OBJECTIVETo quantitatively measure plasma level of human telomerase reverse transcriptase (hTERT) mRNA in patients with nasopharyngeal carcinoma (NPC) and explore its implications for NPC diagnosis and treatment.

METHODSWith 24 healthy volunteers serving as controls, the plasma level of hTERT mRNA was detected in 33 NPC patients by real-time PCR before and after treatments with chemotherapy or radiotherapy, and its association with the clinicopathological parameters of the patients were analyzed.

RESULTSThe NPC patients showed a significantly higher mean plasma level of hTERT mRNA than the healthy volunteers (10.75 ± 4.29 vs 0.95 ± 0.37, P<0.05). The plasma hTERT mRNA level in the NPC patients was significantly correlated with clinical staging, tumor size, and degree of nodal metastasis (P<0.05) but with gender or age (P>0.05). In patients with stage I and II NPC, the plasma hTERT mRNA level decreased significantly after radiotherapy (5.60 ± 2.33 vs 3.43 ± 1.42); in patients in advanced stages (III and IV), plasma hTERT mRNA level decreased significantly from 12.68 ± 3.08 to 10.68 ± 2.48 (P<0.05) after chemotherapy and to 3.13 ± 1.69 (P<0.05) after radiotherapy.

CONCLUSIONRadiotherapy and chemotherapy can effectively suppress elevated plasma hTERT mRNA levels in NPC patients. Plasma hTERT mRNA level is closely related to the clinicopathological factors and provides important information for early diagnosis and therapeutic effect evaluation of NPC.

Carcinoma ; Case-Control Studies ; Humans ; Nasopharyngeal Neoplasms ; blood ; RNA, Messenger ; blood ; Telomerase ; blood

OBJECTIVETraditional prenatal diagnosis for congenital diseases were villus sampling and amniocentesis. These invasive diagnosis methods are not only technical complicated, but also harmful to mother or fetus. Fetus in its different gestational age has its different type of hemoglobin or different amount of hemoglobin, especially epsilon hemoglobin exiting in the body of 10 weeks gestation fetal, however gamma hemoglobin has its high amount before baby to be born. But epsilon and gamma hemoglobin did not exist in the bodies of adults bodies. It is possible to use advanced molecular biological technique to extract the fetal hemoglobin gene from maternal peripheral blood. In articles from domestic and abroad, no report related to fetal hemoglobin extraction from maternal peripheral blood was found. We tried to use non-invasive method to detect fetal hemoglobin epsilon/gamma gene from maternal peripheral blood by molecular biological technique. The purpose was to establish a convenient, sensitive and special method to be a basis of screening prenatal diseases in the population and lay a basis for family planning and clinical application.

METHODSBlood samples were collected and the fetal mRNA extracted from the pregnant women with the use of random primer. We used ultraviolet spectrophotometer to test the concentration and purity of extracted mRNA are suitable for reverse transcription. Reverse transcription of mRNA into cDNA was carried out and cDNA by PCR with the special epsilon/gamma primer being used. Via 1.2% EB in agarose gel electrophoresis, we used "Gel Works System" to scan the electrophoresis image to detect epsilon/gamma gene band.

RESULTSThe peripheral blood of pregnant women was collected. With RT-PCR and agarose gel electrophoresis method, we detected epsilon/gamma gene successfully in 7 samples with 6 positive and 1 negative.

CONCLUSIONThis was the first time that we used non-invasive way to detect expression of fetal epsilon/gamma gene in maternal blood to have found that this was a simple method to separate fetal cells from maternal blood, and could easily be accepted by pregnant women. Success of RT-PCR to detect fetal specific mRNA gave the hint that this method could be used in the field of prenatal diagnosis of hemoglobin disease, predicting fetal gender, predicting Rh blood type and single gene disease and be used widespread in prenatal diagnosis.

Female ; Globins ; genetics ; Humans ; Pregnancy ; blood ; RNA, Messenger ; blood ; Reverse Transcriptase Polymerase Chain Reaction ; methods

Biomarkers, Tumor ; blood ; Carcinoma, Hepatocellular ; diagnosis ; Humans ; Liver Neoplasms ; diagnosis ; RNA, Messenger ; blood ; alpha-Fetoproteins ; analysis

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

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

To explore the expression of heme oxygenase-1 (HO-1) in the peripheral blood mononuclear cells (PBMCs) and its relationship with pulmonary ventilation function in asthmatic patients, 18 asthmatic patients and 18 healthy subjects were selected. HO-1 protein and mRNA levels in PBMCs were measured by immunohistochemical staining and reverse transcription-polymerase chain reaction (RT-PCR), respectively. Blood carbon monoxide Hb (COHb), serum total IgE and pulmonary ventilatory function were observed. Our results showed that the percentage of cells positive for immunohistochemical staining of HO-1 were significantly higher in asthmatic patients (41.72 +/- 7.44) % than that in with healthy subjects (10.45 +/- 4.36) % (P < 0.001) and the optical density of PBMC HO-1 mRNA was higher in asthmatic patients (26.05 +/- 4.14) than that in healthy subjects (10. 82 +/- 4.26) (P < 0.001). The relation analysis showed that PBMC HO-1 protein and mRNA levels had significantly negative relation with FEV1%, PEFR, MEFR50%, respectively (r = -0.51-0.89, P < 0.05-0.001, respectively) and a positive relation with COHb and serum total IgE (r = 0.48-0. 85, 0.05-0.001, respectively). It is concluded that the expression of PBMC HO-1 protein and mRNA increased significantly in asthmatic patients, and HO-1 may play a significant role in the pathogenesis of asthma. The expression of HO-1 may bear a relation with severity of asthma.
Asthma/blood ; Asthma/*enzymology ; Carbon Monoxide/blood ; Heme Oxygenase-1/*biosynthesis ; Heme Oxygenase-1/blood ; Immunoglobulin E/*blood ; Leukocytes, Mononuclear/*enzymology ; RNA, Messenger/blood

OBJECTIVETo observe the relationship between variation of IL-2, IL-4, IL-18 and IP10 mRNA expressions in peripheral blood and the occurrence of acute graft-versus-host disease (aGVHD), and investigate whether some cytokines combined expression profiles could improve the diagnostic accuracy of aGVHD.

METHODSA total of 58 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT) were enrolled for the study. Peripheral blood samples were collected at different time points after transplantation. The mRNA expression levels of 4 kinds of cytokines (IL-2, IL-4, IL-18, IP10) were measured by real-time quantitative PCR (RQ-PCR). The relationship between mRNA expression level and the occurrence of aGVHD was analyzed with clinical features.

RESULTSThe expression levels of IL-2 and IL-18 at the onset of aGVHD were much higher than those after engraftment, being 2.69-fold and 3.12-fold increase, respectively (P = 0.000 & P = 0.000). The expressions of IL-2 and IL-18 mRNAs were slightly increased in patients with infection, but not statistically significant (P = 0.208 & P = 0.123). There was a slight but not statistically significant decrease of IL-4 and IP10 mRNA expressions at the onset of aGVHD (P = 0.230 & P = 0.325). Either IL-2 or IL-18 expression level could diagnose aGVHD as an independent factor (P = 0.000 & P = 0.000). The multivariate logistic regression analysis showed that the main factors related to aGVHD were IL-2, IL-18 and IL-4 (β = 1.13, P = 0.068 & β = 1.339, P = 0.047 & β = -0.600, P = 0.008 respectively). A composite panel of these three cytokines produced a better model for the diagnosis of aGVHD (AUC: 0.862, 95%CI: 0.768 - 0.957, P = 0.000), and the sensitivity and specificity were 75.0% & 83.3% respectively.

CONCLUSIONThe diagnosis of aGVHD can be optimized with a composite cytokines panel.

Cytokines ; blood ; Graft vs Host Disease ; diagnosis ; Hematopoietic Stem Cell Transplantation ; Humans ; Interleukin-18 ; blood ; RNA, Messenger

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

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