MMP-9 levels between the plaque and non-plaque groups. Current smoking has a significant influence on unstable angina patients without documented plaques. The serum MMP-9 level may be a significant biomarker which can help differentiate patients with unstable angina with plaques from those with unstable angina but without plaques.

Aged ; Angina, Unstable ; blood ; metabolism ; physiopathology ; C-Reactive Protein ; metabolism ; Coronary Angiography ; Coronary Artery Disease ; blood ; metabolism ; physiopathology ; Coronary Vessels ; metabolism ; pathology ; Female ; Humans ; Male ; Matrix Metalloproteinase 9 ; blood ; Middle Aged ; Multivariate Analysis ; Risk Factors ; Smoking ; adverse effects

OBJECTIVETo investigate the operative reduction techniques and clinical results of surgical treatment of type C1 (AO/ASIF) acetabular fracture by posteroproximal-posteroanterior sequential reduction and internal fixation.

METHODSFrom August 2004 to January 2012, 13 patients with type C1 (AO/ASIF) acetabular fracture were treated by posteroproximal-posteroanterior sequential reduction and internal fixation. Of them, 8 cases were male and 5 cases were female with an average age of 42 years years old (ranged, 18 to 64). Pelvis 3-dimentional CT reconstruction were used to confirmed the classification of fracture, and the operation were performed during from 5 to 20 days with an average of 9.5 days. Operation time, blood loss, complications and reduction were recorded and evaluated. The function of hip joint were accessed at the final follow-up.

RESULTSThe operation time ranged from 190 to 290 min with an average of 240 min. The mean blood loss was 1 800 ml (ranged, 1 300 to 3 000 ml). One case had superficial infection and healed after 3 weeks. According to Matta reduction criteria, 8 cases obtained anatomical reduction, 4 cases got satisfied results and 1 cases got unsatisfied results. Eleven cases were followed up with an average of (24.0 +/- 8.0) months, and 2 cases were lost to follow-up. According to revised Mede d'Aubingne and Postel evaluation system, 7 cases got excellent results, 2 good, 1 moderate and 1 poor.

CONCLUSIONPosteroproximal-posteroanterior sequential reduction and internal fixation for the treatment of type C1 (AO/ASIF) acetabular fracture can achieve satisfied surgical proces and operation quality.

Acetabulum ; diagnostic imaging ; injuries ; surgery ; Adolescent ; Adult ; Female ; Fracture Fixation, Internal ; Hip Fractures ; diagnostic imaging ; surgery ; Hip Joint ; diagnostic imaging ; surgery ; Humans ; Male ; Middle Aged ; Radiography ; Treatment Outcome ; Young Adult