OBJECTIVE: To detect the expression of nucleolin in cardiac hypertrophy rats induced by pressure overload. METHODS: A total of 40 SD rats with body weight 180 g and 220 g were recruited and randomly divided into 2 groups: a transverse aortic constriction (TAC) group and a sham surgery group. Cardiac hypertrophy model was employed by transverse aortic constriction surgery. Then 2 weeks and 4 weeks after the experiment, the heart mass index (HMI), left ventricle mass index (LVMI) were measured. β-MHC mRNA in the heart tissue was detected with RT-PCR. Nucleolin in the heart, brain and kidney was respectively detected with Western blot. RESULTS: Compared with the sham surgery group, HMI, LVMI in the TAC group increased significantly (P<0.01) 4 weeks after the surgery; the expression of β-MHC mRNA in the heart tissue increased (P<0.05) in the TAC group 4 weeks after the surgery; and the expression of nucleolin protein in the heart tissue of the TAC group was remarkably upregulated (P<0.05) 2 weeks after the surgery, with no change in the brain and kidney tissue between the 2 groups. CONCLUSION Expression of nucleolin protein has been upregulated in response to pressure overload, which may suggest that nucleolin plays a role in cardiac hypertrophy induced by pressure overload.
Animals ; Blood Pressure ; Cardiomegaly ; metabolism ; Myocardium ; metabolism ; Phosphoproteins ; metabolism ; RNA, Messenger ; RNA-Binding Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley

This study was purposed to identify endothelial nitric oxide synthase (eNOS) mRNA in human RBCs during storage and to investigate the relationship of its changing profile and preservation time at 4°C. RT-PCR and gene sequencing were applied to identify eNOS-mRNA in banked RBC. Real time PCR was used to study the relationship of eNOS-mRNA expression and preservation time. The results showed that eNOS mRNA was detected in RBC. Compared with fresh RBC, the content of eNOS mRNA in RBC was 0.868 ± 0.119 stored for 1 week, which was 0.379 ± 0.289, 0.108 ± 0.134, 0.141 ± 0.141, 0.125 ± 0.12 stored for 2, 3, 4 and 5 weeks respectively. It is concluded that eNOS mRNA exists in human RBC and its content is decreasing gradually along with the prolongation of storage time in banked RBC. Stored for 3 weeks, the content of eNOS-mRNA remains to be at lower level of concentration in human RBC.
Blood Donors ; Blood Preservation ; Erythrocytes ; metabolism ; Humans ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism

BACKGROUNDAs one of the intercellular adhesion molecules, CD58 plays important roles in promotion of the adhesion between T cells and target cells, hyperplasia, activation of T cells and natural killer cells, and balance between Th1 and Th2. We studied the relationship between the levels of CD58 expression in peripheral blood mononuclear cells (PBMCs) and severity of HBV infection.

METHODSThe levels of CD58 mRNA in PBMCs were detected using quantitative reverse transcription PCR. The percentage of CD58 positive cells was detected by flow cytometry in patients and healthy controls.

RESULTSThe levels of CD58 mRNA and the percentage of CD58 positive cells in patients infected with HBV were significantly higher than that in the control. Based on severity of HBV infection, the patients were classified into four groups. The expression of CD58 increased significantly in an order from mild chronic, moderate chronic, severe chronic to severe hepatitis groups. The levels of CD58 mRNA and the percentage of CD58 positive cells in PBMCs from patients with HBV infection were both positively correlated with serum levels of ALT and AST.

CONCLUSIONThe level of CD58 expression is related with the severity of HBV infection and the degree of liver damage.

Alanine Transaminase ; blood ; Aspartate Aminotransferases ; blood ; CD58 Antigens ; genetics ; Hepatitis B ; blood ; physiopathology ; Humans ; Leukocytes, Mononuclear ; metabolism ; RNA, Messenger ; analysis

OBJECTIVETo investigate the mRNA expression profiles of megakaryocytes (MKs) from human cord blood CD34+ cells ex vivo expanded using Solexa technique.

METHODSCD34+ Cells were isolated using density gradient centrifugation and magnetic activated cell sorting. Cultures were stimulated with recombinant human thrombopoietin (100 ng/ml). After 12 days, the MKs fraction was separated from the non-MKs fraction using an anti-CD41 monoclonal antibody by immunomagnetic sorting. The mRNA expression of MKs and non-MKs was detected by Solexa sequencing.

RESULTSWe obtained 3 773 147 and 3 533 805 Tags from MKs and non-MKs, respectively. The amounts of unambiguous tags were 3 291 132 and 2 967 947 and those of distinct tags were 197 769 and 245 318. The expression of 1161 genes was up-regulated and that of 902 genes down-regulated. The expression of 2717 tags was up-regulated and that of 1519 tags down-regulated.

CONCLUSIONSMKs and non-MKs have remarkably different mRNA expression profiles. The differential gene-encoded products may be involved in cellular development, adhesion, apoptosis metabolism, intra- and intercellular signal transduction, and immune response. Further studies on this topic may clarify the expression mechanism, signal transduction, and regulation mechanisms.

Antigens, CD34 ; Cells, Cultured ; Fetal Blood ; cytology ; Humans ; Megakaryocytes ; cytology ; metabolism ; RNA, Messenger ; genetics ; Transcriptome

OBJECTIVETo investigate the effects of hydrodynamics-mediated RNAi for Mfn2 gene expression in liver and the levels of blood sugar and fat in mice.

METHODSFifty-six male BALB/c mice were randomly divided into normal control group (NC, n = 8), negative control group (HK, n = 24) and transfection group (Mfn2, n = 24) according to random digits table. 1.5 ml plasmid (negative control or Mfn2 shRNA, 75mug for each mouse) diluted into phosphate buffered solution (PBS) was injected into the HK and Mfn2 groups mice via hydrodynamic intravascular injection. Mfn2 mRNA and protein expression in hepatic tissue was detected by RT-PCR and Western-blot 24 hours, 72 hours and 120 hours respectively after injection. At the same time, the levels of fasted blood sugar (FBS) and triglyceride (TG) were measured.

RESULTSCompared with HK mice, the expressions of Mfn2 mRNA (1.00+/-0.03 vs 1.14+/-0.07, t = 4.027, P = 0.007; 1.01+/-0.053 vs 1.18+/-0.07, t = 4.234, P = 0.006) and protein (7.81+/-0.80 vs 8.01+/-0.08, t = 2.941, P = 0.042; 8.05+/-0.15 vs 8.56+/-0.014, t = 4.883, P = 0.039) decreased markedly in Mfn2 mice in 72 and 120 hours after injection. In the fasting state, in 24 hours after injection, FBS in Mfn2 group was significantly lower than that in HK group [(2.65+/-0.70 vs 5.28+/-0.82) mmol/L, t = 6.879, P value less than 0.01] and TG was also significantly higher than that in HK group [(1.96+/-0.32 vs 1.12+/-0.16) mmol/L, t = -6.711, P value less than 0.01]. No statistical differences found between the NC and HK groups for FBS and TG (F = 1.412, P = 0.26; F = 2.711, P = 0.14). The plasma glucose level in Mfn2 mice was significantly higher than that in HK mice [(7.23+/-0.82 vs 5.18+/-0.69) mmol/L, t = 2.050, P value less than 0.01; (7.00+/-0.67 vs 6.05+/-0.76) mmol/L, t = 3.57, P = 0.023] in 72 and 120 hours after injection. However, no differences found between the two groups for blood TG [(1.53+/-0.27 vs 1.37+/-0.18) mmol/L, t = 0.160, P = 0.23; (1.84+/-0.30 vs 1.52+/-0.37) mmol/L, t = 0.330, P = 0.503].

CONCLUSIONThe data indicate that hydrodynamics- mediated RNAi for Mfn2 gene can effectively inhibit the expression of target gene in mice liver in 72 and 120 hours after shRNA administration, and the inhibition of hepatic Mfn2 can induce glycometabolic and fat metabolic disorder.

Animals ; Blood Glucose ; metabolism ; GTP Phosphohydrolases ; genetics ; metabolism ; Gene Expression ; Hydrodynamics ; Lipids ; blood ; Liver ; chemistry ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; RNA Interference ; RNA, Messenger ; genetics

MicroRNAs (miRNAs) are genomically encoded non-protein-encoding small RNAs, which negatively regulate target gene expression at post-transcriptional level. The present study aimed to investigate whether disorders of miRNAs system were involved in the pathogenesis of hypertension in spontaneously hypertensive rats (SHR). MiRanda, Target Scan and PicTar were utilized for predictive analysis of miRNAs and target genes. MiR-1, miR-133a, miR-155 and miR-208 were selected as the candidate miRNAs potentially related to blood pressure. The expression levels of miR-1, miR-133a, miR-155 and miR-208 in the aorta of 4-, 8-, 16- and 24-week-old SHR and age-matched Wistar-Kyoto (WKY) rats were detected by real-time RT-PCR. The mRNA levels of angiotensin II receptor type 1 (AGTR1a), angiotensin II receptor associated protein (AGTRAP), divalent metal transporter 1 (DMT1), low-density lipoprotein-related protein 1B (LRP1B), fibroblast growth factor-7 (FGF-7), protocadherin 9 precursor (PCDH9), chloride channel protein 5 (CLCN-5), small conductance calcium activated potassium channel protein 3 (KCNN3) and thyroid hormone receptor associated protein 1 (THRAP1), which were predicted to be target genes of differentially expressed miRNAs, were further detected by real-time RT-PCR. The results obtained showed that the expression levels of miR-1, miR-155 and miR-208 in the aorta were significantly different from those in the heart of WKY rats. The miR-155 level was significantly lower in aorta of 16-week-old SHR than that of age-matched WKY rats (P<0.05), but there was no difference between SHR and WKY rats in other age groups. In addition, miR-155 level was negatively correlated to blood pressure (r=-0.525, P<0.05). Both in WKY rats and SHR, miR-208 was most abundantly expressed in 4-week-old rats, but declined significantly in 8-, 16- and 24-week-old rats (P<0.05). No difference in miR-208 levels was observed between age-matched SHR and WKY rats. Moreover, miR-208 expression in aorta was negatively correlated with blood pressure (r=-0.400, P<0.05) and age (r=-0.684, P<0.0001). Neither miR-1 nor miR-133a was differentially expressed in SHR and WKY rats in different age groups. The mRNA levels of predicted target genes were not correlated to miR-155 or miR-208 levels. These results indicate that miR-155 is less expressed in the aorta of adult SHR compared with that of WKY rats and is negatively correlated with blood pressure, suggesting it is possibly involved in the development and pathologic progress of hypertension. The miR-208 expression in rat aorta declines with aging and it may play a role in the blood vessel development.
Animals ; Aorta ; metabolism ; Blood Pressure ; Hypertension ; metabolism ; MicroRNAs ; metabolism ; RNA, Messenger ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY

OBJECTIVETo investigate the relationship between visceral fat depot and adiponectin level in OLETF rats.

METHODSTwenty male OLETF rats and 10 male Long-Evans Tokushima Otsuka (LETO) rats were subjected to regular oral glucose tolerance test (OGTT). The rats were sacrificed at the ages of 8, 32 and 40 weeks for measurements of the body weight, blood glucose, blood lipid level, blood insulin, and weight of the visceral fat.

RESULTSCompared with LETO rats, OLETF rats had significantly higher body weight and visceral fat with impaired glucose tolerance (P<0.05). OLETF rats also had higher blood insulin, TG, FFA and CHOL levels (P<0.05). The plasma adiponectin level was significantly lower in OLETF rats than in LETO rats at different ages (P<0.05). The adiponectin mRNA level in the adipose tissue of OLETF rats was comparable with that in LETO rats, but significantly decreased at 32 and 40 weeks of age (P<0.01).

CONCLUSIONPlasma adiponectin level is significantly correlated to insulin sensitivity and visceral fat depots in OLETF rats, but a lowered APN mRNA expression level is not the main reason for a decreased plasma adiponectin level in the early stage.

Adiponectin ; blood ; genetics ; metabolism ; Animals ; Insulin Resistance ; Intra-Abdominal Fat ; metabolism ; Male ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Inbred OLETF

To investigate TLR2 (Toll-like receptor 2) mRNA expression in ischemic hepatic lobes under the condition of partial hepatic ischemia/reperfusion injury in BALB/c mice and its relationship with liver function impairment. A partial ischemia/reperfusion injury model was established. The portal vein and hepatic artery supply to the median and left lobes of the liver were obstructed by an atraumatic artery micro-clip, with the obstruction lasting for about 60 min. Then reperfusion was fulfilled by removal of the clip. The liver samples were collected at the 4th h after the restoration of blood inflow. Total RNA was extracted from the liver samples and analyzed quantitatively by method of real-time PCR. At the same time, portal vein serum and plasma were taken respectively for further detection of the level of endotoxin, tumor necrosis factor alpha (TNF-alpha) and plasmic alanine aminotransferase (pALT). The results indicated that TLR2 mRNA in ischemic lobe was up-regulated markedly in mice partial liver ischemia/reperfusion injury model compared to that in sham operation group (deltaCt: 1.05 +/- 1.02 vs 5.08 +/- 1.36, P<0.001). The level of portal vein pALT and TNF-alpha increased significantly (112.32 +/- 17.56 pg/ml vs 6.07 +/- 5.33 pg/ml, P<0.01; 890 +/- 127 microm/L vs 30 +/- 5 microm/L, P<0.001) . However, the level of portal vein endotoxin remained below the normal line, suggesting a state of non-endotoxemia. TLR2 mRNA expression in ischemic lobe, as well as portal vein pALT and TNF-alpha, was up-regulated in the model of mice partial ischemia/reperfusion injury, suggesting the involvement of TLR2 in ischemia/reperfusion pathological process.
Liver/*blood supply ; Liver/metabolism ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; RNA, Messenger/physiology ; Reperfusion Injury/etiology ; Reperfusion Injury/*metabolism ; Toll-Like Receptor 2/*biosynthesis ; Toll-Like Receptor 2/genetics ; Toll-Like Receptor 2/physiology ; Up-Regulation

OBJECTIVETo investigate the relationship between the expression of murine double minute 2 (MDM2) oncogene and non-Hodgkin lymphoma (NHL) in childhood.

METHODSThirty-one cases of NHL were enrolled in this study as patient group and 8 cases of lymphadenitis as control group. (1) Immunohistochemistry ultrasensitive S-P assay was used to detect the expression of MDM2 protein in pathological tissues in all cases. Positive cells were dyed yellow or brown in nuclei. MDM2 positive cell was defined as >/= 10% of the tumor cells were positive, which was overexpression of MDM2 protein. (2) RT-PCR (reverse transcription-polymerase chain reaction) was performed to value the overexpression of MDM2 mRNA in the pathological tissues and mononuclear cells in peripheral blood. While the ratio of MDM2/beta-actin was >16% was defined as overexpression of MDM2 mRNA.

RESULTS(1) Rates of overexpression of MDM2 protein and MDM2 mRNA were 64.5% and 61.3%, respectively, which were significantly different as compared to that of control group (P < 0.05 and P < 0.01, respectively). (2) The relationship analysis among subgroups in the experiment group showed that the overexpression of MDM2 protein did not correlate with classifications of working formulation, cellular origin, sex, clinical stage and involved extranodal sites (P > 0.05), but significantly correlated with classifications of B status and the increased serum LDH level (P < 0.05). It was shown that the overexpression of MDM2 mRNA did not correlate with classifications of working formulation, cellular origin, sex and clinical stage (P > 0.05), significantly correlated with B status (P < 0.05), and was remarkably significantly correlated with the involved extranodal sites and the increased serum LDH level (P < 0.01). (3) It was demonstrated that the overexpression of MDM2 mRNA in the pathological tissues was similar to the overexpression of MDM2 protein in the pathological tissues and MDM2 mRNA in peripheral blood (P > 0.05, kappa = 0.655 and 0.571), and the overexpression of MDM2 protein in the pathological tissues was similar to that of MDM2 mRNA in peripheral blood (P > 0.05, kappa = 0.609).

CONCLUSIONS(1) The rate of MDM2 oncogene overexpression was quite high. (2) The overexpression of MDM2 protein in pathological tissues determined by using immunohistochemistry ultrasensitive S-P assay was similar to that of MDM2 mRNA in pathological tissues detected by using RT-PCR method. Both methods might be used to detect the overexpression of MDM2 oncogene in the cases of childhood NHL. (3) The overexpression of MDM2 oncogene related to the poor status and poor prognosis of patients with childhood NHL.

Biomarkers, Tumor ; analysis ; blood ; Child ; Humans ; Immunohistochemistry ; Lymphoma, Non-Hodgkin ; blood ; genetics ; metabolism ; Neoplasm Proteins ; blood ; genetics ; Oncogenes ; Proto-Oncogene Proteins c-mdm2 ; blood ; genetics ; metabolism ; RNA, Messenger

The objective of this study was to investigate the expressions of TPO receptor (c-mpl) proteins on CD34 positive bone marrow cells (CD34+ BMCs), platelets and the expression of c-mpl mRNA in bone marrow cells of the patients with polycythemia vera (PV). The expressions of c-mpl proteins on CD34+ bone marrow hematopoietic cells of 13 PV patients and 15 normal controls were assessed by bicolor flow cytometry and the expressions of c-mpl proteins on platelets of 15 PV patients and 15 normal controls were assessed by monocolor flow cytometry, and the expressions of c-mpl mRNA in bone marrow hematopoietic cells (BMHCs) were assessed by RT-PCR. The results showed that no difference was found between the expression of c-mpl proteins on CD34+ BMHCs of PV patients (0.99% +/- 0.14%) and that of normal controls (0.92% +/- 0.12%) (p > 0.05). There was no difference too between the expression of c-mpl protein on platelets in PV patients (20.33% +/- 4.84%) and that in normal controls (23.50% +/- 3.64%) (p > 0.05). No difference between the expression of c-mpl mRNA in BMHCs of PV patients and that in normal controls was seen. In conclusion, the expressions of c-mpl proteins on CD34+ BMHCs, platelets and c-mpl mRNA in BMHCs of PV patients were not obviously abnormal.
Antigens, CD34 ; analysis ; Blood Platelets ; metabolism ; Bone Marrow Cells ; metabolism ; pathology ; Hematopoietic Stem Cells ; metabolism ; pathology ; Humans ; Polycythemia Vera ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Receptors, Thrombopoietin ; metabolism