OBJECTIVETo find sensitive and specific micro-metastic markers for prostate cancer.

METHODSUsing nested reverse transcription-PCR, we examined the expression of PSA, hK2 and PSMA mRNA in peripheral blood mononuclear cells of 51 patients with prostate cancer, 33 patients with benign prostate hyperplasia (BPH) and 32 normal young people.

RESULTSThe expression rates of PSA, hK2 and PSMA mRNA were 52.9%, 43.1% and 64.7%, respectively in prostate cancer group, and 6.2%, 7.7% and 4.6%, respectively in control group (BPH patients and normal young people) with statistical significance (P < 0.01). Although the expression rate of PSA and hK2 mRNA increased with cancer progression, there was no statistical significance among patients in different stages. The expression rate of PSMA mRNA was higher than that of PSA and hK2 mRNA in each clinical stage.

CONCLUSIONPSMA mRNA expression detected by nested RT-PCR is of greater value for the diagnosis, therapy choice and prognostic evaluation of prostate cancer patients.

Aged ; Antigens, Surface ; blood ; Biomarkers, Tumor ; blood ; Glutamate Carboxypeptidase II ; blood ; Humans ; Male ; Middle Aged ; Neoplasm Invasiveness ; Neoplasm Staging ; Prostate-Specific Antigen ; blood ; Prostatic Hyperplasia ; blood ; pathology ; Prostatic Neoplasms ; blood ; pathology ; Tissue Kallikreins ; blood

OBJECTIVETo investigate the influence of human C-type natriuretic peptide (hCNP) on proliferation of vascular endothelial cells (HUVECs).

METHODSReconstructed pcDNA3.1 (+)/hCNP was transfected into HUVECs with polyethylenimine and its plasmid expression was examined with RT-PCR, immunohistochemistry and Western blot. MTT method was used to determine the effect of expressed protein on proliferation of HUVECs. pcDNA3.1 (+)/hCNP transfection was used for control.

RESULTSThe proliferation of HUVEC 48 h after pcDNA3.1 (+)/hCNP transfection was (0.301 +/- 0.096), which was obviously higher than that with pcDNA3.1 (+) transfection (0.164 +/- 0.012). Reconstructed pcDNA3.1 (+)/hCNP might be expressed in HUVECs effectively and its protein expression was capable of promoting HUVECs proliferation markedly.

CONCLUSIONThe successive expression of reconstructed pcDNA3.1 (+)/hCNP and the promoting activity of its expressed protein on HUVECs lay the foundation potential therapeutic value of C-type natriuretic peptide.

Cell Line ; Cell Proliferation ; Endothelial Cells ; cytology ; Humans ; Natriuretic Peptide, C-Type ; genetics ; Plasmids ; RNA, Messenger ; genetics ; Transfection

OBJECTIVETo investigate the influence of hypoxia induction factor-1alpha (HIF-1alpha) on glycosis of rat myocardial cell under hypoxic condition.

METHODSThe myocardial cells of the rats were routinely isolated and cultured. The cells were divided into single hypoxia (H) and HIF-1alpha inhibiting (I) groups. The cells in H group were cultured in glucose-free medium with mixed low-oxygen gas [1% O2, 94% N2 and 5% CO2 (v/v)]. While the cells in I group were cultured with low-oxygen gas after the cell model of low expression of HIF-1alpha protein constructed by RNAi technique. The cells in both groups were all observed before hypoxia (routine culture) and at the time points of 1, 3, 6, 12 and 24 hours of hypoxia. The LA (lactate acid ) content in the supernatant of the culture and the activity of the key enzymes in glycolysis such as hexokinase (HK), phosphofructokinase (PFK) and lactate dehydrogenase (LDH) of both groups of cells were determined at all the time points.

RESULTS(1) After hypoxia, the HK and PFK activities of the rat myocardial cells in H and I groups were obviously increased at the beginning and decreased thereafter when compared with that before hypoxia. While the activities of HK and PFK in H group at 1, 3 and 6 hours after hypoxia were evidently higher than those in I group (P <0.05 or 0.01), and the peak activity of them in H and I groups was 159 +/- 13 U/g vs 133 +/- 55 U/g, and 298 +/- 44 U/g vs 188 +/- 55 U/g, respectively. (2) Compared with normal control (92 +/- 12 U/g), the LDH activity of the cells in H group after hypoxia increased significantly, reaching the peak at 6 hours after hypoxia (2 568 +/- 125 U/g, P < 0. 01), and it decreased thereafter, while that in I group peaked at 3 hours after hypoxia (2125 +/- 126 U/g, P <0.01). The LA content in the culture supernatant in H group increased significantly after hypoxia with the passage of time, while that in I group increased in smaller magnitude (P <0.01).

CONCLUSIONHigh expression of HIF-1alpha in the rat myocardial cells after hypoxia could directly cause continuous enhancement of cell glycolysis, which was beneficial to the protection of myocardial cells under hypoxic condition.

Animals ; Cell Hypoxia ; Cells, Cultured ; Glycolysis ; Hexokinase ; metabolism ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Myocytes, Cardiac ; metabolism ; Phosphofructokinase-1 ; metabolism ; RNA Interference ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the changes in the expression of hypoxia induction factor-1alpha (HIF1-alpha) in myocardial tissues in severely scalded rats during early postburn stage.

METHODSMale Wistar rats inflicted with 40% TBSA III degree scald were employed as the model. The myocardial tissue samples were harvested from the left and right ventricles at different postburn time points, and samples were also obtained from normal rats as control. The mRNA and protein expressions of HIF-1alpha in rat myocardial tissue were determined by RT-PCR and Western blot analysis respectively.

RESULTSThere was a difference of HIF-1alpha expression between left and right ventricles of the normal rats at both transcriptional and translational levels, and the mRNA and protein expressions of HIF-1alpha in the myocardial tissue of scalded rats were increased dramatically at early postburn stage.

CONCLUSIONThe tolerance to ischemia and hypoxia of the rat left ventricle was higher than that of the right ventricle under normal condition. An increase in HIF1alpha expression in rat myocardial tissue could be induced in severely scalded rats.

Animals ; Burns ; metabolism ; Heart Ventricles ; metabolism ; Hypoxia ; physiopathology ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Male ; Myocardial Ischemia ; physiopathology ; Myocardium ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effects of glycine on apoptosis in murine cardiomyocyte suffering from ischemia and hypoxia.

METHODSThe primary passage of cultured cardiomyocytes from neonatal rats were subjected to ischemia and hypoxia, and the cells were divided into IH (without other treatment), and G (with treatment of 5 mmol/L glycine) groups. Normal murine cardiomyocytes served as control (C group). Cardiomyocytes were cultured for 6 hours in vitro. Apoptosis, mitochondrial membrane potential and its distribution, the condition of mitochondria permeability transition pore (mPTP) were observed with expression of fluorescence intensity. The activity of caspase-3 was observed by Laser Scanning staining.

RESULTS(1) Apoptosis: the fluorescence intensity in IH group was obviously higher than that in G and C groups (P < 0.01). (2) Mitochondrial membrane potential: the fluorescence intensity in IH group was 32 +/- 7, which was obviously lower than that in G and C groups (52 +/- 4, 73 +/- 4, respectively, P < 0.01). (3) The condition of mPTP: the intensity in IH group was 27 +/- 4, which was obviously lower than that in G and C groups (62 +/- 8, 90 +/- 7, respectively, P < 0.01). (4) The activity of caspase-3: the activity of caspase-3 in IH group was obviously higher than that in G and C groups (P < 0.01).

CONCLUSIONGlycine can inhibit apoptosis in cardiomyocytes subjected to ischemia and hypoxia,and the effect may be attributable to changes in mitochondrial membrane potential, lessening opening of mPTP, alleviation of calcium overload , and decrease in activity of caspase-3.

Animals ; Apoptosis ; Caspase 3 ; metabolism ; Cell Hypoxia ; drug effects ; Cells, Cultured ; Glycine ; pharmacology ; Ischemia ; metabolism ; Myocytes, Cardiac ; cytology ; drug effects ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the influence of microtubule intervention drugs on glycolytic key enzymes in myocardial cells after hypoxia.

METHODSThe primary passage of cultured myocardial cells from neonatal rats were divided into A group (with hypoxia), B group (with hypoxia and administration of l0 micromol/L colchicine), C group (with hypoxia and administration of 5 micromol/L taxol), D group (with hypoxia and administration of 10 micromol/L taxol), E group (with hypoxia and administration of 15 micromol/L taxol). The morphology of microtubule was observed with laser scanning microscope (LSM). The cell vitality was assayed by cell counting kit (CCK). The activities of hexokinase (HK), pyruvate kinase (PK), phosphofructokinase (PFK) and lactate dehydrogenase (LDH) were assayed with colorimetry.

RESULTSIn group B and E, the microtubule structure was damaged heavily, and the cell vitality was decreased significantly [The cell vitality was (89.99 +/- 3.47)% in B group and (84.56 +/- 6.61)% in E group, respectively, at 1.0 post hypoxia hour (PHH), and hoth values were obviously lower than that in A group (97.44 +/- 1.76)%, P < 0.01]. The HK, PK and PFK activities decreased obviously. The activities of HK, PK and PFK in group C were similar to those of the A group. Compared with that in other groups, the degree of damage of microtubule structure in D group was milden. The activities of HK, PK and PFK in D group during 0.5 - 6.0 PHH were significantly higher than those in A group. The activity of LDH in each group was increased after hypoxia.

CONCLUSIONProper concentration of microtubule-stabilizing drugs can alleviate the damages to microtubule structure, and enhance the activity of glycolytic key enzymes of myocardial cells at early stage of hypoxia.

Animals ; Cell Hypoxia ; Cells, Cultured ; Glycolysis ; drug effects ; Hexokinase ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Microtubules ; drug effects ; metabolism ; Myocytes, Cardiac ; enzymology ; metabolism ; Phosphofructokinase-1 ; metabolism ; Pyruvate Kinase ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effects of the Chinese herbal medicine of Longbixiao (LBX) Capsule on the expressions of TGF-beta1 and Smoothelin in human prostatic stromal cells cultured in vitro.

METHODSBlood serum medicated with LBX was incubated with the stromal cells isolated from men with benign prostatic hyperplasia (BPH) and cultured in vitro. The mRNA expression levels of TGF-beta1 and Smoothelin were detected by real-time RT-PCR and other relevant techniques.

RESULTSIn the high and low concentration groups, the gene relative expressions of TGF-beta1 were (0.158 +/- 0.020) and (0.169 +/- 0.020) , while those of Smoothelin were (0.035 +/- 0.007) and (0.036 +/- 0.007) respectively, both significantly decreased in comparison with the control group(P < 0.01).

CONCLUSIONLBX reduces the mRNA expressions of TGF-beta1 and Smoothelin in human prostatic stromal cells and can be used in the treatment of BPH.

Animals ; Capsules ; Cells, Cultured ; Cytoskeletal Proteins ; genetics ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Gene Expression ; drug effects ; Humans ; Male ; Muscle Proteins ; genetics ; Prostatic Hyperplasia ; pathology ; Rabbits ; Reverse Transcriptase Polymerase Chain Reaction ; Serum ; chemistry ; Stromal Cells ; drug effects ; metabolism ; pathology ; Transforming Growth Factor beta1 ; genetics

OBJECTIVETo construct hypoxic induction factor-1alpha (HIF-1alpha) siRNA expression cassette containing U6 promoter, alpha HIF-1alpha sense or antisense target sequence, and to observe its influence on the expression of cardiomyocytic HIF-1alpha during hypoxic state.

METHODSNeonatal murine cardiomyocytes cultured in the mixed gas were employed as the hypoxic model and were divided into normal control (cultured in normal oxygen), RNAi control (invalidated transfection interference sequence IV) and RNAi effective inhibition (effective transfection interference sequence, which was further divided into I, II and III groups according to the difference of downstream primer) groups. Three pairs (I, II and III) of PCR downstream primer containing HIF-1alpha encoded gene fragments (sense and antisense) and one pair of randomize sequence (IV) PCR downstream primer were designed and synthesized. U6 starter expression frame was constructed by PCR method. The cardiomyocytes were transfected simultaneously by sense and antisense sequence expression frame. Five plates of the cells were set at each time points in each group. The expression of HIF-1alpha mRNA was detected by RT-PCR at 6 hours of hypoxia. The change in the protein expression level at 1 hour of hypoxia was determined by Western blot, and the interference effects were monitored by immunohistochemistry.

RESULTSThe best inhibition fragment screened was group II sequence. After the transfection and hypoxic culture, it was found that the cardiomyocytic HIF1alpha mRNA and protein levels in RNAi effective inhibition group were evidently lower than those in normal control and RNAi control groups (P < 0.01). While the protein inhibition rate (60% - 80%) between the former group and normal and RNAi control groups was no difference (P > 0.05).

CONCLUSIONThe expression of the HIF1alpha in hypoxic rat cardiomyocytes could be effectively inhibited by our constructed HIF1alpha siRNA expression cassette group II.

Animals ; Cell Hypoxia ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Myocytes, Cardiac ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the influence of microtubule intervention drugs on the energy metabolism of myocardial cells after hypoxia.

METHODSThe primary passage of cultured myocardial cells from neonatal rats were divided into A (with hypoxia), B (with hypoxia and administration of 10 micromol/ml colchicine), C (with hypoxia and administration of 5 micromol/ml taxol), D (with hypoxia and administration of 10 micromol/ml taxol) and E (with hypoxia and administration of 15 micromol/ml taxol) groups. The creatine kinase (CK) activity and contents of ATP and ADP were assayed with colorimetry and HPLC, respectively, and the vitality of myocardial cells were determined by trypan blue method at 0.5, 1.0, 3.0, 6.0, 12.0, 24.0 post-hypoxia hours (PHH).

RESULTSThe mortality was obviously higher in B and E groups than those in A group( P < 0.05) at each time-points, but that in C and D groups were markedly lower than those in A group during 6.0 to 24.0 PHH (P < 0.01). The CK activity was significantly higher in B group than that in A group during 1.0 to 24.0 PHH, while that in E group was evidently higher, but it was lower in C and D groups than that in A group at each time-points (P < 0.05 or 0.01). The ATP contents in C group during 0.5 to 6.0 PHH were [(49.9 +/- 2.8), (40.7 +/- 2.0), (25.8 +/- 1.9), (19.1 +/- 1.2) microg/10(6) cells, respectively], which were obviously higher than those in A group [(42.9 +/- 5.8), (29.5 +/- 1.8), (18.2 +/- 0.9), (14.1 +/- 0.7) microg/10(6) cells, respectively, P < 0.05 or P < 0.01, and those in E group at each time-point were significantly lower than those in A and D groups (P < 0.01). The changes in the contents of ADP were on the contrary to the above.

CONCLUSIONMicrotubule-destabilizing drugs and high concentration microtubule-stabilizing drugs can sharply decrease ATP content in myocardiocytes under hypoxic conditions, while suitable amount of microtubule-stabilizing drugs can protect myocardiocytes by promoting its energy production.

Animals ; Cell Hypoxia ; Cells, Cultured ; Colchicine ; pharmacology ; Energy Metabolism ; drug effects ; Microtubules ; drug effects ; metabolism ; Myocytes, Cardiac ; drug effects ; metabolism ; Paclitaxel ; pharmacology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the role of p38 mitogen activated protein kinase ( p38 MAPK) in the regulation of cytosolic phospholipase A2 ( cPLA2 ) expression and degradation of membrane phospholipids in myocardium in early stage of burn rats.

METHODSWistar rats were randomized into normal group (n = 8), burn(n =40) , burn and SB203580(n = 16), burn and isotonic saline( n = 16) groups, with 8 rats at each time-points. There were 5 time-points in burn group, and 2 time-points in other groups. The rats in the latter 3 groups were inflicted with 40% TBSA full-thickness burns, and those in burn and SB203580, burn and isotonic saline groups were administered with SB203580 (p38 MAPK inhibitor) or isotonic saline, respectively. The levels of cPLA2 mRNA and membrane phospholipids in myocardium were detected with RT-PCR. In the same experiment, the effect of SB203580 on cPLA2 expression in rat myocardial cells was determined after hypoxia and burn serum treatment in vitro.

RESULTSThe level of myocardial cPLA2 mRNA in burn group at each time-point was obviously higher than those in normal group (0. 280 +/- 0. 020) , and it reached the peak value at 3 PBH. In contrast, the level of cardiac membrane phospholipids was lowered immediately after burns, and it reached the lowest level at 6 PBH [(0. 052 +/- 0. 017) mg phosphorus/mg protein]. Herein, a significant negative correlation was showed between the levels of cPLA2 mRNA and cardiac membrane phospholipids ( r = - 0. 53, P < 0. 05). Administration of SB203580, however, inhibited the increased activity of p38 MAP kinase, suppressed the upregulation of cPLA2(72% and 51% of those in burn and saline group, P <0. 01) , and markedly increased the levels of membrane phospholipids in myocardium at 6 and 12 PBH. In addition, treatment of cardiac myocytes with SB203580 also abolished the upregulation of cPLA2 mRNA elicited by hypoxia and burn serum challenge.

CONCLUSIONp38 MAP kinase play an important role in the burn-induced degradation of cardiac membrane phospholipids in rat through the upregulation of myocardial expression of cPLA2 mRNA in the myocardial cells.

Animals ; Burns ; metabolism ; Disease Models, Animal ; Myocytes, Cardiac ; metabolism ; Phospholipases A2 ; metabolism ; Phospholipids ; metabolism ; RNA, Messenger ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; p38 Mitogen-Activated Protein Kinases ; metabolism